Lipopolysaccharide potentiates platelet responses via toll-like receptor 4-stimulated Akt-Erk-PLA2 signalling

Lipopolysaccharide (LPS) from the cell envelope of Gram-negative bacteria is a principal cause of the symptoms of sepsis. LPS has been reported to modulate the function of platelets although the underlying mechanisms of LPS action in these cells remain unclear. Platelets express the Toll-like receptor 4 (TLR4) which serves as a receptor for LPS, although the potential role of TLR4 and associated cell signalling in controlling platelet responses to LPS has not been extensively explored. In this study, we therefore investigated the actions of LPS prepared from different strains of Escherichia coli on platelet function, the underlying signalling mechanisms, and the potential role of TLR4 in orchestrating these. We report that LPS increased the aggregation of washed platelets stimulated by thromboxane (U46619) or GPVI collagen receptor agonists, effects that were prevented by a TLR4 antagonist. Associated with this, LPS enhanced fibrinogen binding, P-selectin exposure and reactive oxygen species (ROS) release. Increase of ROS was found to be important for the actions of LPS on platelets, since these were inhibited in the presence of superoxide dismutase or catalase. The effects of LPS were associated with phosphorylation of Akt, ERK1/2 and PLA2 in stimulated platelets, and inhibitors of PI3-kinase, Akt and ERK1/2 reduced significantly LPS enhanced platelet function and associated ROS production. Furthermore, inhibition of platelet cyclooxygenase or the thromboxane receptor, revealed an important role for thromboxane A2. We therefore conclude that LPS increases human platelet activation through a TLR4-PI3K-Akt-ERK1/2-PLA2 -dependent pathway that is dependent on ROS and TXA2 formation

Androgens by immunoassay and mass spectrometry in children with 46,XY disorder of sex development

Objective: Steroid measurement is a challenge in pediatric endocrinology. Currently, liquid chromatography with tandem mass spectrometry (LC-MS/MS) is considered a gold standard for this purpose. The aim of this study was to compare both LC-MS/MS and immunoassay (IA) for androgens before and after human recombinant chorionic gonadotropin (rhCG) stimulus in children with 46,XY disorders of sex development (DSD). Methods: Nineteen patients with 46,XY DSD were evaluated; all of them were prepubertal and non-gonadectomized. Testosterone, dihydrotestosterone (DHT), DHEA and androstenedione were measured by IA and LC-MS/MS before and 7 days after rhCG injection. The correlation between IA and LC-MS/MS was analyzed by the intraclass correlation coefficient (ICC) and Spearman’s rank correlation coefficient (SCC). For concordance analysis the Passing and Bablok (PB) regression and the Bland and Altman (BA) method were used. Results: Testosterone showed excellent correlation (ICC = 0.960 and SCC = 0.964); DHT showed insignificant and moderate correlations as indicated by ICC (0.222) and SCC (0.631), respectively; DHEA showed moderate correlation (ICC = 0.585 and SCC = 0.716); and androstenedione had poor and moderate correlations in ICC (0.363) and SCC (0.735), respectively. Using the PB method, all hormones showed a linear correlation, but proportional and systematic concordance errors were detected for androstenedione, systematic errors for testosterone and no errors for DHEA and DHT. By the BA method, there was a trend of IA to overestimate testosterone and androstenedione and underestimate DHEA and DHT when compared to LC-MS/MS. Conclusion: Traditional IA should be replaced by LC-MS/MS for the androgens measurement in prepubertal children whenever is possible

Leydig and Sertoli cell function in individuals with genital ambiguity, 46,XY karyotype, palpable gonads and normal testosterone secretion : a case-control study

BACKGROUND: Because normal male sexual differentiation is more complex than normal female sexual differentiation, there are more cases of disorders of sex development (DSDs) with 46,XY karyotype that have unclear etiology. However, Leydig and Sertoli cell markers are rarely used in distinguishing such individuals. OBJECTIVES: To evaluate the function of Leydig and Sertoli cells in individuals with genital ambiguity, 46,XY karyotype, palpable gonads and normal testosterone secretion. STUDY DESIGN AND SETTING: Case-control study with 77 patients, including eight with partial androgen insensitivity syndrome, eight with 5α-reductase deficiency type 2 (5ARD2) and 19 with idiopathic 46,XY DSD, and 42 healthy controls, from the Interdisciplinary Study Group for Sex Determination and Differentiation (GIEDDS), at the State University of Campinas (UNICAMP), Campinas, Brazil. METHODS: Baseline levels of gonadotropins, anti-Müllerian hormone (AMH), inhibin B, insulin-like 3 (INSL3), testosterone and dihydrotestosterone in cases, and AMH, inhibin B, and INSL3 levels in controls, were assessed. RESULTS: There was no significant difference in age between cases and controls (P = 0.595). AMH and inhibin B levels were significantly lower in cases than in controls (P = 0.031 and P < 0.001, respectively). INSL3 levels were significantly higher in cases than in controls (P = 0.003). Inhibin B levels were lower in 5ARD2 patients (P = 0.045) and idiopathic patients (P = 0.001), in separate comparisons with the controls. CONCLUSION: According to our findings, we can speculate that inhibin B levels may be used to differentiate among DSD cases

Analysis of anti-Mullerian hormone (AMH) and its receptor (AMHR2) genes in patients with persistent Mullerian duct syndrome

Objective: To screen for mutations in AMH and AMHR2 genes in patients with persistent Mullerian duct syndrome (PMDS). Patients and method: Genomic DNA of eight patients with PMDS was obtained from peripheral blood leukocytes. Directed sequencing of the coding regions and the exon-intron boundaries of AMH and AMHR2 were performed. Results: The AMH mutations p.Arg95*, p.Arg123Trp, c.556-2A&gt;G, and p. Arg502Leu were identified in five patients; and p.Gly323Ser and p.Arg407* in AMHR2 of two individuals. In silico analyses of the novel c.556-2A&gt;G, p.Arg502Leu and p.Arg407* mutations predicted that they were harmful and were possible causes of the disease. Conclusion: A likely molecular etiology was found in the eight evaluated patients with PMDS. Four mutations in AMH and two in AMHR2 were identified. Three of them are novel mutations, c.556-2A&gt;G, and p. Arg502Leu in AMH; and p.Gly323Ser in AMHR2. Arq Bras Endocrinol Metab. 2012;56(8):473-8Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) [305743/2011-2, 314392/2009-2, 302084/2009-6]Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (Fapesp) [06/50999-0]Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP

Analysis of anti-Mullerian hormone (AMH) and its receptor (AMHR2) genes in patients with persistent Mullerian duct syndrome

Objective: To screen for mutations in AMH and AMHR2 genes in patients with persistent Mullerian duct syndrome (PMDS). Patients and method: Genomic DNA of eight patients with PMDS was obtained from peripheral blood leukocytes. Directed sequencing of the coding regions and the exon-intron boundaries of AMH and AMHR2 were performed. Results: The AMH mutations p.Arg95*, p.Arg123Trp, c.556-2A&gt;G, and p. Arg502Leu were identified in five patients; and p.Gly323Ser and p.Arg407* in AMHR2 of two individuals. In silico analyses of the novel c.556-2A&gt;G, p.Arg502Leu and p.Arg407* mutations predicted that they were harmful and were possible causes of the disease. Conclusion: A likely molecular etiology was found in the eight evaluated patients with PMDS. Four mutations in AMH and two in AMHR2 were identified. Three of them are novel mutations, c.556-2A&gt;G, and p. Arg502Leu in AMH; and p.Gly323Ser in AMHR2. Arq Bras Endocrinol Metab. 2012;56(8):473-8Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) [305743/2011-2, 314392/2009-2, 302084/2009-6]Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (Fapesp) [06/50999-0]Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP

408 Cases of Genital Ambiguity Followed by Single Multidisciplinary Team during 23 Years: Etiologic Diagnosis and Sex of Rearing

Objective. To evaluate diagnosis, age of referral, karyotype, and sex of rearing of cases with disorders of sex development (DSD) with ambiguous genitalia. Methods. Retrospective study during 23 years at outpatient clinic of a referral center. Results. There were 408 cases; 250 (61.3%) were 46,XY and 124 (30.4%) 46,XX and 34 (8.3%) had sex chromosomes abnormalities. 189 (46.3%) had 46,XY testicular DSD, 105 (25.7%) 46,XX ovarian DSD, 95 (23.3%) disorders of gonadal development (DGD), and 19 (4.7%) complex malformations. The main etiology of 46,XX ovarian DSD was salt-wasting 21-hydroxylase deficiency. In 46,XX and 46,XY groups, other malformations were observed. In the DGD group, 46,XY partial gonadal dysgenesis, mixed gonadal dysgenesis, and ovotesticular DSD were more frequent. Low birth weight was observed in 42 cases of idiopathic 46,XY testicular DSD. The average age at diagnosis was 31.7 months. The final sex of rearing was male in 238 cases and female in 170. Only 6.6% (27 cases) needed sex reassignment. Conclusions. In this large DSD sample with ambiguous genitalia, the 46,XY karyotype was the most frequent; in turn, congenital adrenal hyperplasia was the most frequent etiology. Malformations associated with DSD were common in all groups and low birth weight was associated with idiopathic 46,XY testicular DSD

Glaucoma congenito primario : uma entidade genetica heterogenea

Orientador: Bernardo BeiguelmanDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: A visão clássica sobre o mecanismo de herança do glaucoma congênito primário é a que essa anomalia é sempre transmitida de modo autossômico recessivo monogênico. A análise dos dados familiais de 1408 portadores dessa anomalia mostra que o glaucoma congênito primário é, na verdade, uma entidade genética heterogênea, visto que foi possível detectar pelo menos duas formas autossômicas monogênicas dessa doença, sendo uma dominante e outra recessiva. Há, ainda, um grande contingente de anômalos cuja etiologia (genética) não fui passível determinar. Assim sendo é fundamental, tanto para o geneticista quanto para o o oftalmologista, que se procure distinguir as diferentes entidades genético-clínicas ao nível anatômico e/ou bioquímicoMestrad

Estudo genetico-clinico de glaucoma congenito primario

Orientador: Antonio Sergio RamalhoTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: O Glaucoma Congênito Primário (GCP) é uma entidade genética heterogênea, geralmente considerada distinta da Megalocórnea e do glaucoma congênito de manifestação tardia ou juvenil. A fim de detectar indicações da heterogeneidade genética do GCP a nível clínico, foram examinados 67 portadores dessa anomalia, dos quais se obtiveram dados anamnésticos e de exame oftalmológico. Os resultados da análise se segregação, bem como a alta freqüência de consangüinidade observada nessa amostra indicam que o padrão de herança autossômico recessivo predomina entre nossos pacientes. A associação significativa encontrada entre a recorrência familial do GCP e a existência de consangüinidade entre os genitores, o início das manifestações ao nascimento e a bilateralidade e simetria da doença indica que os casos que manifestem essas três características sejam considerados de alto risco de recorrência na irmandade, enquanto que aqueles em que nenhuma dessas características é observada devam ser considerados os de mais baixo risco. A existência de megalocórnea ou de glaucoma congênito de manifestação tardia ou juvenil em um dos olhos do portador de GCP ou em outros membros da família sugere fortemente que essas entidades nosológicas correspondam a diferentes formas de expressão de uma mesma anomalia básica do ângulo iridocorneal, indicando que devam ser analisadas em conjunto. Estudos aprofundados do glaucoma congênito se fazem necessários, portanto, para elucidação de seus diversos aspectos genéticos e clínicosAbstract: Primary Congenital Glaucoma (PCG) is a heterogeneous genetic entity which is usuall_ considered distinct from Megalocornea and congenital glaucoma of late or juvenil onset. History and ophthalmological data were obtained from 67 PCG cases in order to find out indications of the genetic heterogeneity_ of this disease at the clinical level. The results obtained from a complex segregation analysis, as well as the high frequency of parental consanguinity in this sample, indicate that the autosomal recessive pattern of inheritance predominate among our patients. A high recurrence risk is expected to the sibs of PCG cases with parental consanguinity, onset of the disease at birth and bilateral and symmetrical involviment, while those which exhibit none of these features seem to be at the lowest risk of having affected sibs. Since megalocornea and congenital glaucoma of late or juvenile onset can be found in the other eye of a PCG patient or in other individuals in the same family, these diseases appear to be manifestations of the same basic abnormality of the iridocornea1 angle. Hence, the_ should be analyzed together with PCG. Comprehensive studies of congenital glaucoma are thus necessary to elucidate its clinical and genetical peculiaritiesDoutoradoDoutor em Ciência

Idiopathic Male Pseudohermaphroditism Is Associated With Prenatal Growth Retardation.

About 50% of intersex cases are due to male pseudohermaphroditism, and of these cases, 50% are not clarified aetiologically. The association of idiopathic male pseudohermaphroditism and prenatal growth retardation has been recently reported. The aim of this study was to verify whether there was a difference in weight and/or length at birth between idiopathic and non-idiopathic male pseudohermaphroditism patients. A total of 70 patients with male pseudohermaphroditism were recruited; 35 non-idiopathic and 35 idiopathic. Birth weight and length were converted to z scores, and the severity of genital ambiguity was classified according to Prader grades: less virilized (Prader 1 to 3) and more virilized (Prader 4 or 5). Data were analysed using a Mann-Whitney test, odds ratio and logistic regression analysis. Birth weight (P = 0.028) and length (P = 0.01) z scores were lower in the idiopathic male pseudohermaphroditism group compared to the non-idiopathic group and were also significantly decreased among the less virilized patients, both in the sample as a whole (weight z score, P = 0.002; length z score, P = 0.0008) and in the group of idiopathic patients (weight z score, P = 0.013; length z score, P = 0.007). According to logistic regression analysis, only birth length z score significantly predicted the severity of the genital ambiguity in patients with idiopathic male pseudohermaphroditism ( P = 0.0007). There is an association between prenatal growth retardation and male pseudohermaphroditism which may be due to genetic factors not clarified yet or to environmental factors which act early in gestation.164287-9

Parents' experiences of having a baby with ambiguous genitalia

CNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOHealth professionals must be aware of the impact on parents of the birth of children with ambiguous genitalia. This study aimed to analyze the experiences and perceptions of such parents. Parents of 30 children who were evaluated in a reference center for disorders of sex development (DSD) were interviewed. The questionnaire covered the prenatal period, the moment they were told about the disorder, initial management by health professionals, and problems they experienced. Only two cases were detected during pregnancy. The news was usually given to the mother alone by pediatricians. Most parents kept it secret and avoided exposing the baby to the prejudice of others. Parents of children who were referred without sex assignment usually held a personal belief of their child's sex. Previous assignment was based on clinical examination and/or karyotype. Spreading knowledge about DSD could increase awareness of this issue, thus reducing parents' shock and societal stigma. Training of neonatal care teams is required to avoid assignment before evaluation.Health professionals must be aware of the impact on parents of the birth of children with ambiguous genitalia. This study aimed to analyze the experiences and perceptions of such parents. Parents of 30 children who were evaluated in a reference center for disorders of sex development (DSD) were interviewed. The questionnaire covered the prenatal period, the moment they were told about the disorder, initial management by health professionals, and problems they experienced. Only two cases were detected during pregnancy. The news was usually given to the mother alone by pediatricians. Most parents kept it secret and avoided exposing the baby to the prejudice of others. Parents of children who were referred without sex assignment usually held a personal belief of their child's sex. Previous assignment was based on clinical examination and/or karyotype. Spreading knowledge about DSD could increase awareness of this issue, thus reducing parents' shock and societal stigma. Training of neonatal care teams is required to avoid assignment before evaluation287-8833838CNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOCNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOsem informaçãoHealth professionals must be aware of the impact on parents of the birth of children with ambiguous genitalia. This study aimed to analyze the experiences and perceptions of such parents. Parents of 30 children who were evaluated in a reference center for disorders of sex development (DSD) were interviewed. The questionnaire covered the prenatal period, the moment they were told about the disorder, initial management by health professionals, and problems they experienced. Only two cases were detected during pregnancy. The news was usually given to the mother alone by pediatricians. Most parents kept it secret and avoided exposing the baby to the prejudice of others. Parents of children who were referred without sex assignment usually held a personal belief of their child's sex. Previous assignment was based on clinical examination and/or karyotype. Spreading knowledge about DSD could increase awareness of this issue, thus reducing parents' shock and societal stigma. Training of neonatal care teams is required to avoid assignment before evaluation