Placental cortisol and cord serum IGFBP-2 concentrations are important determinants of postnatal weight gain.

There is a need to identify simple biochemical markers at birth that may predict subjects at risk of growth failure and metabolic complications in later life. Limited research to date has been performed on relationships of specific biochemical determinants at birth with postnatal weight gain and growth. We proposed to establish whether placental cortisol and IL-6 concentrations and cord serum IGF-II and IGFBP-2 concentrations influenced postnatal growth. We followed up from pregnancy 23 IUGR and 37 AGA subjects, and determined placental cortisol and IL-6 concentrations, and cord serum IGF-II, and IGFBP-2 concentrations at birth. We obtained height and weight measurements at 3, 6, 12, 24 months and 5 years of age in 20 IUGR and 15 AGA subjects of comparable gestational age. A multiple linear regression model was designed to establish the effect of the placental and cord serum peptides on postnatal linear growth and weight gain. All IUGR subjects had catch-up growth before 2 years of age. Placental cortisol concentration correlated positively with weight gain during the first 5 years of postnatal growth (P<0.05). Subjects with the highest placental cortisol concentrations were those who showed a greater increase in weight. Cord serum IGFBP-2 concentrations correlated positively with weight gain throughout the 5 year observation period (P:0.003). The subjects with the highest concentrations showed a greater weight gain. Placental cortisol and cord serum IGFBP-2 concentrations were related to postnatal weight gain, suggesting that the fetal environment has long-term effects on growth.

Insulin production and resistance in cystic fibrosis: effect of age, disease activity, and genotype.

AIM: To assess the major determinants of glucose tolerance between age, genotype, and clinical status in cystic fibrosis (CF) patients, and study if defects of insulin secretion and insulin sensitivity were associated with the onset of CF-related diabetes (CFRD). SUBJECTS AND METHODS: One hundred and nineteen patients, in stable clinical condition were studied. They were subdivided into 3 groups based on age, and 2 groups based on Schwachman-Kulczycki clinical score. All patients were genotyped, and subsequently divided into 3 groups. Ninety-four healthy normal-weight controls, comparable for sex and age were also studied. All subjects had baseline blood samples taken for glucose and insulin, C-peptide, and glycated hemoglobin. Homeostasis model assessment of insulin resistance (HOMA-IR), fasting glucose/insulin ratio (FGIR) were calculated as indices of IR and insulinogenic index as a marker of pancreatic ß-cell function. All patients underwent an oral glucose tolerance test, and 57 underwent an IVGTT for the calculation of first-phase (FPIR) and acute insulin responses (AIR). RESULTS: The F508del homozygous patients had an increased chance of developing impaired glucose tolerance (IGT) and significantly lower FPIR, decreased HOMA-IR, and insulinogenic index. Heterozygote F508del patients had an increased chance of having normal glucose tolerance. HOMA-IR, FGIR, and insulinogenic index did not change with age or clinical score. HOMAIR correlated with FPIR. FPIR correlated positively with insulinogenic index. AIR correlated negatively with FGIR, and positively with C-reactive protein. In multiple linear regression analyses, glucose tolerance was related to the agegroup, and to the HOMA-IR and insulinogenic indexes. CONCLUSIONS: IGT and CFRD were related mainly to genotype, although, as expected, the prevalence increased with age. The data suggested a possible combined contribution of insulin deficiency, ß-cell function, and reduced insulin sensitivity to the onset of CFRD; however, further studies are warranted to better elucidate this aspect.

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.

Interleukin-6 and insulin-like growth factor system relationships and differences in the human placenta and fetus from the 35th week of gestation.

The integrity of the insulin-like growth factor (IGF) system is essential for normal fetal growth. Cytokine and IGF-IGFBP relationships have been shown in specific tissues, but it is unknown whether these occur in the placenta. We aimed to assess possible differences in the IGF system depending on gestational age (GA) from week 35 to 40, and to study relationships of IL-6 with components of the IGF system in the placenta and newborn infant. We followed 32 normal births and collected whole villous tissue and cord serum. Total RNA was extracted from the placenta samples, reverse transcribed and then real-time quantitative (TaqMan) RT-PCR was performed to quantify cDNA for IGF-I, IGF-II, IGFBP-1, IGFBP-2 and IL-6. The corresponding proteins were assayed in placenta lysates and cord serum using specific commercial kits. Two groups of subjects (Group 1, 35-37 weeks GA, N=12 and Group 2, 38-40 weeks GA, N=20) were studied. In placenta, IGF-I mRNA was more abundant than IGF-II mRNA at all times and together with IGFBP-1mRNA were less expressed at term. IGFBP-2 and IL-6 mRNAs were higher after week 37 GA. IL-6 and IGFBP-2 gene expression were closely related. The corresponding proteins showed similar differences to the genes but IGF-I was undetectable in the lysates, whereas IGF-II was abundant. IGFBP-2 concentrations were very high and greater than those of IGFBP-1. In the newborn, no difference was seen in any cord serum protein after week 35 GA. IGFBP-1 was negatively correlated with parameters of neonatal size. In conclusion, this study reports new insights into IL-6, IGF-IGFBP relationships within the human placenta and shows the importance of comparing subjects with the same GA.

Aromatase is differentially expressed in peripheral blood leukocytes from children, and adult female and male subjects.

OBJECTIVE: Aromatase, the key enzyme involved in estrogen synthesis, is expressed in a variety of cells and tissues including human peripheral blood leukocytes (PBLs). The present study was designed to evaluate PBL aromatase gene expression in male and female subjects of different age groups. In addition, differences in gene expression during the follicular and luteal phase of the menstrual cycle in women, and before and after testosterone administration in men, were estimated. DESIGN: Aromatase mRNA and protein were measured in PBLs obtained from young (n = 10) and postmenopausal women (n = 10), men (n = 15), and prepubertal children (n = 10). Aromatase mRNA and protein were also measured during the follicular and luteal phases of the menstrual cycle in women, and before and after the intramuscular administration of 250 mg testosterone enanthate in men. METHODS AND RESULTS: Aromatase mRNA measured by real-time PCR in PBLs from women during the follicular phase was significantly higher than during the luteal phase of the menstrual cycle (P < 0.05). In men, PBL aromatase mRNA values increased significantly following testosterone administration (P < 0.05). PBL mRNA aromatase levels in women during the follicular phase and men after testosterone administration were significantly higher (one-way ANOVA; P < 0.05) than in any other group. Children, postmenopausal women, and women during the luteal phase showed the lowest aromatase mRNA expression. The results of the immunoblot analysis confirmed the data obtained by real-time PCR. A positive correlation between PBL aromatase mRNA values and plasma estradiol and estrone levels during the follicular phase of the menstrual cycle was observed in the group of adult women. No other correlations were found. CONCLUSIONS: The aromatase gene is differentially expressed in PBLs from women, men, and prepubertal children, indicating a sexual dimorphism in the enzyme expression and an important role of sex steroids in the modulation of aromatase gene expression.

Decreased androgen receptor gene methylation in premature pubarche: a novel pathogenetic mechanism?

CONTEXT: Several studies found links between DNA methylation and gene expression. In patients with idiopathic hirsutism, a preferential methylation of the of shorter androgen receptor (AR) alleles was hypothesized to be responsible for the abnormal hair growth. OBJECTIVE: The objective of this study was to assess whether abnormalities in the AR function in both peripheral blood leukocytes (PBLs) and androgen target tissues are present in children with premature pubarche (PP). DESIGN: Human DNA was extracted from PBLs and pubic hair and CAG repeats length and methylation status of the AR gene were analyzed. SETTING: The study was performed at a Pediatric Endocrinology referral clinic. PATIENTS: Twenty-five girls with PP, 23 prepubertal children, and 10 girls with Tanner stage II pubertal development were studied. MAIN OUTCOME MEASURE: The main outcome measures were CAG repeat length and AR methylation pattern in PBLs and pubic hair. RESULTS: In PBLs from PP patients, AR gene methylation was significantly lower (P < 0.01) than that of prepubertal children and similar to that of girls with Tanner II stage pubertal development. A negative correlation between AR gene methylation in PBLs and the age of normal children was detected. PATIENTS with PP exhibited a hair follicle AR methylation pattern similar to that of Tanner stage II girls. The mean number of CAG repeats was lower in PP patients than in prepubertal and Tanner stage II girls, although it was within the normal range for the general population in both groups. CONCLUSIONS: The increased AR gene activity observed in PP patients, as indicated by the reduced AR gene methylation pattern, together with the presence of shorter CAG repeats, might lead to hypersensitivity of the hair follicles to steroid hormones and therefore to the premature development of pubic hair.

Usefulness of genus-specific PCR and Southern blot species-specific hybridization for the detection of imported malaria cases in Italy.

A PCR method involving a genus-specific oligonucleotides set and Southern blot hybridization with four species-specific probes to P. falciparum, P. vivax, P. malariae and P. ovale was evaluated for the detection of malaria parasites in blood samples from 101 patients with clinically suspect malaria infection imported to Italy. Plasmodium falciparum was the main species detected. As determined by microscopy, 53 (52.4%) patients had malaria and of these: 40 (75.5%) were infected with P. falciparum; 7 (13.2%) with P. vivax; 1 (1.9%) with P. ovale; 3 (5.7%) with P. malariae; 1 (1.9%) with P. vivax or P. ovale; and 1 (1.9%) with P. falciparum or P. vivax. Ninety-seven out 101 blood samples were submitted to ParaSight-F test which showed a sensitivity of 94.73%, and a specificity of 93.22%, as compared to microscopy. The PCR assay using the genus-specific oligonucleotide primer set (pg-PCR) was able to detect 53 (52.4%) infections and showed a sensitivity of 100% and a specificity of 100%, when compared to microscopy. The parasite species were identified by Southern blot hybridization using species-specific probes and 40 (75.5%) samples were P. falciparum positive, 5 (9.4%) P. vivax positive, 4 (7.5%) P. ovale positive, and 2 (3.8%) P. malariae positive. When the Southern blot results were compared to those of blood-film diagnosis, we observed some disagreement. In particular, compared to Southern blot, microscopy underestimated P. ovale infection; blood film analysis recognised only 1 P. ovale sample, whereas Southern blot recognised 4 P. ovale positive samples (by microscopy, 2 of these were detected as P. vivax, 1 as P. ovale or P. vivax, and the other as P. falciparum or P. vivax). Southern blot hybridization was unable to identify one P. falciparum and one P. vivax positive case detected by microscopy. We also plan to use a reference nested-PCR assay to clarify the disagreement observed between microscopy and Southern blot hybridization.

Evaluation of OptiMAL Assay test to detect imported malaria in Italy.

This study evaluated a newly developed rapid malaria diagnostic test, OptiMAL Assay, to detect "Plasmodium falciparum malaria" and "non Plasmodium falciparum malaria" in blood samples from 139 individuals with a presumptive clinical diagnosis of imported malaria in Italy. OptiMAL Assay utilizes a dipstick coated with monoclonal antibodies against the intracellular metabolic enzyme, plasmodium Lactate Dehydrogenase (pLDH) present in and released from parasite-infected erythrocytes. Blood samples from 56 cases out of 139 were found "Plasmodium falciparum malaria" positive by microscopy; with these samples OptiMAL Assay and the ParaSight-F test, which is a kit detecting the P. falciparum histidin-rich protein 2 (HRP-2), showed an overall sensitivity of 83% and 94%, respectively, in comparison with microscopy. Parasitemia levels tested in the 56 P. falciparum positive blood samples by microscopy ranged from <0.004% to 20%. A correlation between sensitivity and parasitemia was evident and OptiMAL Assay and ParaSight-F test were more sensitive (96-100%; 100%) with samples with 0.1%-20% levels of parasitemia, while proved less sensitive (0-44%; 50-88%) with <0.004-0.01% levels of parasitemia.

Search for malaria parasites by PCR and Southern blot in patients with imported malaria in Italy.

The present study evaluates the sensitivity, specificity and usefulness of a PCR method with Southern blot hybridization to detect malaria parasites in blood samples from subjects with a suspect clinical diagnosis of malaria imported to Italy. Plasmodia were detected by PCR using a genus-specific primer-set corresponding to the sequences common to P. falciparum, P. vivax, P. malariae and P. ovale, as described by Arai (Arai et al., Nucleosides Nucleotides, 1994, 13, 1363-1364) and Kimura (Kimura et al., Journal of Clinical Microbiology, 1995, 33, 2342-2346). In addition, four distinct tandemly repetitive species-specific probes, described by Kawai (Kawai et al., Analytical Biochimestry, 1993, 209, 63-69), were synthesized to specifically detect the four malaria parasites species by Southern blot hybridization. Fifteen blood samples from 12 patients (7 with malaria) were tested and the genus-specific PCR method showed a sensitivity of 100% and a specificity of 100%, when compared to microscopy, in detecting malaria parasites in the tested blood samples. Fourteen samples (nine were positive and five negative by PCR) were confirmed by Southern blot, whereas only one P. vivax positive sample was not hybridized with the species-specific probes. We conclude that this PCR method with Southern blot hybridization may be useful in detecting malaria parasites in patients with malaria imported to Italy.

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.