Isabella Viani

Ghrelin Inhibits Steroid Biosynthesis by Cultured Granulosa-Lutein Cells

CONTEXT Growing evidence indicates that ghrelin may participate in the regulation of different aspects of reproductive function. The genes encoding for this peptide and its receptor are expressed in the human ovary, but their functional role is still unknown. OBJECTIVE The aim of our study was to assess whether ghrelin has any effect on steroid synthesis by human granulosa-lutein cells and to identify the receptor isoform through which this potential effect is exerted. DESIGN, PATIENTS, AND METHODS Thirty-five women with spontaneous ovulatory cycles undergoing in vitro fertilization for infertility due to uni- or bilateral tubal impatency or male factor were studied. Granulosa-lutein cells obtained from follicular fluid were incubated with increasing amounts of human acylated ghrelin (10(-11) to 10(-7) mol/liter) either alone or together with a 1:500 concentration of a specific anti-ghrelin receptor antibody [GH secretagogue receptor 1a (GHS-R1a)]. Culture media were tested for estradiol (E(2)) and progesterone (P(4)). The expression of GHS-R1a and GHS-R1b in human granulosa-lutein cells was also studied by real-time quantitative PCR. RESULTS E(2) and P(4) concentrations in the culture media were significantly reduced by ghrelin in a dose-dependent fashion. The maximal decrease in E(2) (25%) and P(4) (20%) media concentrations was obtained with the 10(-7) and 10(-8) mol/liter ghrelin concentrations, respectively. The inhibitory effect of all ghrelin concentrations used was antagonized by the specific anti-ghrelin receptor-1a antibody added to the culture media and not by the specific anti-ghrelin receptor-1b antibody. Both 1a and 1b isoforms of the GHS-R were expressed in human granulosa-lutein cells, with the latter exceeding the formers expression (GHS-R1b/GHS-R1a ratio, 143.23 +/- 28.15). CONCLUSIONS Ghrelin exerts an inhibitory effect on granulosa-lutein cells steroidogenesis by acting through its functional GHS-R1a. This suggests that ghrelin may serve an autocrine-paracrine role in the control of gonadal function and be part of a network of molecular signals responsible for the coordinated control of energy homeostasis and reproduction.

Evidence for epigenetic abnormalities of the androgen receptor gene in foreskin from children with hypospadias.

CONTEXT Hypospadias is a malformation of the penis due to an incomplete development of the male urethra, the exact etiology of which in the majority of cases remains unknown. OBJECTIVE The objective of the study was to assess whether defects of the androgen receptor (AR) gene (CAG repeats and methylation pattern) and DNA methyltransferases (DNMT) family are present in hypospadic patients. DESIGN CAG repeats length, methylation status, and expression of the AR gene were analyzed. The DNMT family was studied at the protein level and the DNMT3A sequenced. SETTING The study was performed at a pediatric endocrinology referral clinic. PATIENTS OR OTHER PARTICIPANTS Twenty boys with isolated glandular hypospadias and 20 age-matched control children undergoing a surgical procedure for circumcision were studied. MAIN OUTCOME MEASURE(S) CAG repeats length and AR methylation pattern in PBLs and foreskin tissue, DNMT expression and sequencing in patients and controls, and in vitro studies in cultured fibroblasts were measured. RESULTS AR gene methylation in foreskin tissues from patients with hypospadias was higher than in normal children. AR expression in foreskin tissue of hypospadic patients was lower than in controls, whereas the DNMT3A protein level was significantly higher in patients than controls. In cultured fibroblasts, both dihydrotestosterone and testosterone significantly reduced AR gene methylation and DNMT3A expression in a dose-dependent fashion and increased AR expression. CONCLUSION The AR gene in target tissues from patients with hypospadias is more methylated than in control children, resulting in a decreased expression of the AR. The mechanism underlying the modulation of the AR gene expression seems to be mediated by DNMT3A. This epigenetic alteration of the AR gene might be involved in the pathogenesis of hypospadias.

Impairment of insulin receptor signal transduction in placentas of intra-uterine growth-restricted newborns and its relationship with fetal growth

OBJECTIVE Intra-uterine growth restriction (IUGR) is related to a higher incidence of type 2 diabetes mellitus. We previously reported reduced adiponectin and increased interleukin 6 (IL6) concentrations in IUGR placentas, which are features of insulin resistance. We aimed to investigate placental insulin receptor (IR) function and activation in human placenta and subsequently the relationships of insulin signalling peptides with placental protein content in IL6, insulin, resistin and adiponectin, and with parameters of fetal growth. DESIGN AND METHODS Whole villous tissue was collected from 18 IUGR and 24 appropriate for gestational age (AGA) placentas of comparable gestational age. Insulin signalling peptides, suppressors of cytokine signalling-2 (SOCS2), insulin, adiponectin, resistin, and IL6 concentrations were determined by using western immunoblotting or specific research kits. RESULTS The amount of total IR was similar in both groups but activated IR significantly higher in IUGR. Total IR substrate-1 (IRS1) was increased in IUGR, whereas total IRS2 and activated IRS1 were similar. AKT content was reduced and activated AKT was undetectable in IUGR placentas. c-Jun N-terminal kinase content was reduced in IUGR. Total and activated ERK1/2 was similar in IUGR and AGA groups, and total SOCS2 was increased in IUGR. IL6 lysate concentrations correlated with AKT content and activated IR. Correlations were found also with adiponectin and resistin. SOCS2 correlated negatively with all growth parameters at birth. CONCLUSIONS IR was more activated in placentas of IUGR compared with AGA; however, signal transduction downstream of the receptor was impaired. The increase in activated IR could be in favour of a compensatory mechanism to increase insulin sensitivity. Close relationships of insulin action in placenta with fetal growth were shown.

Changes and relationships of IGFS and IGFBPS and cytokines in coeliac disease at diagnosis and on gluten-free diet

Objective  To evaluate changes and relationships of IGFs and IGFBPs, serum interleukin 6 (IL‐6) and tumour necrosis factor (TNF)‐α, and auxological parameters at diagnosis of coeliac disease (CD) and at 6 months and 12 months after starting a gluten‐free diet (GFD), compared with a control population.

Markers of insulin sensitivity in placentas and cord serum of intrauterine growth-restricted newborns.

Objective  Intrauterine growth restriction (IUGR) has been related to a higher incidence of insulin resistance in adult life, which is associated with low adiponectin and high resistin, insulin and interleukin (IL)‐6 serum concentrations. This study assessed cortisol, insulin, total insulin receptor, resistin, adiponectin and IL‐6 concentrations, as markers of insulin sensitivity, in placental lysates and cord serum of IUGR and appropriate for gestational age (AGA) newborns, to establish relationships among peptides and with growth parameters at birth.

The IGF system and cytokine interactions and relationships with longitudinal growth in prepubertal patients with cystic fibrosis

Objective  Growth delay is a feature of patients with cystic fibrosis (CF). CF is a condition characterized by chronic inflammation that has been shown to modify the IGF system, which is essential for normal growth, and is related to pulmonary function in CF patients. We aimed to verify whether circulating levels of tumour necrosis factor (TNF)‐α, interleukin (IL)‐6, insulin and the IGF system were related and/or had relationships with linear growth in children with CF.

Differentiation of leptospires of the serogroup Pomona by monoclonal antibodies, pulsed-field gel electrophoresis and arbitrarily primed polymerase chain reaction

All reference strains described as representing separate serovars belonging to the serogroup Pomona and a clinical leptospiral isolate (LP2) from this serogroup were analyzed using a battery of 9 monoclonal antibodies, pulsed-field gel electrophoresis (PFGE) and arbitrarily primed polymerase chain reaction (AP-PCR). Monoclonal antibody analysis provided taxonomic results which were in agreement with the current classification of the serogroup Pomona into six serovars and allowed the classification of the isolate LP2 in the serovar pomona. PFGE and AP-PCR, although in general agreement with monoclonal antibody analysis, also were able to demonstrate some differences in the restriction patterns of strains Pomona, Monjakov and CB. These results indicate that these strains, grouped within serovar pomona after the introduction of bacterial restriction endonuclease analysis as the typing method, but formerly described as representing separate serovars (pomona, monjakov and cornelli, respectively), are similar but not identical to one another. This was also the case with strains 5621, the serovar mozdok reference strain, and K1, formerly described as serovar dania reference strain, but currently recognized to be a mozdok-like strain. These findings suggest that the deletion of some serovars within the serogroup Pomona, namely mozdok, cornelli, and dania, should be reconsidered. Thus, PFGE appears to be a useful tool for the serovar identification of leptospires belonging to the serogroup Pomona and for shedding light on the problem of their classification.

Cooperative haemolysis between weakly-beta haemolytic human intestinal spirochaetes and Clostridium perfringens.

Interactions between human intestinal spirochaetes (HIS) related to intestinal spirochaetosis and intestinal pathogenic anaerobic bacteria were investigated by searching for the presence of cooperative haemolysis among 39 strains of weakly beta-haemolytic human intestinal spirochaetes and Clostridium perfringens alpha-toxin producers on plates carrying six different sheep blood agar media. An area of intense cooperative haemolysis (about 3-10 mm) was observed between all tested spirochaetal strains and C. perfringens where the clostridial alpha-toxin diffused toward the colonies of the spirochaetes overlapping part of their growth zone. The cooperative haemolysis was a potentiation of the haemolysis due to the single cultivation of human intestinal spirochaetes and C. perfringens and was observed after anaerobic incubation for 24-48 hours when both bacteria at a concentration range of 10(8)-10(3) CFU/ml were streaked at a distance of 3-10 mm to each other. A cooperative haemolysis was also observed between C. perfringens and weakly beta-haemolytic spirochaetes related to porcine and avian intestinal spirochaetosis and the spirochaete causing swine dysentery. The present study indicated that the damage produced in vitro by the clostridial alpha-toxin was enhanced only on the red blood cells which were in proximity to the HIS colonies causing the complete lysis of the erythrocytes. It is hence possible that the potentiation of the damage to red blood cells observed in vitro mimics an in vivo damage on the membranes of enterocytes to which HIS are attached when intestinal spirochaetosis occurs and when cytolysins similar to the alpha-toxin are available in the intestine of the host.

A novel mutation in the NR0B1 gene in a family with monozygotic twin sisters and congenital adrenal hypoplasia affected children.

OBJECTIVE: Congenital adrenal hypoplasia (CAH) is a rare disorder that can be inherited in an X-linked or autosomal recessive pattern. CAH is frequently associated with hypogonadotropic hypogonadism (HHG) with absent or arrested puberty and impaired fertility caused by abnormalities in spermatogenesis. It is estimated that more than 50% of boys with idiopathic adrenal insufficiency have mutations in the NR0B1 gene product, DAX1. CASE REPORT: The proband is a young boy born after an uneventful pregnancy and delivery to non-consanguineous parents. At age 4 years and 4 months he came to our attention because of severe vomiting, abdominal pain, dehydration, and asthenia. The proband underwent a detailed clinical investigation including genetic testing. Sequencing analysis of the NR0B1 gene coding region from the affected child revealed a novel hemizygous deletion [c.385delC; p.(Leu129Cysfs*135)]. This mutation was also present in the heterozygous healthy mother and in her twin sister and in the first cousin of the proband. Monozygosity of the twin sisters was demonstrated. This suggests a de novo mutation and gonadal mosaicism for the deletion. CONCLUSIONS: Adrenal hypoplasia typically presents as adrenal insufficiency during the first few months of life, however, not necessarily as shown by our index case. HHG is thought to affect all NR0B1 mutated patients who reach puberty and, as understanding of the disease has improved, more of these patients survive while presenting different features of the disease, this emphasizing the value of genetic testing in boys with primary adrenal insufficiency and suspected X-linked CAH.