Etiopathogenesis of Hepatic Osteodystrophy in Wistar Rats with Cholestatic Liver Disease

The pathophysiology of hepatic osteodystrophy (HO) remains poorly understood. Our aim was to evaluate bone histomorphometry, biomechanical properties, and the role of the growth hormone (GH)/insulin-like growth factor-I (IGF-I) system in the onset of this disorder. Forty-six male Wistar rats were divided into two groups: sham-operated (SO, n = 23) and bile duct-ligated (BDL, n = 23). Rats were killed on day 30 postoperatively. Immunohistochemical expression of IGF-I and GH receptor was determined in liver tissue and in the proximal growth plate cartilage of the left tibia. Histomorphometric analysis was performed in the right tibia, and the right femur was used for biomechanical analysis. The maximal force at fracture and the stiffness of the mid-shaft femur were, respectively, 53% and 24% lower in BDL compared to SO. Histomorphometric measurements showed low cancellous bone volume and decreased cancellous bone connectivity in BDL, compatible with osteoporosis. This group also showed increased mineralization lag time, indicating disturbance in bone mineralization. Serum levels of IGF-I were lower in BDL (basal 1,816 +/- A 336 vs. 30 days 1,062 +/- A 191 ng/ml, P < 0.0001). BDL also showed higher IGF-I expression in the liver tissue but lower IGF-I and GH receptor expression in growth plate cartilage than SO. Osteoporosis is the most important feature of HO; BDL rats show striking signs of reduced bone volume and decreased bone strength, as early as after 1 month of cholestasis. The endocrine and autocrine-paracrine IGF-I systems are deeply affected by cholestasis. Further studies will be necessary to establish their role in the pathogenesis of HO.

MicroRNAs Differentially Expressed in ACTH-Secreting Pituitary Tumors

Context: MicroRNAs (miRNAs) are small noncoding RNAs, functioning as antisense regulators of gene expression by targeting mRNA and contributing to cancer development and progression. More than 50% of miRNA genes are located in cancer-associated genomic regions or in fragile sites of the genome. Objective: The aim of the study was to analyze the differential expression of let-7a, miR-15a, miR-16, miR-21, miR-141, miR-143, miR-145, and miR-150 in corticotropinomas and normal pituitary tissue and verify whether their profile of expression correlates with tumor size or remission after treatment. Material and Methods: ACTH-secreting pituitary tumor samples were obtained during transphenoidal surgery from patients with Cushing disease and normal pituitary tissues from autopsies. The relative expression of miRNAs was measured by real-time PCR using RNU44 and RNU49 as endogenous controls. Relative quantification of miRNA expression was calculated using the 2(-Delta Delta Ct) method. Results: We found underexpression of miR-145 (2.0-fold; P = 0.04), miR-21 (2.4-fold; P = 0.004), miR-141 (2.6-fold; P = 0.02), let-7a (3.3-fold; P = 0.003), miR-150 (3.8-fold; P = 0.04), miR-15a (4.5-fold; P = 0.03), miR-16 (5.0-fold; P = 0.004), and miR-143 (6.4-fold; P = 0.004) in ACTH-secreting pituitary tumors when compared to normal pituitary tissues. There were no differences between miRNA expression and tumor size as well as miRNA expression and ratio of remission after surgery, except in patients presenting lower miR-141 expression who showed a better chance of remission. Conclusion: Our results support the possibility that altered miRNA expression profile might be involved in corticotrophic tumorigenesis. However, the lack of knowledge about miRNA target genes postpones full understanding of the biological functions of down-regulated or up-regulated miRNAs in corticotropinomas. (J Clin Endocrinol Metab 94: 320-323, 2009)

Loss-of-function mutations in the genes encoding prokineticin-2 or prokineticin receptor-2 cause autosomal recessive Kallmann syndrome

Context: Physiological activation of the prokineticin pathway has a critical role in olfactory bulb morphogenesis and GnRH secretion in mice. Objective: To investigate PROK2 and PROKR2 mutations in patients with hypogonadotropic hypogonadism (HH) associated or not with olfactory abnormalities. Design: We studied 107 Brazilian patients with HH (63 with Kallmann syndrome and 44 with normosmic HH) and 100 control individuals. The coding regions of PROK2 and PROKR2 were amplified by PCR followed by direct automatic sequencing. Results: In PROK2, two known frameshift mutations were identified. Two brothers with Kallmann syndrome harbored the homozygous p. G100fsX121 mutation, whereas one male with normosmic HH harbored the heterozygous p. I55fsX56 mutation. In PROKR2, four distinct mutations (p. R80C, p. Y140X, p. L173R, and p. R268C) were identified in five patients with Kallmann syndrome and in one patient with normosmic HH. These mutations were not found in the control group. The p. R80C, p. L173R, and p. R268C missense mutations were identified in the heterozygous state in the HH patients and in their asymptomatic first-degree relatives. In addition, nomutations of FGFR1, KAL1, GnRHR, KiSS-1, or GPR54 were identified in these patients. Notably, the new nonsense mutation (p. Y140X) was identified in the homozygous state in an anosmic boy with micropenis, bilateral cryptorchidism, and high-arched palate. His asymptomatic parents were heterozygous for this severe defect. Conclusion: We expanded the repertoire of PROK2 and PROKR2 mutations in patients with HH. In addition, we show that PROKR2 haploinsufficiency is not sufficient to cause Kallmann syndrome or normosmic HH, whereas homozygous loss-of-function mutations either in PROKR2 or PROK2 are sufficient to cause disease phenotype, in accordance with the Prokr2 and Prok2 knockout mouse models.

Ovarian recovery after stem cell transplantation

Autologous or allogeneic SCT with conventional conditioning (chemotherapy with or without irradiation) has emergedas an effective and potentially curative therapy in patients with hematologic malignancies and in other selected solid tumors; however, several patients experience significant early and delayed side effects, including long-term endocrine imbalance and infertility. In spite of several reproductive recovery and pregnancy reports published in the oncology literature, review of medical literature reveals a paucity of comparable information in the SCT field. We report here four cases of ovarian recovery in patients who received hormonal replacement therapy after diagnosis of primary ovarian failure due to high-dose chemotherapy and SCT.

TAC3/TACR3 Mutations Reveal Preferential Activation of Gonadotropin-Releasing Hormone Release by Neurokinin B in Neonatal Life Followed by Reversal in Adulthood

Context: Mutations in TAC3 and TACR3 (encoding neurokinin B and its receptor) have been identified in Turkish patients with idiopathic hypogonadotropic hypogonadism (IHH), but broader populations have not yet been tested and genotype-phenotype correlations have not been established. Objective: A broad cohort of normosmic IHH probands was screened for mutations in TAC3/TACR3 to evaluate the prevalence of such mutations and define the genotype/phenotype relationships. Design and Setting: The study consisted of sequencing of TAC3/TACR3, in vitro functional assays, and neuroendocrine phenotyping conducted in tertiary care centers worldwide. Patients or Other Participants: 345 probands, 18 family members, and 292 controls were studied. Intervention: Reproductive phenotypes throughout reproductive life and before and after therapy were examined. Main Outcome Measure: Rare sequence variants in TAC3/TACR3 were detected. Results: In TACR3, 19 probands harbored 13 distinct coding sequence rare nucleotide variants [three nonsense mutations, six nonsynonymous, four synonymous (one predicted to affect splicing)]. In TAC3, one homozygous single base pair deletion was identified, resulting in complete loss of the neurokinin B decapeptide. Phenotypic information was available on 16 males and seven females with coding sequence variants in TACR3/TAC3. Of the 16 males, 15 had microphallus; none of the females had spontaneous thelarche. Seven of the 16 males and five of the seven females were assessed after discontinuation of therapy; six of the seven males and four of the five females demonstrated evidence for reversibility of their hypogonadotropism. Conclusions: Mutations in the neurokinin B pathway are relatively common as causes of hypogonadism. Although the neurokinin B pathway appears essential during early sexual development, its importance in sustaining the integrity of the hypothalamic-pituitary-gonadal axis appears attenuated over time. (J Clin Endocrinol Metab 95: 2857-2867, 2010)

THE MEDIAL AMYGDALOID NUCLEUS MODULATES CARDIOVASCULAR RESPONSES TO ACUTE RESTRAINT IN RATS

The medial amygdaloid nucleus (MeA) modulates several physiological and behavioral processes and among them, the cardiovascular correlates of behavioral responses to stressful stimuli. Acute restraint evokes cardiovascular responses, which are characterized by both elevated blood pressure (BP) and intense heart rate (HR) increase. We presently report effects of MeA pharmacological manipulations on BP and HR responses evoked by acute restraint in rats. Bilateral microinjection of 100 nL of the unspecific synaptic blocker COCl(2) (1 mM) into the MeA increased HR response to acute restraint, without significant effect on the BP response. This result indicates an inhibitory influence of MeA on restraint-evoked HR changes. Injections of the non-selective muscarinic receptor antagonist atropine (3 nmol); the inhibitor of choline uptake hemicholinium (2 nmol) or the selective M(1)-receptor antagonist pirenzepine (6 nmol) caused effects that were similar to those caused by cobalt. These results suggest that local cholinergic neurotransmission and M(1)-receptors mediate the MeA inhibitory influence on restraint-related HR responses. Pretreatment with the M3 receptor antagonist 4-DAMP (4-Diphenylacetoxy-N-methylpiperidine methiodide-2 nmol) did not affect restraint-related cardiovascular responses, reinforcing the idea that M(1)-receptors mediate MeA-related inhibitory influence on restraint-evoked HR increase. (C) 2009 IBRO. Published by Elsevier Ltd. All rights reserved.

The paraventricular nucleus of the hypothalamus is involved in cardiovascular responses to acute restraint stress in rats

The paraventricular nucleus of the hypothalamus (PVN) has been implicated in several aspects of cardiovascular control. Stimulation of the PVN evokes changes in blood pressure and heart rate. Additionally, this brain area is connected to several limbic structures implicated in behavioral control, as well as to forebrain and brainstem structures involved in cardiovascular control. This evidence indicates that the PVN may modulate cardiovascular correlates of behavioral responses to stressful stimuli. Acute restraint is an unavoidable stressor that evokes marked and sustained cardiovascular changes, which are characterized by elevated mean arterial pressure (MAP) and an intense heart rate (HR) increase. We report on the effect of inhibition of PVN synapses on MAP and HR responses evoked by acute restraint in rats. Bilateral microinjection of the nonspecific synaptic blocker cobalt (CoCl2, 1mM/100nl) into the PVN did not change the HR response or the initial peak of the MAP response to restraint stress, but reduced the area under the curve of the MAP response. Moreover, bilateral microinjection of cobalt in areas surrounding the PVN did not change the cardiovascular response to restraint. These results indicate that synapses in the PVN are involved in the neural pathway that controls blood pressure changes evoked by restraint.

Clinical Criteria as Predictive Factors of Response to Primary Hormone Therapy in Locally Advanced Breast Cancer

This study investigates the efficacy of clinical criteria in selecting patients for primary tamoxifen therapy. A total of 60 breast cancer patients with large primary tumors and unknown hormonal receptor status were subjected to primary hormone therapy. Inclusion criteria were age over 60 years old or menopausal status for at least 10 years and no clinical evidence of inflammatory disease and fast tumor growth. The objective response rate was 55%. There was a positive correlation between the lack of clinical response and axillary lymph node metastasis (p = 0.009). Patients with objective response had significantly improved disease-free (p = 0.045) and overall (p = 0.0002) survival over those who did not have response to hormonal therapy. In multivariate analysis, the clinical response to therapy was the most powerful prognostic factor. This analysis demonstrates that clinical criteria were very effective predictor of response to neo-adjuvant hormone therapy in large breast tumors for postmenopausal women. Response to therapy is the major prognostic factor in primary tamoxifen-treated breast cancer.

Sonographic evaluation of endothelial function in letrozole and tamoxifen users

Objectives: Studies have shown that women previously treated for breast cancer present fewer cardiovascular events, indicating a possible protective effect of tamoxifen treatment. The effects of these aromatase inhibitors on cardiovascular protection remain controversial. The aim of this study was to compare some cardiovascular risk markers among breast cancer survivors following treatment with tamoxifen group (TMXg), letrozole group (LTZg) or no endocrine treatment group (NETg). Methods: A total of 103 breast cancer survivors: 35 using TMXg, 34 using letrozole group (LTZg) and 34 using no endocrine treatment group (NETg) were evaluated. Ultrasonographic evaluation of brachial artery flow-mediated dilation (FMD), carotid intima-media thickness (IMT) and stiffness index (beta); blood total cholesterol, HDL and triglycerides were assessed. Results: All three groups presented similar values of HDL and IMT. TMXg showed the lowest total cholesterol (219.29 +/- 36.31 mg/dL vs. 250.59 +/- 38.37 mg/dL vs. 245.09 +/- 35.35 mg/dL; TMXg vs. LTZg vs. NETg, respectively; p < 0.01-ANOVA), the highest triglycerides (139.34 +/- 41.82 mg/dL vs. 111.35 +/- 28.22 mg/dL vs.122.09 +/- 33.42 mg/dL; p < 0.01), the highest FMD (6.32 +/- 2.33% vs. 4.10 +/- 2.06% vs. 4.66 +/- 2.52%; p < 0.01) and the lowest stiffness index (beta) (5.08 +/- 1.68 vs. 6.28 +/- 1.75 vs. 5.99 +/- 1.86; p=0.01). LTZg did not differ significantly from NETg on any evaluated parameter. Conclusions: We did not observe any effect of LTZg on the evaluated cardiovascular risk parameters compared to NETg. As such, the observed difference on lipid values, stiffness index (beta) and FMD between women receiving tamoxifen anti letrozole might be best attributed to the beneficial effect of tamoxifen than to a detrimental effect of letrozole. (C) 2008 Elsevier Ireland Ltd. All rights reserved.

Aspirin prevents diabetic oxidative changes in rat lacrimal gland structure and function

The aim of this study is to evaluate whether aspirin reduces Diabetis Mellitus (DM) oxidative damage in the lacrimal gland (LG), and ocular surface (OS). Ten weeks after streptozotocin induced DM and aspirin treatment, LG and OS of rats were compared for tear secretion, hidtology, peroxidase activity, and expression of uncoupling proteins (UCPs). DM reduction of tear secretion was prevented by aspirin (P < 0.01). Alterations of LG morphology and increased numbers of lipofucsin-like inclusions were observed in diabetic but not in aspirin-treated diabetic rats. Peroxidase activity levels were higher and UCP-2 was reduced in DM LG but not in aspirin treated (P = 0.0025 and P < 0.05, respectively). The findings prevented by aspirin indicate a direct inhibitory effect on oxidative pathways in LG and their inflammatory consequences, preserving the LG structure and function against hyperglycemia and/or insulin deficiency damage.

Dry eye in childhood: Epidemiological and clinical aspects

Because dry eye disease is rare in children and its pathogenesis is less well known than in adults, its diagnosis is often overlooked. It can occur in association with a number of congenital, autoimmune, endocrine, and inflammatory disorders, or under certain environmental and nutritional conditions. In some cases, early detection allows the underlying cause of the dry eye to be successfully treated and eliminated. In other cases, the disease may represent a lifelong problem, whose proper management can prevent ulceration and scarring of the ocular surface. Because of the association of pediatric dry eye with other conditions, a multidisciplinary approach to diagnosis and treatment is usually required. The purpose of this review is to enhance physician awareness of dry eye in children, to describe the most frequently associated conditions, and to discuss the diagnostic and therapeutic options available.

Mineralocorticoid Receptor Mutations Differentially Affect Individual Gene Expression Profiles in Pseudohypoaldosteronism Type 1

Context: Type 1 pseudohypoaldosteronism (PHA1), a primary form of mineralocorticoid resistance, isdueto inactivating mutations of the NR3C2 gene, coding for the mineralocorticoid receptor (MR). Objective: The objective of the study was to assess whether different NR3C2 mutations have distinct effects on the pattern of MR-dependent transcriptional regulation of aldosterone-regulated genes. Design and Methods: Four MR mutations affecting residues in the ligand binding domain, identified in families with PHA1, were tested. MR proteins generated by site-directed mutagenesis were analyzed for their binding to aldosterone and were transiently transfected into renal cells to explore the functional effects on the transcriptional activity of the receptors by cis-trans-cotrans-activation assays and by measuring the induction of endogenous gene transcription. Results: Binding assays showed very low or absent aldosterone binding for mutants MR(877Pro), MR(848Pro), and MR(947stop) and decreased affinity for aldosterone of MR(843Pro). Compared with wildtype MR, the mutations p.Leu843Pro and p.Leu877Pro displayed half-maximal aldosterone-dependent transactivation of reporter genes driven by mouse mammary tumor virus or glucocorticoid response element-2 dependent promoters, whereas MR(848Pro) and MR(947stop) nearly or completely lost transcriptional activity. Although MR(848Pro) and MR(947stop) were also incapable of inducing aldosterone-dependent gene expression ofendogenoussgk1, GILZ, NDRG2, and SCNN1A, MR(843Pro) retained complete transcriptional activity on sgk1 and GILZ gene expression, and MR(877Pro) negatively affected the expression of sgk1, NDRG2, and SCNN1A. Conclusions: Our data demonstrate that MR mutations differentially affect individual gene expression in a promoter-dependent manner. Investigation of differential gene expression profiles in PHA1 may allow a better understanding of the molecular substrate of phenotypic variability and to elucidate pathogenic mechanisms underlying the disease. (J Clin Endocrinol Metab 96: E519-E527, 2011)

Obesity due to Melanocortin 4 Receptor (MC4R) Deficiency Is Associated with Increased Linear Growth and Final Height, Fasting Hyperinsulinemia, and Incompletely Suppressed Growth Hormone Secretion

Context: Melanocortin receptor 4 (MC4R) deficiency is characterized by increased linear growth greater than expected for the degree of obesity. Objective: The objective of the investigation was to study the somatotroph axis in obese MC4R-deficient patients and equally obese controls. Patients and Methods: We obtained anthropometric measurements and insulin concentrations in 153 MC4R-deficient subjects and 1392 controls matched for age and severity of obesity. We measured fasting IGF-I, IGF-II, IGF binding protein (IGFBP)-1, IGFBP-3, and acid-labile subunit levels in a subset of 33 MC4R-deficient patients and 36 control subjects. We examined pulsatile GH secretion in six adult MC4R-deficient subjects and six obese controls. Results: Height so score was significantly greater in MC4R-deficient children under 5 yr of age compared with controls (mean +/- SEM: 2.3 +/- 0.06 vs. 1.8 +/- 0.04, P < 0.001), an effect that persisted throughout childhood. Final height (cm) was greater in MC4R-deficient men (mean +/- SEM 173 +/- 2.5 vs. 168 +/- 2.1, P < 0.001) and women (mean 165 +/- 2.1 vs. 158 +/- 1.9, P < 0.001). Fasting IGF-I, IGF-II, acid-labile subunit, and IGFBP-3 concentrations were similar in the two groups. GH levels were markedly suppressed in obese controls, but pulsatile GH secretion was retained in MC4R deficiency. The mean maximal GH secretion rate per burst (P < 0.05) and mass per burst (P < 0.05) were increased in MC4R deficiency, consistent with increased pulsatile and total GH secretion. Fasting insulin levels were markedly elevated in MC4R-deficient children. Conclusions: In MC4R deficiency, increased linear growth in childhood leads to increased adult final height, greater than predicted by obesity alone. GH pulsatility is maintained in MC4R deficiency, a finding consistent with animal studies, suggesting a role for MC4R in controlling hypothalamic somatostatinergic tone. Fasting insulin levels are significantly higher in children carrying MC4R mutations. Both of these factors may contribute to the accelerated growth phenotype characteristic of MC4R deficiency. (J Clin Endocrinol Metab 96: E181-E188, 2011)

The-202 A Allele of Insulin-Like Growth Factor Binding Protein-3 (IGFBP3) Promoter Polymorphism Is Associated with Higher IGFBP-3 Serum Levels and Better Growth Response to Growth Hormone Treatment in Patients with Severe Growth Hormone Deficiency

Context: Genetic factors that influence the response to recombinant human GH (rhGH) therapy remain mostly unknown. To date, only the GH receptor gene has been investigated. Objective: The aim of the study was to assess the influence of a polymorphism in the IGF-binding protein-3 (IGFBP-3) promoter region (-202 A/C) on circulating IGFBP-3 levels and growth response to rhGH therapy in children with GH deficiency (GHD). Design and Patients: -202 A/C IGFBP3 genotyping (rs2854744) was correlated with data of 71 children with severe GHD who remained prepubertal during the first year of rhGH treatment. Main Outcome Measures: We measured IGFBP-3 levels and first year growth velocity (GV) during rhGH treatment. Results: Clinical and laboratory data at the start of treatment were indistinguishable among patients with different -202 A/C IGFBP3 genotypes. Despite similar rhGH doses, patients homozygous for the A allele presented higher IGFBP-3 SD score levels and higher mean GV in the first year of rhGH treatment than patients with AC or CC genotypes (first year GV, AA = 13.0 +/- 2.1 cm/yr, AC = 11.4 +/- 2.5 cm/yr, and CC = 10.8 +/- 1.9 cm/yr; P = 0.016). Multiple linear regression analyses demonstrated that the influence of -202 A/C IGFBP3 genotype on IGFBP-3 levels and GV during the first year of rhGH treatment was independent of other variables. Conclusion: The -202 A allele of IGFBP3 promoter region is associated with increased IGFBP-3 levels and GV during rhGH treatment in prepubertal GHD children. (J Clin Endocrinol Metab 94: 588-595, 2009)

C-Peptide Levels and Insulin Independence Following Autologous Nonmyeloablative Hematopoietic Stem Cell Transplantation in Newly Diagnosed Type 1 Diabetes Mellitus

Context In 2007, the effects of the autologous nonmyeloablative hematopoietic stem cell transplantation (HSCT) in 15 patients with type 1 diabetes mellitus (DM) were reported. Most patients became insulin free with normal levels of glycated hemoglobin A(1c) (HbA(1c)) during a mean 18.8-month follow-up. To investigate if this effect was due to preservation of beta-cell mass, continued monitoring was performed of C-peptide levels after stem cell transplantation in the 15 original and 8 additional patients. Objective To determine C-peptide levels after autologous nonmyeloablative HSCT in patients with newly diagnosed type 1 DM during a longer follow-up. Design, Setting, and Participants A prospective phase 1/2 study of 23 patients with type 1 DM(aged 13-31 years) diagnosed in the previous 6 weeks by clinical findings with hyperglycemia and confirmed by measurement of serum levels of anti glutamic acid decarboxylase antibodies. Enrollment was November 2003-April 2008, with follow-up until December 2008 at the Bone Marrow Transplantation Unit of the School of Medicine of Ribeirao Preto, Ribeirao Preto, Brazil. Hematopoietic stem cells were mobilized via the 2007 protocol. Main Outcome Measures C-peptide levels measured during the mixed-meal tolerance test, before, and at different times following HSCT. Secondary end points included morbidity and mortality from transplantation, temporal changes in exogenous insulin requirements, and serum levels of HbA1c. Results During a 7- to 58-month follow-up (mean, 29.8 months; median, 30 months), 20 patients without previous ketoacidosis and not receiving corticosteroids during the preparative regimen became insulin free. Twelve patients maintained this status for a mean 31 months (range, 14-52 months) and 8 patients relapsed and resumed insulin use at low dose (0.1-0.3 IU/kg). In the continuous insulin-independent group, HbA(1c) levels were less than 7.0% and mean (SE) area under the curve (AUC) of C-peptide levels increased significantly from 225.0 (75.2) ng/mL per 2 hours pretransplantation to 785.4 (90.3) ng/mL per 2 hours at 24 months posttransplantation (P<.001) and to 728.1 (144.4) ng/mL per 2 hours at 36 months (P=.001). In the transient insulin-independent group, mean (SE) AUC of C-peptide levels also increased from 148.9 (75.2) ng/mL per 2 hours pretransplantation to 546.8 (96.9) ng/mL per 2 hours at 36 months (P=.001), which was sustained at 48 months. In this group, 2 patients regained insulin independence after treatment with sitagliptin, which was associated with increase in C-peptide levels. Two patients developed bilateral nosocomial pneumonia, 3 patients developed late endocrine dysfunction, and 9 patients developed oligospermia. There was no mortality. Conclusion After a mean follow-up of 29.8 months following autologous nonmyeloablative HSCT in patients with newly diagnosed type 1 DM, C-peptide levels increased significantly and the majority of patients achieved insulin independence with good glycemic control. Trial Registration clinicaltrials.gov Identifier: NCT00315133