The TALE homeodomain of PBX1 is involved in human primary testis-determination.

Human sex-determination is a poorly understood genetic process, where gonad development depends on a cell fate decision that occurs in a somatic cell to commit to Sertoli (male) or granulosa (female) cells. A lack of testis-determination in the human results in 46,XY gonadal dysgenesis. A minority of these cases is explained by mutations in genes known to be involved in sex-determination. Here, we identified a de novo missense mutation, p.Arg235Gln in the highly conserved TALE homeodomain of the transcription factor Pre-B-Cell Leukemia Transcription Factor 1 (PBX1) in a child with 46,XY gonadal dysgenesis and radiocubital synostosis. This mutation, within the nuclear localization signal of the protein, modifies the ability of the PBX1 protein to localize to the nucleus. The mutation abolishes the physical interaction of PBX1 with two proteins known to be involved in testis-determination, CBX2 and EMX2. These results provide a mechanism whereby this mutation results specifically in the absence of testis-determination.

Virilization of a young girl caused by concomitant ectopic and intra-adrenal adenomas of the adrenal cortex.

BACKGROUND: Adenomas of the adrenal gland are rare causes of virilization in childhood. CASE REPORT: A girl aged 2 years and 4 months presented with pubarche, distinct clitoral hypertrophy, tall stature, and increased height velocity. Plasma testosterone and dehydroepiandrosterone were elevated. Androgens remained unchanged after adrenocorticotropic hormone, and dexamethasone administrations. Ultrasound examination and magnetic resonance imaging indicated an extra-adrenal mass adjacent to the left adrenal gland, which was removed by endoscopic surgery. However, plasma androgens remained elevated and (131)I-iodomethyl-norcholesterol scintigraphy revealed tracer enhancement in the right adrenal gland, which was consecutively removed. Virilization regressed after extirpation of the adenomas and height velocity normalized. RESULTS: Histology revealed a circumscribed adenoma in the right adrenal gland and an epithelial mass with adrenal cortical cells in the left-sided ectopic tumor. In the ectopic tumor, melanocortin 2 receptor expression was augmented threefold compared to the control, indicating adrenal origin. CONCLUSIONS: In this young girl, virilization is due to concomitant ectopic and intra-adrenal adenomas of the adrenal cortex. By melanocortin 2 receptor expression, it was confirmed that the ectopic adenoma derived from the adrenal cortex. Specific scintigraphy, if available, assists in allocating the source of androgen hypersecretion.

Anti insulin-like growth factor I receptor immunoliposomes: a single formulation combining two anticancer treatments with enhanced therapeutic efficiency.

CONTEXT: Through overexpression and aberrant activation in many human tumors, the IGF system plays a key role in tumor development and tumor cell proliferation. Different strategies targeting IGF-I receptor (IGFI-R) have been developed, and recent studies demonstrated that combined treatments with cytostatic drugs enhance the potency of anti-IGFI-R therapies. OBJECTIVE: The objective of the study was to examine the IGFI-R expression status in neuroendocrine tumors of the gastroenteropancreatic system (GEP-NETs) in comparison with healthy tissues and use potential overexpression as a target for novel anti-IGFI-R immunoliposomes. EXPERIMENTAL DESIGN: A human tumor tissue array and samples from different normal tissues were investigated by immunohistochemistry. An IGFI-R antagonistic antibody (1H7) was coupled to the surface of sterically stabilized liposomes loaded with doxorubicin. Cell lines from different tumor entities were investigated for liposomal association studies in vitro. For in vivo experiments, neuroendocrine tumor xenografts were used for evaluation of pharmacokinetic and therapeutic properties of the novel compound. RESULTS: Immunohistochemistry revealed significant IGFI-R overexpression in all investigated GEP-NETs (n = 59; staining index, 229.1 +/- 3.1%) in comparison with normal tissues (115.7 +/- 3.7%). Furthermore, anti-IGFI-R immunoliposomes displayed specific tumor cell association (44.2 +/- 1.6% vs. IgG liposomes, 0.8 +/- 0.3%; P < 0.0001) and internalization in human neuroendocrine tumor cells in vitro and superior antitumor efficacy in vivo (life span 31.5 +/- 2.2 d vs. untreated control, 19 +/- 0.6, P = 0.008). CONCLUSION: IGFI-R overexpression seems to be a common characteristic of otherwise heterogenous NETs. Novel anti-IGFI-R immunoliposomes have been developed and successfully tested in a preclinical model for human GEP-NETs. Moreover in vitro experiments indicate that usage of this agent could also present a promising approach for other tumor entities.

Thyroid hormone replacement for central hypothyroidism: a randomized controlled trial comparing two doses of thyroxine (T4) with a combination of T4 and triiodothyronine.

BACKGROUND: Dosage of T(4) in central hypothyroidism is primarily guided by the free serum T(4) level (fT4). However, the optimum fT4 range is ill defined, and subtle hypothyroidism might be missed using this approach. OBJECTIVES: Our aim was to investigate the effects of a body weight (bw)-adapted T(4) treatment, alone or in combination with T(3), on metabolism, well-being, and cognitive function in comparison to a regimen leading to normal fT4. DESIGN: This was a placebo-controlled trial (double-blind, crossover). PATIENTS: A total of 29 patients (age 52 +/- 2 yr; females/males, 8/21) with hypopituitarism, including TSH deficiency, participated in the study. INTERVENTIONS: Three regimens were compared (5 wk each): "EMPIRICAL-T4," empirical T(4) dosage (1 +/- 0.05 microg/kg bw) leading to normal fT4; BW-ADAPTED-T4 (1.6 microg/kg bw T(4)); and "BW-ADAPTED-T3T4," bw-adapted combination of T(3) and T(4) (ratio of 1:10). RESULTS: BW-ADAPTED-T4 administration increased mean fT4 concentrations to the upper limit of the normal range (peak levels). Compared with EMPIRICAL-T4, BW-ADAPTED-T4 treatment resulted in a lower body mass index (BMI) (29.0 +/- 0.7 vs. 29.5 +/- 0.7 kg/m(2); P < 0.03), lower total cholesterol (198 +/- 9 vs. 226 +/- 7 mg/dl; P < 0.01), and lower low-density lipoprotein (LDL) cholesterol (116 +/- 5 vs. 135 +/- 7 mg/dl; P < 0.01). BW-ADAPTED-T3T4 treatment was associated with additional beneficial effects on ankle reflex time and working memory but resulted in supraphysiological free serum T(3) (fT(3)) levels. LIMITATIONS: Long-term side effects may have been missed. CONCLUSIONS: Using a dose of 1.6 microg/kg bw improved markers commonly associated with central hypothyroidism. This suggests that T(4) dosage based on bw and aiming at fT4 in the upper reference range is superior to titration of T(4) aiming at middle normal fT4 concentrations in those patients.

Sex-specific association of PTPN22 1858T with type 1 diabetes but not with Hashimoto's thyroiditis or Addison's disease in the German population.

BACKGROUND: Endocrine autoimmune disorders share genetic susceptibility loci, causing a disordered T-cell activation and homeostasis (HLA class II genes, CTLA-4). Recent studies showed a genetic variation within the PTPN22 gene to be an additional risk factor. MATERIALS AND METHODS: Patients with type 1 diabetes (n = 220), Hashimoto's thyroiditis (n = 94), Addison's disease (n = 121) and healthy controls (n = 239) were genotyped for the gene polymorphism PTPN22 1858 C/T. RESULTS: Our study confirms a significant association between allelic variation of the PTPN22 1858 C/T polymorphism and type 1 diabetes mellitus (T1D). 1858T was observed more frequently in T1D patients (19.3% vs 11.3%, P = 0.0009; odds ratio for allele T = 1.88, 95% confidence interval [1.3-2.7]). Furthermore, we found a strong association in female patients with T1D (P = 0.0003), whereas there was no significant difference between male patients with type 1 diabetes and male controls. No significant difference was observed between the distribution of PTPN22 C/T in patients with Hashimoto's thyroiditis or Addison's disease and healthy controls. CONCLUSION: The PTPN22 polymorphism 1858 C/T may be involved in the pathogenesis of type 1 diabetes mellitus by a sex-specific mechanism that contributes to susceptibility in females.

Genetic influence of an ACTH receptor promoter polymorphism on adrenal androgen secretion.

OBJECTIVE: Adrenocorticotropic hormone (ACTH) is the primary secretagogue stimulating secretion of adrenal androgens (AA). Yet, genetic and environmental factors are assumed to play a determining role in the regulation of their biosynthesis and thus might explain the high variability of AA levels. Here we investigate the influence of an ACTH receptor promoter polymorphism affecting ACTH receptor gene transcription on ACTH-dependent dehydroepiandrosterone (DHEA) secretion. DESIGN: We recently reported a polymorphism within the transcription initiation site of the ACTH receptor gene promoter that alters the consensus sequence from CTC to CCC at -2 bp. This results in lower promoter activity in vitro and is associated with impaired cortisol response to ACTH stimulation in vivo. We now studied 14 normal, lean volunteers aged 20-35 years (eight CTC/CTC and six CCC/CCC carriers) in a 6-h ACTH stimulation test. METHODS: After overnight dexamethasone suppression, ACTH1-24 was administered continuously in each subject with hourly increasing doses (120-3840 ng/m2 body surface area/h) within a 6-h period. On a separate day, baseline DHEA samples were collected. RESULTS: In the 6-h ACTH stimulation test, CTC/CTC carriers showed a significantly higher DHEA response than CCC/CCC carriers (area under the curve: 19 367 +/- 2919 vs 11 098 +/- 1241 nmol/l per min; P < 0.04, Mann-Whitney U-test). In contrast, baseline DHEA concentrations did not differ between groups. CONCLUSION: These data demonstrate that genetic variations within the ACTH receptor promoter result in decreased DHEA secretion. Thus, we might have identified one of the genetic factors responsible for variation in ACTH-dependent DHEA secretion.

ACTH 1-24 inhibits proliferation of adrenocortical tumors in vivo.

OBJECTIVES: Although several lines of evidence suggest that the overall effects of the ACTH receptor, melanocortin 2 receptor (MC2-R), mediated signal transduction on adrenocortical growth and tumorigenesis are anti-proliferative, activation of MC2-R induces mitogens like jun, fos, and myc and activates the MAPK pathway. In vivo, potential effects of endogenous ACTH on adrenal tumori-genesis can not be separated from effects of other POMC derived peptides. METHODS: Murine adrenocortical tumor cells that lack MC2-R expression (Y6(pcDNA)) and Y6 cells stablely transfected with MC2-R (Y6(MC2-R)) were generated. Presence of functional MC2-R was demonstrated by RT-PCR and Western blot using an antibody for phosphorylated CREB. As a syngenic tumor model, LaHeF1/J mice simultaneously received 10(7) Y6(MC2-R) and Y6(pcDNA) subcutaneously, giving rise to MC2-R positive and negative tumors within the same animal. Animals were treated for 3 weeks in groups of 12 according to the following schedule: group A, control animals receiving saline injection; group B, animals receiving 5.7 ng/injection of a slow release formula of ACTH 1-24 administered i.p. three times a week (aiming at a low physiologic dose); and group C, animals receiving 57 ng/injection of ACTH 1-24 (high physiological dose). RESULTS: Twenty days of ACTH 1-24 treatment did not significantly affect corticosterone levels, endogenous ACTH levels or adrenal and thymus weight compared with saline injection. However, ACTH 1-24 treatment of group B and C mice significantly reduced tumor weight in MC2-R positive tumors in a dose dependent manner (P = 0.03), while no significant difference in tumor mass was observed in MC2-R negative tumors. PCNA and TUNEL staining, together with morphological characterization, demonstrated that these in vivo effects were due to reduced proliferation, while apoptosis and cellular hypertrophy within the tumor remained unchanged. CONCLUSION: MC2-R expression is associated with a less aggressive adrenal tumor phenotype and anti-proliferative effects can be amplified through stimulation with physiological doses of ACTH.

Clinical and molecular evidence for DAX-1 inhibition of steroidogenic factor-1-dependent ACTH receptor gene expression.

BACKGROUND: The ACTH receptor (ACTH-R) is a member of the seven transmembrane domain receptor super-family. In non-functional adrenal adenomas and adrenocortical carcinomas, ACTH-R expression is low. However, no inhibitory factor for ACTH-R expression has been defined to date. DAX-1 (dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene-1) is a general repressor of steroid production, inhibiting steroidogenic factor-1 (SF-1)-dependent expression of multiple steroidogenic enzymes. The aim of this study was to investigate whether ACTH-R gene transcription is affected by DAX-1 and whether this mechanism is involved in down-regulation of ACTH-R expression in adrenocortical tumors. METHODS: We screened 22 adrenocortical tumors for ACTH-R and DAX-1 mRNA expression by Northern blot. For in vitro analyses we co-transfected mouse Y1 adrenocortical carcinoma cells with the luciferase reporter gene vector pGL3 containing full-length constructs of human (h) or mouse (m) ACTH-R promoter together with a DAX-1 expression plasmid. These experiments were also performed using ACTH-R promoter 5'-deletion constructs and constructs mutated at the SF-1-binding sites. RESULTS: We found a negative correlation between DAX-1 and ACTH-R mRNA expression (R=-0.47, P<0.02). Accordingly, in vitro expression of DAX-1 significantly reduced hACTH-R and mACTH-R promoter activity by 89 and 55% respectively. DAX-1 inhibition was also present in the shortest construct of a series of 5'-deletion constructs of the human promoter extending from -64 to +40 bp relative to the transcription start site. Mutation of the SF-1-binding sites within the hACTH-R promoter resulted in reduced or abolished DAX-1 inhibition, arguing for a mechanism that involves SF-1 for DAX-1 inhibition. CONCLUSIONS: These data support the concept that DAX-1 is a major repressor of ACTH-R gene expression in vitro and in vivo.

A promoter polymorphism of the CYP27B1 gene is associated with Addison's disease, Hashimoto's thyroiditis, Graves' disease and type 1 diabetes mellitus in Germans.

BACKGROUND: CYP27B1 hydroxylase catalyzes the conversion of 25 hydroxyvitamin D(3) (25OHD(3)) to 1,25(OH)(2)D(3), the most active natural vitamin D metabolite, which plays a role in the regulation of immunity and cell proliferation. We therefore investigated two single nucleotide polymorphisms in the CYP27B1 hydroxylase gene for an association with Addison's disease, Hashimoto's thyroiditis, Graves' disease and type 1 diabetes mellitus. METHODS: Patients with Addison's disease (n=124), Hashimoto's thyroiditis (n=139), Graves' disease (n=334), type 1 diabetes mellitus (n=252) and healthy controls (n=320) were genotyped for the promoter (-1260) C/A polymorphism and for the intron 6 (+2838) C/T polymorphism of the CYP27B1 gene. Patients and controls were compared using genotype-wise and allele-wise X(2) testing. RESULTS: A significant association was found between allelic variation of the promoter (-1260) C/A polymorphism and Addison's disease, Hashimoto's thyroiditis, Graves' disease and type 1 diabetes mellitus (P=0.0062, P=0.0173, P=0.0094 and P=0.0028 respectively). Significant differences were also observed for the intron 6 (+2838) C/T polymorphism (P=0.0058) in Hashimoto's thyroiditis but not for the other autoimmune endocrine diseases. CONCLUSIONS: The CYP27B1 promoter (-1260) C/A polymorphism appears to be associated with endocrine autoimmune diseases but the CYP27B1 intron 6 (+2838) C/T polymorphism appears to be associated only with Hashimoto's thyroiditis. These results imply a regulatory difference of the CYP27B1 hydroxylase to predispose to endocrine autoimmunity.

Characterization of an adrenocorticotropin (ACTH) receptor promoter polymorphism leading to decreased adrenal responsiveness to ACTH.

The ACTH receptor has a pivotal role in the regulation of adrenal cortisol secretion. Here, we describe a polymorphism within the transcription initiation site of the ACTH receptor promoter altering the consensus sequence from CTC to CCC. The prevalence of the polymorphism in 1266 unrelated healthy men was 80.2% for CTC/CTC, 19.0% for CTC/CCC, and 0.8% for CCC/CCC, respectively. In vitro studies using luciferase assays demonstrated a lower basal (CCC, 73 +/- 4%; CTC, 100 +/- 5%; P = 0.02) and forskolin-stimulated (CCC, 143 +/- 13%; CTC, 194 +/- 15%; P = 0.0008) promoter activity in the CCC construct compared with CTC. The clinical significance of the in vitro findings was investigated by a 6-h ACTH stimulation test with increasing ACTH(1-24) doses in normal subjects, demonstrating a blunted cortisol response in CCC/CCC subjects compared with CTC/CTC individuals (area under the curve, 12176 +/- 966; 16334 +/- 1051 nmol/liter.min; P < 0.03). Accordingly, after CRH stimulation, subjects with CCC/CCC showed a higher ACTH/cortisol ratio (P < 0.05) suggesting a decreased adrenal responsiveness to endogenous ACTH. In conclusion, we describe an ACTH receptor promoter polymorphism that results in a lower promoter activity in vitro and is associated with a lower cortisol secretion to prolonged ACTH stimulation in vivo. This polymorphism might influence cortisol homeostasis under stress conditions.