Urocortin 1 expression and secretion by human umbilical vein endothelial cells: In vitro effects of interleukin 8, interferon γ, lipopolysaccharide, endothelin 1, prostaglandin F-2α, estradiol, progesterone and dexamethasone.

Urocortin 1 (Ucn1) is a 40-amino-acid peptide that has vasodilatory activity and displays immunomodulatory and antioxidant properties. Maternal and cord plasma Ucn1 levels are increased in preeclampsia and preterm labor, but the mechanisms of such increase are poorly known. Thus, we investigated Ucn1 localization in human umbilical cord and assessed some potential stimuli to Ucn1 release by human umbilical vein endothelial cells (HUVEC). Human umbilical cords were obtained at uncomplicated term pregnancy (n=11). Ucn1 localization was assessed by immunohistochemistry and quantified. HUVEC were grown in vitro to confluence, then incubated with serial concentrations of interleukin (IL)-8, interferon (INF)-γ, lipopolysaccharide (LPS), endothelin (ET)-1, prostaglandin (PG)F-2α, estradiol, progesterone and dexamethasone and Ucn1 concentrations were measured in the supernatants. Ucn1 was immunolocalized with similar intensity in umbilical cord arteries, vein and Wharton's jelly. Ucn1 mRNA was detected in all HUVEC cultures and Ucn1 peptide was detectable in culture medium from untreated cells at different time points. Incubation with IFN-γ increased Ucn1 secretion in a dose-dependent manner. Treatments with IL-8, LPS, ET-1 and dexamethasone were able to increase three to fourfold Ucn1 release from cultured endothelial cells. In conclusion, umbilical vessels express Ucn1 and may be a contributive source of Ucn1 release into fetal-placental circulation. IL-8, IFN-γ, LPS, ET-1 and dexamethasone promote Ucn1 secretion from cultured HUVEC.

Reversal of autoimmune diabetes by restoration of antigen-specific tolerance using genetically modified Lactococcus lactis in mice.

Current interventions for arresting autoimmune diabetes have yet to strike the balance between sufficient efficacy, minimal side effects, and lack of generalized immunosuppression. Introduction of antigen via the gut represents an appealing method for induction of antigen-specific tolerance. Here, we developed a strategy for tolerance restoration using mucosal delivery in mice of biologically contained Lactococcus lactis genetically modified to secrete the whole proinsulin autoantigen along with the immunomodulatory cytokine IL-10. We show that combination therapy with low-dose systemic anti-CD3 stably reverted diabetes in NOD mice and increased frequencies of local Tregs, which not only accumulated in the pancreatic islets, but also suppressed immune response in an autoantigen-specific way. Cured mice remained responsive to disease-unrelated antigens, which argues against excessive immunosuppression. Application of this therapeutic tool achieved gut mucosal delivery of a diabetes-relevant autoantigen and a biologically active immunomodulatory cytokine, IL-10, and, when combined with a low dose of systemic anti-CD3, was well tolerated and induced autoantigen-specific long-term tolerance, allowing reversal of established autoimmune diabetes. Therefore, we believe this method could be an effective treatment strategy for type 1 diabetes in humans.

Viral infections and diabetes.

Type 1 diabetes mellitus (T1DM) is a multi-factorial autoimmune disease determined by the interaction of genetic, environmental and immunologic factors. One of the environmental risk factors identified by a series of independent studies is represented by viral infection, with strong evidence showing that viruses can indeed infect pancreatic beta cells with consequent effects ranging from functional damage to cell death. In this chapter we review the data obtained both in man and in experimental animal models in support of the potential participation of viral infections to Type 1 diabetes pathogenesis, with a particular emphasis on virus-triggered islet inflammation, beta-cell dysfunction and autoimmunity.

Increased expression of microRNA miR-326 in type 1 diabetic patients with ongoing islet autoimmunity.

BACKGROUND: The current paradigm that microRNAs represent a new layer of gene regulation has generated much interest in this field. MicroRNAs have emerged as important regulatory factors involved in the developmental processes and in the regulation of insulin secretion and signalling. Furthermore, recent studies revealed an altered microRNA profiling in lymphocytes of patients with autoimmune diseases like multiple sclerosis, in which a hyperexpression of miR-326 was reported. Here, we analysed the expression levels of miR-326 in peripheral blood lymphocytes from type 1 diabetic (T1D) patients in relationship with ongoing islet autoimmunity. METHODS: Peripheral blood lymphocytes were obtained from 19 T1D patients; 4/19 patients were positive for both glutamic acid decarboxylase (GAD) and islet cell antigen 512 autoantibodies; 10/19 were single GAD or IA-2 Ab positive and 5/19 were GAD antibodies and IA-2 antibodies (IA-2A) negative. Quantitative analysis of miR-326 was performed using specific stem-loop primers followed by real-time polymerase chain reaction. All values were normalized to endogenous control U6. RESULTS: miR-326 resulted increased in Ab-positive versus Ab-negative T1D subjects. Its expression levels were 2.05±0.38-fold increased in peripheral blood lymphocytes from patients expressing both GADA and IA-2A and 2.93±0.46-fold increased in single Ab-positive versus Ab-negative individuals (p<0.05). CONCLUSION: In conclusion, we have shown that miR-326 is expressed at higher levels in T1D subjects with ongoing islet autoimmunity, similar to what has been observed in multiple sclerosis, in which levels of this microRNA were highly correlated with disease severity. Interestingly, an online search of miR-326 predicted targets revealed vitamin D receptor and Erythroblastosis virus E26 oncogene homologue 1, two molecules highly involved in immune regulation.

Virus infections: lessons from pancreas histology.

Type 1 diabetes mellitus is a chronic autoimmune disease resulting from the progressive immune-mediated destruction of pancreatic beta cells in genetically susceptible individuals, with the likely contribution of environmental factors, among which viruses have been extensively studied. The pathologic hallmark of the disease is insulitis-a process characterized by islet infiltration of immunocompetent cells that has been well characterized in animal models of islet autoimmunity, and to a lesser extent, in humans. Insulitis characterization has provided valuable information to gain insights into the disease pathogenesis. We review the recent literature on the viral contribution to beta-cell destruction and dysfunction in type 1 diabetes, with particular reference to the pathology of the pancreatic islet in humans and in animal models of the disease.

Activin A in asphyxiated full-term newborns with hypoxic ischemic encephalopathy.

Activin-A is a protein over-expressed and secreted by the brain after neuronal destruction. We evaluated whether serum activin-A increases in asphyxiated full-term newborns (AFTNs) at risk of hypoxic-ischemic-encephalopathy (HIE). 105 consecutive infants (35 affected by perinatal asphyxia due to acute fetal distress; 70 healthy gestational-age matched newborns) underwent cranial assessment and neurologic examination at 12, 24 and 72 hours after birth and, on discharge from the hospital and; activin-A and monitoring laboratory variables assessment at birth. According to the occurrence of HIE within 7-days after birth, AFTNs were subdivided in Group A (n= 20; no/mild HIE with good prognosis) and Group B (n= 15; moderate/severe HIE with a greater risk of neurological handicap). Activin-A was significantly (P less than 0.0001) higher in Groups A and B than controls and highest (P less than 0.001) in Group B. At 0.66 ng/L activin-A achieved a sensitivity of 93.33 per cent and a specificity of 96.63 per cent, respectively, as HIE diagnostic test. These findings show that activin A increased in AFTNs with HIE before the appearance of related signs.

Evaluation of endometrial activin A secretion for prediction of pregnancy after intrauterine insemination.

OBJECTIVE: To measure activin A concentrations in uterine washing fluid collected from women with couple infertility undergoing intrauterine insemination (IUI). DESIGN: Retrospective case-control study. SETTING: Tertiary university center for women's care. PATIENT(S): Fifty women, of whom 25 became pregnant after up to three IUI attempts and 25 did not. INTERVENTION(S): Endocrine and clinical evaluation, semen analyses and hypo-osmotic swelling test, ovarian stimulation, endometrial thickness measurement by ultrasound, uterine washing fluid collection by sonohysterography, and IUI. MAIN OUTCOME MEASURE(S): Activin A measurement by enzyme-linked immunosorbent assay, receiver operating characteristics curve analysis, and pregnancy rates after IUI; sensitivity, specificity, positive and negative likelihood ratios of activin A, and endometrial thickness for pregnancy prediction. RESULT(S): Activin A was measurable in all samples evaluated, and the levels (mean +/- SEM) were statistically significantly higher in the pregnant (0.08 +/- 0.01 ng/mL) than in the nonpregnant (0.022 +/- 0.001 ng/mL) group. Activin A at the cut off of 0.04 ng/mL achieved a sensitivity of 76.0 % (95% CI, 54.9%-90.6%) and a specificity of 100% (95% CI, 86.2%-100%) as a single marker for prediction of pregnancy. CONCLUSION(S): Activin A is secreted into uterine lumen; the levels in endometrial washing fluids were higher in women who subsequently became pregnant after IUI, so its measurement may be useful in predicting successful implantation.

Amniotic fluid urocortin, CRF, oestriol, dehydroepiandrosterone sulfate and cortisol concentrations at mid-trimester: putative relationship with preterm delivery.

OBJECTIVE: Stress-related peptide and steroid hormones are involved in the pathogenesis of preterm delivery, even though their clinical usefulness as predictive markers of preterm delivery remains unclear. The present study evaluated whether mid-trimester amniotic fluid concentrations of stress-related peptides, that is corticothophin-releasing factor (CRF) and urocortin (Ucn) and feto-placental steroids (oestriol, DHEA-S and cortisol) correlated with preterm delivery. STUDY DESIGN: It is a retrospective case-control study. Healthy women (n=130) undergoing amniocentesis at mid-gestation for genetic indications, of whom 15 had a preterm delivery (cases) and 115 delivered at term (controls). CRF, urocortin, cortisol, DHEA-S and oestriol concentrations were measured by specific and sensitive immunoenzymatic assays. RESULTS: Amniotic fluid urocortin concentrations in the cases (0.50+/-0.07 ng/ml) (M+/-SD) were significantly lower (P<0.0001) than in the control group (0.90+/-0.26 ng/ml), while CRF concentrations did not differ between the cases (1.52+/-0.39 ng/ml) and control group (1.64+/-0.68 ng/ml). Amniotic fluid cortisol (17.71+/-3.72 ng/ml vs. 17.32+/-3.17 ng/ml), DHEA-S (0.16+/-0.06 ng/ml vs. 0.17+/-0.09 ng/ml) and oestriol (4.68+/-1.95 ng/ml vs. 4.79+/-1.84 ng/ml) concentrations were similar in the two groups. CONCLUSIONS: The low amniotic fluid concentrations of urocortin at mid-trimester may be a signal of predisposition to preterm delivery, while the unchanged CRF and steroid hormones concentrations in women delivering preterm suggest that this mechanisms are not yet activated at mid-trimester.

Activin A, activin receptor type II, nodal, and cripto mRNA are expressed by eutopic and ectopic endometrium in women with ovarian endometriosis.

Activin A is a dimeric protein that regulates endometrial functions by signaling at its receptors, namely type I (ActRI) and type II (ActRII). Nodal is an activin competitor that requires the coreceptor cripto to assemble its signaling pathway through ActRI and ActRII. In the current study, we evaluated the expression of activin A, ActRII, nodal, and cripto in eutopic and ectopic endometrium collected from women with ovarian endometrioma (n = 15) and in eutopic endometrium of healthy participants (n = 15). Eutopic endometrial samples were evaluated according to the stage of menstrual cycle. Total RNA was extracted from tissue homogenates and analyzed by real-time polymerase chain reaction (PCR). Activin A messenger RNA (mRNA) expression in eutopic endometrium of patients with endometriosis was significantly higher than in controls (P < .001) with a 10.2-fold and 7.3-fold increase in the proliferative and secretory phases, respectively. ActRII and nodal mRNA expression were found to be similar in patients with and without endometriosis, while cripto mRNA was markedly lower in eutopic (fold change = 0.03 at proliferative phase, P < .001) and ectopic endometrium (fold change = 0.14, P < .001) of women with endometriosis compared with eutopic endometrium from healthy controls. In conclusion, the altered endometrial expression of activin A and cripto during the menstrual cycle and the differences observed in the endometriotic tissue support the involvement of the activin system in endometrial changes of women with endometriosis.

Low serum and peritoneal fluid concentration of interferon-gamma-induced protein-10 (CXCL10) in women with endometriosis.

OBJECTIVE: To evaluate serum and peritoneal fluid concentrations of interferon-gamma-inducible protein-10 (CXCL10), a chemokine involved in local immune function, in women with endometriosis. DESIGN: Prospective study. SETTING: Division of Obstetrics and Gynecology, University of Siena. PATIENT(S): A total of 147 women were divided in two groups: women with (n = 77) and without (n = 70) endometriosis. INTERVENTION(S): Serum and peritoneal fluid were collected from all patients undergoing laparoscopy. MAIN OUTCOME MEASURE(S): CXCL10 concentrations were measured by a specific ELISA. RESULT(S): Serum CXCL10 concentrations in women with endometriosis were significantly lower than in those without endometriosis. No statistically significant difference between women with early endometriosis and those with advanced endometriosis was found. CXCL10 concentrations in peritoneal fluid of women with advanced endometriosis were significantly lower than in that of women with an early stage of, or without, endometriosis. CONCLUSION(S): The decreased concentrations of CXCL10 in serum and peritoneal fluid of women with endometriosis indicate an impaired immune activity in women with endometriosis.

Changes of placental Kiss-1 mRNA expression and maternal/cord kisspeptin levels at preterm delivery.

Kisspeptin, a placental polypeptide secreted throughout pregnancy, is suggested to play a role at parturition. Here we evaluated whether its placental mRNA expression and maternal/fetal plasma levels change at term and preterm delivery, and its effect on oxytocin secretion from placental explants. Samples were collected from 40 women with singleton pregnancies who underwent elective cesarean section at term (TNL), term vaginal delivery (TD), and preterm vaginal delivery (PTD). Plasma Kisspeptin and oxytocin levels were assessed by ELISA; placental mRNA expression by Real-time quantitative RT-PCR analysis. Placental expression was significantly (P < 0.0001) higher in PTD than TNL and TD and significantly (P < 0.001) higher in TD than TNL. Maternal/fetal plasma concentrations did not differ among the groups, and maternal were significantly higher than fetal levels (P < 0.05). In placental explants increasing doses of kisspeptin did not modify oxytocin secretion. In conclusion, labor is associated with increased placental KiSS-1 expression without changes in maternal/fetal circulation.

Evaluation of endometrial urocortin secretion for prediction of pregnancy after intrauterine insemination.

BACKGROUND: Urocortin is a neuropeptide produced by the human endometrium and has biological effects putatively important for promoting blastocyst implantation. We measured urocortin concentrations in samples of endometrial wash fluid collected from women with unexplained infertility who underwent intrauterine insemination (IUI). METHODS: Patients 28-42 years of age (n = 71) were consecutively enrolled after a complete clinical evaluation. Endometrial wash fluid was retrieved before IUI, at the time of ultrasound evaluation of endometrial thickness. Urocortin concentrations were assayed with a specific ELISA. RESULTS: After IUI, 28 patients (39%) became pregnant. Urocortin concentrations were significantly higher in women who became pregnant than in those who did not (0.38 microg/L vs 0.13 microg/L, P <0.0001). At a cutoff of 0.321 microg/L, urocortin results were positive in 61% [95% confidence interval (CI), 41%-78%] of women who had successful implantation and negative in 98% (95% CI, 88%-99.6%) of those who did not. The pregnancy rate for women with urocortin concentrations >0.32 microg/L was 94%, which differed significantly (P <0.05) from the overall pregnancy rate of 39% in the study population. CONCLUSIONS: Urocortin is measurable in endometrial wash fluid, and its concentrations before IUI are higher in women who subsequently achieve pregnancy. These data suggest that the probability of having a successful pregnancy-producing IUI may be better estimated by measuring urocortin in endometrial wash fluid.

Urocortin 2 and urocortin 3 are expressed by the human placenta, deciduas, and fetal membranes.

OBJECTIVE: Urocortin 2 (UCN2) and urocortin 3 (UCN 3) are recently identified neuropeptides showing homology to corticotropin-releasing factor (CRF). In the present study, we evaluated their expression and localization in gestational tissues (placenta, decidua, fetal membranes), and their effect on placental adrenocorticotropic hormone secretion. STUDY DESIGN: The study was performed in a tertiary clinical care center. Tissues were obtained at first (n = 8; 8-11 weeks of pregnancy) and third (n = 8; 38-40 gestational weeks) trimester. The mRNA expression was evaluated by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR); the cellular localization by immunohistochemistry; ACTH levels were measured in media collected from cultured placental villi. RESULTS: All tissues analyzed expressed UCN2 and UCN3 mRNA. UCN2 and UCN3 were localized in cytotrophoblast and syncytiotrophoblast cells; UCN2 was present in maternal and fetal vessels and in amniotic cells, while UCN3 was absent. Finally, UCN2 and UCN3 did not stimulate ACTH secretion. CONCLUSION: Gestational tissues differentially express UCN2 and UCN3 and, despite their homology to CRF, they did not stimulate placental ACTH secretion.

Estrogen receptor gene polymorphisms are associated with recurrence of endometriosis.

The presence of gene polymorphisms of the estrogen receptors ERalpha (PvuII and XbaI) and ERbeta (AluI) in 61 women with endometriosis was investigated. A statistically significant correlation between PvuII ERalpha gene polymorphism (PvuII), both in homozygosity (PP) and in heterozygosity (Pp), and a recurrence of endometriosis was found. In conclusion, women affected by endometriosis with the ERalpha polymorphic allele, even if heterozygous, have a worse prognosis, and these results suggest that the ERalpha gene polymorphisms may be included among the genetic risk factors for endometriosis.

Inhibin alpha-subunit and the inhibin coreceptor betaglycan are downregulated in endometrial carcinoma.

OBJECTIVE: In the present study we evaluated the protein distribution and mRNA levels of inhibin alpha-subunit and its coreceptor betaglycan in endometrial adenocarcinoma. DESIGN: Two groups of postmenopausal women were studied: the first group had recently diagnosed endometrial adenocarcinoma (n = 16; age range 61-79 years), and the second group (n = 12; age range 64-78 years) had undergone hysterectomy for uterine prolapse and served as control. METHODS: Inhibin alpha-subunit and betaglycan gene expression and tissue distribution were evaluated by semiquantitative RT-PCR and immunohistochemistry respectively. RESULTS: Inhibin alpha-subunit and betaglycan mRNAs were expressed by both healthy and tumoral endometria, but their expression was significantly lower in endometrial carcinoma (P < 0.001, based on Student's t test). Inhibin alpha-subunit expression was much weaker in the glands of tumours than in non-neoplastic specimens. Betaglycan protein was identified in the epithelial cells lining non-tumoral endometrium, and in endothelial cells of both normal and tumoral endometria. Well-differentiated neoplastic cells had a faint and scarce betaglycan staining, and poorly differentiated cells did not express betaglycan at all. CONCLUSIONS: The lower inhibin alpha and betaglycan expression in endometrial adenocarcinoma suggests that the inhibin action may be disrupted. However, the expression of betaglycan in the endothelia of the tumour vasculature suggests that a selective vascular response to inhibin may be possible in these tumours.