Recognition of heat shock protein 60 epitopes in children with type 1 diabetes.

BACKGROUND: Treatment with a specific HSP60 epitope in new onset of type 1 diabetes (T1D) patients has been shown to preserve endogenous insulin production. Previously, recognition of pan HLA-DR-binding HSP60 epitopes in various autoimmune diseases was found; this study investigated recognition of these epitopes in newly diagnosed T1D patients and correlated findings to the occurrence of a partial remission. METHODS: Peripheral blood mononuclear cells of 18 children with T1D were prospectively collected at disease onset and a few months after diagnosis. Epitope-specific T-cell proliferation and cytokine production (intracellular and in culture supernatants) were measured. Results were compared with 31 longstanding T1D patients and ten healthy controls. RESULTS: Although HSP60 epitope-specific T-cell proliferative responses were detected, overall proliferative responses were low. At onset, epitope-specific intracellular IFN-γ production was higher in T1D patients compared with healthy controls (p < 0.05). At follow-up, both IL-10 and IFN-γ production were higher in those without a partial remission than in those with a partial remission (both p < 0.05). Also, IL-10 and IFN-γ production were higher compared with onset for patients without a PR (both p < 0.01). In supernatants of HSP60 epitope-specific T-cell cultures, no substantial differences in cytokine production were found between T1D patients with and without a partial remission, either at onset or a few months after onset. As patient numbers were small, results should be interpreted with caution. CONCLUSIONS: Pan-DR-binding HSP60 peptides induced low peptide-specific proliferative responses and peptide-specific production of some, mainly intracellular, cytokines in T1D patients. Recognition did not differ significantly between patient groups and various time points.

[Antipsychotics and metabolic abnormalities in children and adolescents: a review of the literature and some recommendations]. / Antipsychotica en metabole afwijkingen bij kinderen en adolescenten; een literatuuroverzicht en aanbevelingen voor de praktijk.

BACKGROUND: Adult patients with schizophrenia and bipolar disorder have an increased risk of developing the metabolic syndrome. This is due to their psychiatric illness and to the use of antipsychotic drugs. Children and adolescents are being treated more and more with antipsychotics. The risk of metabolic abnormalities in this age group remains unclear. AIM: To investigate the relationship between psychotic disorders in childhood and metabolic abnormalities and to study the influence of the use of both typical and atypical antipsychotics on this relationship. METHOD: The PubMed database was searched for relevant articles published between 2000 and June 2009. RESULTS: So far, research into the relationship between psychiatric disorders and metabolic abnormalities in children and adolescents has been inadequate. The normal values and meaning of the components of the metabolic syndrome in children and adolescents have not yet been firmly established. Children and adolescents who use antipsychotics run a significantly higher risk of weight gain. The younger the child, the greater the risk. There are no data about the risk of developing diabetes mellitus type 2. The influence of typical antipsychotics on these conditions has not been investigated. CONCLUSION: The risk of significant weight gain due to the use of atypical antipsychotics is greater in younger children. The 'metabolic syndrome' concept is not applicable to children and adolescents. Very little is known about metabolic risks in the long term. Caution is called for in the prescription of antipsychotics for children and adolescents and further research is needed.

An amino-terminal DAX1 (NROB1) missense mutation associated with isolated mineralocorticoid deficiency.

CONTEXT: Mutations in DAX1 (dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome gene 1; NR0B1) cause X-linked adrenal hypoplasia congenita, a disease characterized by primary adrenal failure, testicular dysgenesis, and gonadotropin deficiency. Most DAX1 mutations are deletions, nonsense, or frameshift mutations that markedly impair its transcriptional activity. Missense mutations have been restricted to the carboxy-terminal domain and are associated with more variable clinical phenotypes. OBJECTIVE: The objective was to identify novel clinical phenotypes associated with DAX1 missense mutations. PATIENTS AND DESIGN: We investigated the genetic basis of isolated mineralocorticoid deficiency in a patient who carries a unique missense mutation (W105C) in the amino-terminal region of DAX1. RESULTS: The W105C DAX1 mutation in the proband was present in three asymptomatic hemizygous males, but it was not detected in the general population. Using in vitro studies of DAX1 expression and function in transfected cells, we demonstrate that the mutant DAX1 protein exhibits mild loss of function, whether studied for genes it represses or for genes it activates. Structure-function studies suggest that the W105C and other mutations in the aminoterminus are compensated by the presence of repeated LXXLL motifs that mediate DAX1 interactions with other proteins. CONCLUSIONS: We describe the first missense mutation in the aminoterminus of DAX1 and conclude that mutations in this region may be partially compensated by redundant functional domains. Mild DAX1 mutations may be a cause of isolated mineralocorticoid deficiency.

Modulation of insulinlike growth factor I binding to human fibroblast monolayer cultures by insulinlike growth factor carrier proteins released to the incubation media.

The relative contributions of type I and type II insulinlike growth factor (IGF) receptors and IGF carrier proteins to the binding of IGF-I tracer to cultured human fibroblasts were determined in competitive binding experiments that used unlabeled insulin and synthetic insulin-IGF-I hybrid molecules containing the A chain of insulin and the B domain of IGF-I. Whereas insulin binds only to type I IGF receptors, the B-IGF-I hybrids bind to type I receptors and IGF carrier proteins but not to type II receptors. In suspended human fibroblasts, IGF-I tracer binds predominantly to type I IGF receptors (inhibition by IGF-I much greater than insulin greater than B-IGF-I hybrid molecules). By contrast, in fibroblast monolayers, IGF-I binding was minimally inhibited by insulin or hybrid molecules, suggesting predominant binding to the type II IGF receptor. The type I receptor appears to be masked on fibroblast monolayers, and to require suspension or detergent solubilization of the cells to be demonstrated. In the course of the monolayers binding experiments, we noted that low concentrations of unlabeled IGF-I (5-10 ng/ml) or B-IGF-I hybrids (100 ng/ml) paradoxically increased IGF-I tracer binding up to twofold. We postulated that during the binding incubation (5 h, 15 degrees C), IGF-I tracer partitioned between binding sites on the cell surface and IGF carrier proteins released to the incubation media. Preferential occupancy of binding sites in the media by unlabeled ligand increased the tracer available to bind to the cells. In support of this hypothesis, carrier proteins were demonstrated in the media at the end of the binding incubation with fibroblast monolayers, and the concentration of unsaturated binding sites in the media correlated inversely with tracer binding to the cells. Thus carrier proteins released to the media during the binding incubation modulate the binding of IGF-I tracer to cell receptors, suggesting that the carrier proteins may play an important role in regulating cellular responsiveness to the IGFs.

Mitogenic activity and receptor reactivity of hybrid molecules containing portions of the insulin-like growth factor I (IGF-I), IGF-II, and insulin molecules.

Insulin and the insulin-like growth factors IGF-I and IGF-II are thought to exert their mitogenic effects in cultured chick embryo fibroblasts and human skin fibroblasts via IGF receptors rather than via insulin receptors. These effects appear to be mediated by the type I subtype of IGF receptor, which is structurally similar to the insulin receptor and exhibits significant cross-reactivity with insulin. As a first step in our long-range goal of defining those features of the IGF-I and IGF-II molecules that confer enhanced mitogenic activity and reactivity with these mitogenic type I IGF receptors, we have prepared two hybrid insulin-IGF molecules and examined their mitogenic and binding activities: (1) A27-insulin, containing an elongated 27-residue A-chain (in which the 6-residue D-domain of IGF-II was added to the carboxy-terminus of the 21-residue A-chain of insulin) combined with the B-chain of insulin; and (2) A insulin-B IGF-1, containing the A-chain of insulin and the synthetic 30-residue B-domain of IGF-I. Both hybrid molecules stimulated DNA synthesis and inhibited 125I-IGF-I binding to type I IGF receptors in both chick embryo and human fibroblast cultures. A27-insulin had considerably greater mitogenic potency and binding potency than A insulin-B IGF-I. Neither hybrid molecule was more potent in these assays than insulin, indicating that the presence of D IGF-II or B IGF-I by itself was not sufficient to increase the mitogenic potency of insulin in fibroblasts. By contrast, A insulin-B IGF-I showed enhanced reactivity with an antiserum to IGF-I. A27-insulin retained significant insulin-like metabolic activity despite the presence of the D-domain of IGF-II.

[When is a tall child too tall?]. / Wanneer zijn lange kinderen te lang?

3 children presented with tall stature. A 14-year-old girl of 179.6 cm was found to be within her target height range and was treated with oestrogen. A 15.5-year-old boy of 199,5 cm was beyond his target height range and was found to have a 47,XYY karyo- type; growth was inhibited with epiphysiodesis. A 12-year-old girl of 178.5 cm and very long legs was beyond her target height range but was found to have homocysteinaemia, a contraindication for hormonal- growth inhibition. her final height was 192 cm. Children growing above the 98th percentile of the growth curve are considered too tall. Most children with tall stature are constitutionally tall and remain within their target height range; no additional investigation is needed. In contrast, growth above this range or disproportionate growth and/or the presence of dysmorphic features in the child or parents warrants further investigation and may reveal important diagnoses. Height prediction based on bone age reading plays a key role in the management of tall children. Treatment with sex steroids may be used in an attempt to limit final height, but some conditions underlying tall stature are a contraindication for this treatment.

[From gene to disease; congenital adrenal hypoplasia and the DAX-1 gene]. / Van gen naar ziekte; congenitale bijnierschorshypoplasie en het DAX-1-gen.

Congenital adrenal hypoplasia is an X-linked disorder resulting in adrenocortical deficiency, failure to complete puberty due to hypogonadotrophic hypogonadism, and infertility. The disease is caused by mutations in the DAX-1 gene. The DAX-1 protein is a transcription inhibitor; it represses the transcription of other, as yet mostly unknown, genes. Mutation analysis can confirm a clinical diagnosis of congenital adrenal hypoplasia. An early diagnosis might prevent critical damage due to an adrenal crisis in an undiagnosed patient. Molecular testing can be used for carrier detection and genetic counselling.

Deoxyribonucleic acid synthesis in cultured adult rat pancreatic B cells.

Previous studies in rodent islets have suggested the existence of a small number of proliferating islet cells. Since islet tissue is composed of endocrine as well as nonendocrine cells, we examined whether the DNA synthesis that is detectable in intact islets in vitro corresponds to an activity of islet B cells, islet endocrine non-B cells and/or nonendocrine islet cells. DNA synthesis was quantified by [3H]thymidine incorporation in trichloracetic acid precipitable material, by nuclear thymidine labeling in autoradiographs, and by nuclear bromodeoxyuridine fluorescence. Adult islet endocrine purified B cells, as well as other endocrine islet cells, incorporated 1 to 2 fmol thymidine/1000 cells, which is 3 times lower than intact islet tissue and 30 times lower than nonendocrine islet cells. Addition of 10% fetal calf serum did not increase DNA synthesis in purified endocrine islet cells but doubled it in intact islets and enhanced it 8-fold in nonendocrine islet cells. The higher thymidine incorporation in intact islets was due to the presence of nonendocrine cells. An increase in medium glucose concentration from 100 to 200 mg/100 ml doubled the thymidine incorporation in purified islet B cells, but not in other endocrine islet cells; no concomitant increase in the number of thymidine or bromodeoxyuridine-labeled nuclei was observed. A phenomenon of glucose-stimulated DNA repair was not excluded. Using three different methods, we have found no evidence for a proliferating activity of adult rat islet B cells under the selected in vitro conditions of this study.

Interaction of insulin-like growth factors with a nonfusing mouse muscle cell line: binding, action, and receptor down-regulation.

Insulin and the insulin-like growth factors (IGFs) are chemically related polypeptides that interact with distinct receptors and elicit the same biological responses. We have sought a readily propagated cell line from a potential target tissue in which to probe the multiple and complex interrelationships among receptor and effector pathways for these polypeptides. We now report that the mouse muscle cell line BC3H-1 represents such a model system. BC3H-1 cells differentiate spontaneously at high density to form cells with muscle-specific properties, but do not fuse. Standaert et al. reported that differentiated BC3H-1 myocytes possess insulin receptors that mediate glucose and amino acid uptake and are down-regulated by prolonged incubation with insulin. The present report demonstrates that BC3H-1 myocytes also possess functional and regulated IGF receptors. Two subtypes of IGF receptors, types I and II, differing in structure and peptide specificity, were demonstrated by competitive binding and affinity cross-linking experiments. Low concentrations of IGFs stimulated glucose incorporation and alpha-aminoisobutyric acid uptake by BC3H-1 myocytes, suggesting that these effects were mediated primarily by IGF receptors rather than insulin receptors. Preincubation with IGFs (or high concentrations of insulin) selectively down-regulated type I IGF receptors without affecting type II IGF receptors. Since [125I]IGF-I binds to both type I and type II receptors in BC3H-1 cells, and since type I receptors have a higher affinity for IGF-I, the selective down-regulation of type I IGF receptors results in an apparent decrease in affinity for IGF-I. This difference in the regulation of type I and type II receptors in BC3H-1 myocytes is consistent with observations in other systems in which only one IGF receptor was present or examined. In their ability to be down-regulated by IGFs and insulin, type I IGF receptors are more similar to the structurally homologous insulin receptors than to the structurally dissimilar type II IGF receptors. These findings indicate that the BC3H-1 cell line provides an excellent model system in which to study the structure-function relationships of the receptor and effector pathways for insulin and the IGFs.

Genotype versus phenotype in families with androgen insensitivity syndrome.

Androgen insensitivity syndrome encompasses a wide range of phenotypes, which are caused by numerous different mutations in the AR gene. Detailed information on the genotype/phenotype relationship in androgen insensitivity syndrome is important for sex assignment, treatment of androgen insensitivity syndrome patients, genetic counseling of their families, and insight into the functional domains of the AR. The commonly accepted concept of dependence on fetal androgens of the development of Wolffian ducts was studied in complete androgen insensitivity syndrome (CAIS) patients. In a nationwide survey in The Netherlands, all cases (n = 49) with the presumptive diagnosis androgen insensitivity syndrome known to pediatric endocrinologists and clinical geneticists were studied. After studying the clinical phenotype, mutation analysis and functional analysis of mutant receptors were performed using genital skin fibroblasts and in vitro expression studies. Here we report the findings in families with multiple affected cases. Fifty-nine percent of androgen insensitivity syndrome patients had other affected relatives. A total of 17 families were studied, seven families with CAIS (18 patients), nine families with partial androgen insensitivity (24 patients), and one family with female prepubertal phenotypes (two patients). No phenotypic variation was observed in families with CAIS. However, phenotypic variation was observed in one-third of families with partial androgen insensitivity resulting in different sex of rearing and differences in requirement of reconstructive surgery. Intrafamilial phenotypic variation was observed for mutations R846H, M771I, and deletion of amino acid N682. Four newly identified mutations were found. Follow-up in families with different AR gene mutations provided information on residual androgen action in vivo and the development of the prepubertal and adult phenotype. Patients with a functional complete defective AR had some pubic hair, Tanner stage P2, and vestigial Wolffian duct derivatives despite absence of AR expression. Vaginal length was functional in most but not all CAIS patients. The minimal incidence of androgen insensitivity syndrome in The Netherlands, based on patients with molecular proof of the diagnosis is 1:99,000. Phenotypic variation was absent in families with CAIS, but distinct phenotypic variation was observed relatively frequent in families with partial androgen insensitivity. Molecular observations suggest that phenotypic variation had different etiologies among these families. Sex assignment of patients with partial androgen insensitivity cannot be based on a specific identified AR gene mutation because distinct phenotypic variation in partial androgen insensitivity families is relatively frequent. In genetic counseling of partial androgen insensitivity families, this frequent occurrence of variable expression resulting in differences in sex of rearing and/or requirement of reconstructive surgery is important information. During puberty or normal dose androgen therapy, no or only minimal virilization may occur even in patients with significant (but still deficient) prenatal virilization. Wolffian duct remnants remain detectable but differentiation does not occur in the absence of a functional AR. In many CAIS patients, surgical elongation of the vagina is not indicated.

17Beta-hydroxysteroid dehydrogenase-3 deficiency: diagnosis, phenotypic variability, population genetics, and worldwide distribution of ancient and de novo mutations.

17Beta-hydroxysteroid dehydrogenase-3 (17betaHSD3) deficiency is an autosomal recessive form of male pseudohermaphroditism caused by mutations in the HSD17B3 gene. In a nationwide study on male pseudohermaphroditism among all pediatric endocrinologists and clinical geneticists in The Netherlands, 18 17betaHSD3-deficient index cases were identified, 12 of whom initially had received the tentative diagnosis androgen insensitivity syndrome (AIS). The phenotypes and genotypes of these patients were studied. Endocrine diagnostic methods were evaluated in comparison to mutation analysis of the HSD17B3 gene. RT-PCR studies were performed on testicular ribonucleic acid of patients homozygous for two different splice site mutations. The minimal incidence of 17betaHSD3 deficiency in The Netherlands and the corresponding carrier frequency were calculated. Haplotype analysis of the chromosomal region of the HSD17B3 gene in Europeans, North Americans, Latin Americans, Australians, and Arabs was used to establish whether recurrent identical mutations were ancient or had repeatedly occurred de novo. In genotypically identical cases, phenotypic variation for external sexual development was observed. Gonadotropin-stimulated serum testosterone/androstenedione ratios in 17betaHSD3-deficient patients were discriminative in all cases and did not overlap with ratios in normal controls or with ratios in AIS patients. In all investigated patients both HSD17B3 alleles were mutated. The intronic mutations 325 + 4;A-->T and 655-1;G-->A disrupted normal splicing, but a small amount of wild-type messenger ribonucleic acid was still made in patients homozygous for 655-1;G-->A. The minimal incidence of 17betaHSD3 deficiency in The Netherlands was shown to be 1: 147,000, with a heterozygote frequency of 1:135. At least 4 mutations, 325 + 4;A-->T, N74T, 655-1;G-->A, and R80Q, found worldwide, appeared to be ancient and originating from genetic founders. Their dispersion could be reconstructed through historical analysis. The HSD17B3 gene mutations 326-1;G-->C and P282L were de novo mutations. 17betaHSD3 deficiency can be reliably diagnosed by endocrine evaluation and mutation analysis. Phenotypic variation can occur between families with the same homozygous mutations. The incidence of 17betaHSD3 deficiency is 0.65 times the incidence of AIS, which is thought to be the most frequent known cause of male pseudohermaphroditism without dysgenic gonads. A global inventory of affected cases demonstrated the ancient origin of at least four mutations. The mutational history of this genetic locus offers views into human diversity and disease, provided by national and international collaboration.

Fetal sex determination from maternal plasma in pregnancies at risk for congenital adrenal hyperplasia.

OBJECTIVE: To determine first-trimester fetal sex by isolating free fetal DNA from maternal plasma. METHODS: The index case was a pregnant woman who previously delivered a girl with congenital adrenal hyperplasia. The SRY gene as a marker for the fetal Y chromosome was detected in maternal serum and plasma by quantitative polymerase chain reaction analysis. Simultaneously, we performed the same test in 25 and 19 women in the first and second trimester, respectively, and compared plasma results with fetal gender as assessed by prenatal karyotyping or as seen at ultrasound or birth. RESULTS: In 44 of 45 patients at gestational ages ranging from 8 3/7 to 17 3/7 weeks, we correctly predicted fetal sex using quantitative polymerase chain reaction analysis of the SRY gene in maternal plasma. In one case, the test result was inconclusive. Overall, fetal sex was correctly predicted in 97.8% of cases (95% confidence interval 88.2%, 99.9%). CONCLUSION: Amplification of free fetal DNA in maternal plasma is a valid technique for predicting fetal sex in early pregnancy. In case of pregnancies at risk for congenital adrenal hyperplasia, the technique allows restriction of dexamethasone treatment to female fetuses resulting in a substantial decrease of unnecessary treatment and invasive diagnostic tests.

A special case of congenital adrenal hypoplasia and acute bilateral infantile striatal necrosis.

Isolated mineralocorticoid deficiency is described in a 5-week-old boy. The deficiency progressed to general adrenal insufficiency during the boy's first year of life. The family history suggested X-linked inheritance. At 18 months of age the patient developed acute bilateral infantile striatal necrosis, which might suggest a possible relationship between both entities.

Hybrid molecules containing the B-domain of insulin-like growth factor I are recognized by carrier proteins of the growth factor.

The insulin-like growth factors (IGFs) are polypeptides in plasma that are chemically related to insulin and have mitogenic and insulin-like activity. Unlike insulin, the IGFs circulate in plasma bound to specific high molecular weight carrier proteins that regulate their delivery to target tissues. To define the sites on the IGFs that allow them to be recognized by carrier proteins, we constructed hybrid molecules containing different portions of the insulin, IGF-I, and IGF-II molecules. The presence of the B domain of IGF-I, but not the D domain of IGF-II, enables these insulin-IGF hybrid molecules to be recognized by acid-stripped IGF carrier proteins from rat serum and other sources. By contrast, neither the BIGF-I nor DIGF-II domain is sufficient to enable binding to type II IGF receptors, despite the fact that type II receptors, like the carrier protein, specifically bind IGF-I and IGF-II but do not interact with insulin. By differentiating those sites on the IGF molecule required for binding to IGF carrier protein and receptors, the insulin-IGF hybrid molecules should help delineate the role of the carrier protein in presenting biologically active IGF to target tissues.

Insulin-like growth factor receptors.

There are two types of insulin-like growth factor (IGF) receptors. The type I receptor generally binds IGF-I more tightly than IGF-II and also interacts weakly with insulin. The type II receptor prefers IGF-II over IGF-I and does not recognize insulin. The type I receptor is made up of an alpha binding subunit (Mr 130 000) and a beta subunit (Mr 95 000) probably organized as a heterotetramer (alpha 2 beta 2). The type II receptor consists of a single binding unit (Mr 250 000). IGF stimulates phosphorylation of the beta subunit of the type I receptor in whole cells and solubilized receptor preparations. Tyrosine kinase activity is associated with the type I receptor, resulting in autophosphorylation of the beta subunit and phosphorylation of exogenous substrates. In contrast, phosphorylation of the type II receptor in whole cells is less IGF-dependent, solubilized receptor preparations are not phosphorylated, and purified type II receptors do not exhibit tyrosine kinase activity toward the artificial substrate poly(Glu, Tyr)4:1. There are many similarities between the type I IGF receptor and the insulin receptor; however, different ligand-binding properties, subtle differences in the size of alpha and beta subunits, and immunoreactivity toward anti-receptor antibodies allow us to distinguish between these two receptors. The presence of both IGF receptors as well as insulin receptors on most cells and cross-reactivity of ligands for binding to these receptors present difficulties in assigning a particular biological response to a specific receptor. The type I receptor is down-regulated by ligand while in several cell types the type II receptor is rapidly up-regulated by insulin; the mechanism of up-regulation appears to be a translocation of type II receptors to the cell surface. There are two classes of serum binding proteins for IGF, a Mr 150 000 species found in adult blood and a Mr 40 000 species, which predominates in foetal blood. Like the type II receptor, IGF binding proteins do not bind insulin. The binding site on the type II receptor can be distinguished from the binding protein sites by a hybrid molecule Ainsulin-BIGF-I, which recognizes the binding protein but not the type II receptor. Binding proteins produced by cells in culture may cause confusion in the interpretation of experiments that are designed to study the binding of radiolabelled IGF to cell surface receptors in monolayer culture.

Testicular degeneration in three patients with the persistent müllerian duct syndrome.

UNLABELLED: The persistent müllerian duct syndrome, characterized by the presence of uterus and tubes in males, is a familial disorder due to defects of synthesis or action of anti-müllerian hormone, a Sertoli cell glycoprotein responsible for the regression of müllerian derivatives in normal male fetuses. Patients are normally virilized and testicular production of testosterone is normal. Both testes may be cryptorchid; alternatively, one may be descended into the inguinal canal or scrotum, together with the müllerian derivatives, a condition known as "hernia uteri inguinalis". We have recently observed three patients affected by the persistent müllerian duct syndrome who experienced progressive degeneration of testicular tissue. In two, functional testicular tissue was still present some months after birth, but deteriorated progressively later. In one patient, testicular tissue was already absent at birth, but the normal virilization of external genitalia indicated that testicular degeneration must have occurred late during fetal life, after the expected time of regression of male müllerian ducts. CONCLUSION: The high incidence of degeneration of testicular tissue in the persistent müllerian duct syndrome could be indirectly linked to anatomical abnormalities which could favour testicular torsion, known to induce testicular regression.