bluuebunny/crossref_metadata_2025 huggingface dataset:
Risedronate, a Highly Effective Oral Agent in the Treatment of Patients with Severe Paget’s Disease1
Authors: Singer Frederick R. , Clemens Thomas L. , Eusebio Rachelle A. , Bekker Pirow J.
Published: January 2014

Thirteen patients with severe Paget’s disease of bone [mean serum alkaline phosphatase (SAP) level 17 times the upper limit of normal] were treated with 30 mg oral risedronate daily for 8 weeks. Patients were followed for 16 weeks without treatment. The change from baseline SAP was the primary end point. Those patients whose SAP levels did not reach the normal range were retreated with 30 mg for another 8 weeks. There was a mean percent decrease in SAP of 77% after the first course of risedronate treatment and 87% after the second course of treatment. All patients who completed the study had a decrease in SAP of at least 77% from the baseline. The urinary hydroxyproline/creatinine level was decreased by 64% and 79%, respectively, during the first and second treatment courses. There were transient asymptomatic decreases in serum calcium and phosphorus levels. The urinary calcium/creatinine ratio also decreased in these patients. Serum intact PTH and 1,25-dihydroxyvitamin D levels increased transiently during risedronate treatment. Oral risedronate was well tolerated by the patients. Only one patient discontinued treatment because of an adverse event (diarrhea) thought to be related to risedronate therapy.
Polymerase Chain Reaction-Based Microsatellite Polymorphism Analysis of Follicular and Hürthle Cell Neoplasms of the Thyroid1
Authors: Segev Dorry L. , Saji Motoyasu , Phillips Grace S. , Westra William H. , Takiyama Yumi , Piantadosi Steven , Smallridge Robert C. , Nishiyama Ronald H. , Udelsman Robert , Zeiger Martha A.
Published: January 2014

Follicular and Hürthle cell carcinomas of the thyroid cannot be differentiated from adenomas by either preoperative fine needle aspiration or intraoperative frozen section examination, and yet there exist potentially significant differences in the recommended surgical management. We examined, by PCR-based microsatellite polymorphism analysis, DNA obtained from 83 thyroid neoplasms [22 follicular adenomas, 29 follicular carcinomas, 20 Hürthle cell adenomas (HA), and 12 Hürthle cell carcinomas (HC)] to determine whether a pattern of allelic alteration exists that could help distinguish benign from malignant lesions. Alterations were found in only 7.5% of informative PCR reactions from follicular neoplasms, whereas they were found in 23.3% of reactions from Hürthle cell neoplasms. Although there were no significant differences between follicular adenoma and follicular carcinoma, HC demonstrated a significantly greater percentage of allelic alteration than HA on chromosomal arms 1q (P < 0.001) and 2p (P < 0.05) by Fisher’s exact test. The documentation of an alteration on either 1q or 2p was 100% sensitive and 65% specific in the detection of HC (P < 0.0005, by McNemar’s test).

In conclusion, PCR-based microsatellite polymorphism analysis may be a useful technique in distinguishing HC from HA. Potentially, the application of this technique to aspirated material may allow this distinction preoperatively and thus facilitate more optimal surgical management. Consistent regions of allelic alteration may also indicate the locations of critical genes, such as tumor suppressor genes or oncogenes, that are important in the progression from adenoma to carcinoma. Finally, this study demonstrates that Hürthle cell neoplasms, now considered variants of follicular neoplasms, differ significantly from follicular neoplasms on a molecular level.
Study of a Kindred with Classic Congenital Adrenal Hyperplasia: Diagnostic Challenge due to Phenotypic Variance1
Authors: Chin Daisy , Speiser Phyllis W. , Imperato-McGinley Julianne , Dixit Naznin , Uli Naveen , David Raphael , Oberfield Sharon E.
Published: January 2014

We sought to determine the concordance of the phenotype and genotype in a kindred with classic congenital adrenal hyperplasia due to 21-hydroxylase deficiency. The variation in phenotypic expression within this family underscores the difficulty of establishing the diagnosis in the absence of newborn screening, even with a heightened index of suspicion.

Steroidogenic profiles were obtained for the three affected siblings. The available clinical history of the two affected aunts was retrieved. Genotyping was performed on several members of the kindred. Detailed sequencing of the entire CYP21 gene of two clinically dissimilar subjects in this family was undertaken to explore the possibility of other mutations or polymorphisms.

PCR with ligase detection reaction analysis of CYP21 revealed that the affected family members III-2, III-3, III-4, II-3, and II-4, all were compound heterozygotes carrying the intron 2 point mutation known to interfere with splicing (nucleotide 656 A to G) and the exon 4 point mutation causing a nonconservative substitution of asparagine for isoleucine at codon 172 (I172N). Detailed sequencing of the gene was performed for the two most phenotypically dissimilar subjects. A single silent polymorphism was found in the third nucleotide for codon 248 in patient II-4, but not in patient III-4, and no additional mutations were found.

Classic congenital adrenal hyperplasia remains a difficult diagnosis to make in the absence of newborn screening because of the variability of phenotypic expression. Likewise, the variable degree of genital ambiguity in affected females in this family serves to question universal advocacy of prenatal steroid treatment in pregnancies at risk for congenital adrenal hyperplasia. Extensive molecular exploration did not provide an explanation of the phenotypic heterogeneity and supports the possibility of influences other than the CYP21 gene for the observed divergence.
Relationship of Serum Sex Steroid Levels and Bone Turnover Markers with Bone Mineral Density in Men and Women: A Key Role for Bioavailable Estrogen1
Authors: Khosla Sundeep , Melton L. Joseph , Atkinson Elizabeth J. , O’Fallon W. M. , Klee George G. , Riggs B. Lawrence
Published: January 2014

Estrogen (E) deficiency associated with the menopause is the major cause of bone loss in aging women. However, men also lose significant amounts of bone with age, but they do not have the equivalent of menopause, and serum total testosterone (T) and E levels decline only marginally with age in men. Thus, it has been difficult to attribute bone loss in aging men to either T or E deficiency. Here, we show in a population-based, age-stratified sample of 346 men, aged 23–90 yr, that serum total T and E (estradiol plus estrone) levels decreased over the life span by 30% and 12%, respectively, but bioavailable (or nonsex hormone-binding globulin-bound) T and E levels decreased by 64% and 47%, respectively. In these men and in a parallel cohort of 304 women, aged 21–94 yr, serum PTH increased 84% and 64% over the life span, and urinary N-telopeptide of type I collagen (NTx) excretion, a bone resorption marker, increased 77% and 80% between age 50–85 yr in the men and women, respectively. By univariate analyses, serum bioavailable T and E levels correlated positively with bone mineral density (BMD) at the total body, spine, proximal femur, and distal radius and negatively with urinary NTx excretion in men and women. Urinary NTx excretion was also negatively associated with BMD in both sexes. By multivariate analyses, however, serum bioavailable E level was the consistent independent predictor of BMD in both men and postmenopausal women. Thus, bioavailable E levels decline significantly with age and are important predictors of BMD in men as well as women. These studies suggest that in contrast to traditional belief, age-related bone loss may be the result of E deficiency not just in postmenopausal women, but also in men.
Late-Night Salivary Cortisol as a Screening Test for Cushing’s Syndrome1
Authors: Raff Hershel , Raff Jonathan L. , Findling James W.
Published: January 2014

abstract

The clinical features of Cushing’s syndrome (such as obesity, hypertension, and diabetes) are commonly encountered in clinical practice. Patients with Cushing’s syndrome have been identified by an abnormal low-dose dexamethasone suppression test, elevated urine free cortisol (UFC), an absence of diurnal rhythm of plasma cortisol, or an elevated late-night plasma cortisol. Because the concentration of cortisol in the saliva is in equilibrium with the free (active) cortisol in the plasma, measurement of salivary cortisol in the evening (nadir) and morning (peak) may be a simple and convenient screening test for Cushing’s syndrome. The purpose of this study was to evaluate the usefulness of the measurement of late-night and morning salivary cortisol in the diagnosis of Cushing’s syndrome.

We studied 73 normal subjects and 78 patients referred for the diagnosis of Cushing’s syndrome. Salivary cortisol was measured at 2300 h and 0700 h using a simple, commercially-available saliva collection device and a modification of a standard cortisol RIA. In addition, 24-h UFC was measured within 1 month of saliva sampling.

Patients with proven Cushing’s syndrome (N = 39) had significantly elevated 2300-h salivary cortisol (24.0 ± 4.5 nmol/L), as compared with normal subjects (1.2 ± 0.1 nmol/L) or with patients referred with the clinical features of hypercortisolism in whom the diagnosis was excluded or not firmly established (1.6± 0.2 nmol/L; N = 39). Three of 39 patients with proven Cushing’s had 2300-h salivary cortisol less than the calculated upper limit of the reference range (3.6 nmol/L), yielding a sensitivity of 92%; one of these 3 patients had intermittent hypercortisolism, and one had an abnormal diurnal rhythm (salivary cortisol 0700-h to 2300-h ratio <2). An elevated 2300-h salivary cortisol and/or an elevated UFC identified all 39 patients with proven Cushing’s syndrome (100% sensitivity). Salivary cortisol measured at 0700 h demonstrated significant overlap between groups, even though it was significantly elevated in patients with proven Cushing’s syndrome (23.0 ± 4.2 nmol/L), as compared with normal subjects (14.5± 0.8 nmol/L) or with patients in whom Cushing’s was excluded or not firmly established (15.3 ± 1.5 nmol/L).

Late-night salivary cortisol measurement is a simple and reliable screening test for spontaneous Cushing’s syndrome. In addition, late-night salivary cortisol measurements may simplify the evaluation of suspected intermittent hypercortisolism, and they may facilitate the screening of large high-risk populations (e.g. patients with diabetes mellitus).
Ontogeny of Iodothyronine Deiodinases in Human Liver1
Authors: Richard Kerry , Hume Robert , Kaptein Ellen , Sanders Jo P. , van Toor Hans , de Herder Wouter W. , den Hollander Jan C. , Krenning Eric P. , Visser Theo J.
Published: January 2014

abstract

The role of the deiodinases D1, D2, and D3 in the tissue-specific and time-dependent regulation of thyroid hormone bioactivity during fetal development has been investigated in animals but little is known about the ontogeny of these enzymes in humans. We analyzed D1, D2, and D3 activities in liver microsomes from 10 fetuses of 15–20 weeks gestation and from 8 apparently healthy adult tissue transplant donors, and in liver homogenates from 2 fetuses (20 weeks gestation), 5 preterm infants (27–32 weeks gestation), and 13 term infants who survived up to 39 weeks postnatally. D1 activity was determined using 1μ m [3′,5′-125I]rT3 as substrate and 10 mm dithiothreitol (DTT) as cofactor, D2 activity using 1 nm [3′,5′-125I]T4 and 25 mm DTT in the presence of 1 mm 6-propyl-2-thiouracil (to block D1 activity) and 1 μm T3 (to block D3 activity), and D3 activity using 10 nm [3,5-125I]T3 and 50 mm DTT, by quantitation of the release of 125I−. The assays were validated by high performance liquid chromatography of the products, and kinetic analysis[ Michaelis-Menten constant (Km) of rT3 for D1: 0.5 μm; Km of T3 for D3: 2 nm]. In liver homogenates, D1 activity was not correlated with age, whereas D3 activity showed a strong negative correlation with age (r −0.84), with high D3 activities in preterm infants and (except in 1 infant of 35 weeks) absent D3 activity in full-term infants. In microsomes, D1 activities amounted to 4.3–60 pmol/min/mg protein in fetal livers and to 170–313 pmol/min/mg protein in adult livers, whereas microsomal D3 activities were 0.15–1.45 pmol/min/mg protein in fetuses and <0.1 pmol/min/mg protein in all but one adult. In the latter sample, D3 activity amounted to 0.36 pmol/min/mg protein. D2 activity was negligible in both fetal and adult livers. These findings indicate high D1 and D3 activities in fetal human liver, and high D1 and mostly absent D3 activities in adult human liver. Therefore, the low serum T3 levels in the human fetus appear to be caused by high hepatic (and placental) D3 activity rather than caused by low hepatic D1 activity. The occasional expression of D3 in adult human liver is intriguing and deserves further investigation.
Soy Isoflavones Exert Modest Hormonal Effects in Premenopausal Women1
Authors: Duncan Alison M. , Merz Barbara E. , Xu Xia , Nagel Theodore C. , Phipps William R. , Kurzer Mindy S.
Published: January 2014

Soy isoflavones are hypothesized to be responsible for changes in hormone action associated with reduced breast cancer risk. To test this hypothesis, we studied the effects of isoflavone consumption in 14 premenopausal women. Isoflavones were consumed in soy protein powders and provided relative to body weight (control diet, 10 ± 1.1; low isoflavone diet, 64 ± 9.2; high isoflavone diet, 128 ± 16 mg/day) for three menstrual cycles plus 9 days in a randomized cross-over design. During the last 6 weeks of each diet period, plasma was collected every other day for analysis of estrogens, progesterone, LH, and FSH. Diet effects were assessed during each of four distinctly defined menstrual cycle phases. Plasma from the early follicular phase was analyzed for androgens, cortisol, thyroid hormones, insulin, PRL, and sex hormone-binding globulin. The low isoflavone diet decreased LH (P = 0.009) and FSH (P = 0.04) levels during the periovulatory phase. The high isoflavone diet decreased free T3 (P = 0.02) and dehydroepiandrosterone sulfate (P = 0.02) levels during the early follicular phase and estrone levels during the midfollicular phase (P = 0.02). No other significant changes were observed in hormone concentrations or in the length of the menstrual cycle, follicular phase, or luteal phase. Endometrial biopsies performed in the luteal phase of cycle 3 of each diet period revealed no effect of isoflavone consumption on histological dating. These data suggest that effects on plasma hormones and the menstrual cycle are not likely to be the primary mechanisms by which isoflavones may prevent cancer in premenopausal women.
Androgen Receptor Distribution in Adult Human Testis1
Authors: Suárez-Quian Carlos A. , Martínez-García Francisco , Nistal Manuel , Regadera Javier
Published: January 2014

The distribution of the androgen receptor (AR) in archival human testes was determined immunocytochemically using an affinity-purified peptide-specific rabbit antibody, PG21, and employing a modified biotin-streptavidin-immunoperoxidase method that incorporated a biotin amplification step. In combination with microwave epitope retrieval, the biotin amplification step increased the sensitivity of the immunostaining assay approximately 20-fold. Thus, the useful range at which PG21 rendered a robust, specific immunostaining signal without also increasing nonspecific background was extended dramatically. Broadening the useful range of the PG21 antibody made it possible to resolve the relative amounts of immunopositive AR in different cell types of the human testis. At a high PG21 concentration, for example, all AR-positive cells exhibited a robust immunostaining intensity, but it was not possible to distinguish between nuclei exhibiting either high or moderate immunostaining intensities. In contrast, as the concentration of PG21 was decreased, distinct populations of testicular cells exhibited differential AR immunostaining intensities in their nuclei. AR immunostaining of Sertoli cell nuclei was present at low PG21 concentrations at which no immunostaining of peritubular myoid cells or Leydig cells could be detected. In turn, AR immunostaining of peritubular myoid cells was detected at PG21 concentrations that did not immunostain Leydig cells. Moreover, within the seminiferous epithelium, Sertoli cell nuclear AR staining intensity was less at stages V and VI of the cycle of the seminiferous epithelium than that at stage III, and stage III staining intensity was greater than that at stages I and II. This AR immunostaining pattern in human Sertoli cell nuclei as a function of the cycle of the seminiferous epithelium is reminiscent of the pattern observed in rodent species. Finally, no AR immunostaining of germ cells was observed at any of the PG21 concentrations examined.
Development of a Luminescent Bioassay for Thyroid Stimulating Antibodies
Authors: Evans C. , Morgenthaler N. G. , Lee S. , Llewellyn D. H. , Clifton-Bligh R. , John R. , Lazarus J. H. , Chatterjee V. K. K. , Ludgate M.
Published: January 2014

The hyperthyroidism of Graves Disease (GD) is due to thyroid stimulating antibodies (TSAb) which are thyrotropin (TSH) agonists. They are detected routinely by measuring their ability to inhibit TSH binding to the receptor (TBII), which does not reflect their true biological activity. Current bioassays which measure cAMP by RIA, are not suitable for routine use. We have developed a luminescent bioassay for TSAb, by introducing a cAMP responsive luciferase construct into CHO cells stably expressing the human TSH receptor (TSHR). Clone lulu1 displays dose dependent TSH response detectable from 10 μU/ml and maximal at 10 mU/ml when a >25 fold increase in light output is obtained. 34 euthyroid sera were tested to determine a reference range, with values >1.5 relative light units (R.L.U.) being considered positive. An international TSAb standard responded in a dose dependent manner with 10 mIU/ml giving an R.L.U. of >10. The assay was adapted to a 96 well format for automatic readout and 100 treated GD samples (50 TBII negative and 50 TBII positive) were tested, 73% being positive. In contrast only 4% of 79 control sera from individuals with Hashimoto’s, non-thyroid autoimmunity or multinodular goitre produced R.L.U. >1.5. When 44 of the GD sera were compared in a traditional salt-free bioassay, 61% were positive compared with 75% in the new luminescent assay. In conclusion, we have developed a luminescent bioassay for TSAb, using unfractionated serum which is capable of high throughput suitable for routine use.
Human Leukocyte Antigen-A24 and -DQA1*0301 in Japanese Insulin-Dependent Diabetes Mellitus: Independent Contributions to Susceptibility to the Disease and Additive Contributions to Acceleration of β-Cell Destruction*
Authors: Nakanishi Koji , Kobayashi Tetsuro , Murase Toshio , Naruse Taeko , Nose Yoshisuke , Inoko Hidetoshi
Published: January 2014

The aim of this study is to identify insulin-dependent diabetes mellitus (IDDM)-susceptible HLA antigens in IDDM patients who do not have established risk allele, HLA-DQA1*0301, and analyze relationship of these HLA antigens and the degree of β-cell destruction.

In 139 Japanese IDDM patients and 158 normal controls, HLA-A, -C, -B, -DR and -DQ antigens were typed. Serum C-peptide immunoreactivity response (ΔCPR) to a 100-g oral glucose load ≤ 0.033 nmol/l was regarded as complete β-cell destruction.

All 14 patients without HLA-DQA1*0301 had HLA-A24, whereas only 35 of 58 (60.3%) normal controls without HLA-DQA1*0301 and only 72 of 125 (57.6%) IDDM patients with HLA-DQA1*0301 had this antigen (Pc = 0.0256 and Pc = 0.0080, respectively). ΔCPR in IDDM patients with both HLA-DQA1*0301 and HLA-A24 (0.097 ± 0.163 nmol/L, mean ± sd, n = 65) were lower than in IDDM patients with HLA-DQA1*0301 only (0.219 ± 0.237 nmol/L, n = 45, P < 0.0001) and in IDDM patients with HLA-A24 only (0.187 ± 0.198 nmol/L, n = 14, P = 0.0395).

These results indicate that both HLA-DQA1*0301 and HLA-A24 contribute susceptibility to IDDM independently and accelerate β-cell destruction in an additive manner.
Immediate Causes of Death in Thyroid Carcinoma: Clinicopathological Analysis of 161 Fatal Cases
Authors: Kitamura Yutaka , Shimizu Kazuo , Nagahama Mitsuji , Sugino Kiminori , Ozaki Osamu , Mimura Takashi , Ito Kunihiko , Ito Koichi , Tanaka Shigeo
Published: January 2014

Most patients with thyroid carcinoma have a good prognosis. Due to the small number of fatal cases, it has not been clarified what conditions result in death for patients with thyroid carcinoma. To provide appropriate management for advanced thyroid carcinoma patients, we analyzed causes of death in 161 fatal cases. Clinical characteristics and immediate (final) causes of death based on pathological conditions were analyzed in 62 anaplastic carcinomas and 99 fatal differentiated carcinomas. Single fatal conditions could not be specified in 55 patients. In the remaining 106 patients, respiratory insufficiency (43%) was the most common specific fatal condition, followed by circulatory failure (15%), hemorrhage (15%), and airway obstruction (13%). Respiratory insufficiency due to remarkable pulmonary metastasis replacing lung tissue, massive hemorrhage and airway obstruction due to uncontrolled local tumors, and circulatory failure resulting from compression of the vena cava by extensive mediastinal or sternal metastases were found to be the most important immediate causes of death. Based on this knowledge, several palliative procedures may be worth considering to improve survival and quality of life in patients with advanced thyroid carcinoma.
No Enzyme Activity of 25-Hydroxyvitamin D3 1α-Hydroxylase Gene Product in Pseudovitamin D Deficiency Rickets, Including That with Mild Clinical Manifestation
Authors: Kitanaka Sachiko , Murayama Akiko , Sakaki Toshiyuki , Inouye Kuniyo , Seino Yoshiki , Fukumoto Seiji , Shima Masaaki , Yukizane Shigenori , Takayanagi Masaki , Niimi Hiroo , Takeyama Ken-ichi , Kato Shigeaki
Published: January 2014

Pseudovitamin D deficiency rickets (PDDR) is an autosomal recessive disorder caused by defect in the activation of vitamin D. We recently isolated 25-hydroxyvitamin D3 1α-hydroxylase gene and identified four homozygous inactivating missense mutations in this gene by analysis of four typical cases of PDDR. This disease shows some phenotypic variation, and it has been suspected that patients with mild phenotypes have mutations that do not totally abolish the enzyme activity. To investigate the molecular defects associated with the phenotypic variation, we analyzed six additional unrelated PDDR patients: one with mild and five with typical clinical manifestation. By sequence analysis, all six patients were proven to have mutations in both alleles. The mutations varied, and we identified four novel missense mutations, a nonsense mutation, and a splicing mutation for the first time. The patient with mild clinical symptoms was compound heterozygous for T321R and a splicing mutation. The splice site mutation caused intron retention. Enzyme activity of the T321R mutant was analyzed by overexpressing the mutant 1α-hydroxylase in Escherichia coli cells to detect the subtle residual enzyme activity. No residual enzyme activity was detected in T321R mutant or in the other mutants. These results indicate that all of the patients, including those of mild phenotype, are caused by 1α-hydroxylase gene mutations that totally abolish the enzyme activity.
Growth and Pubertal Development in Elite Female Rhythmic Gymnasts
Authors: Georgopoulos N. , Markou K. , Theodoropoulou A. , Paraskevopoulou P. , Varaki L. , Kazantzi Z. , Leglise M. , Vagenakis A. G.
Published: January 2014

Optimal growth depends upon both environmental and genetic factors. Among environmental factors that could alter growth and sexual maturation are stress and intensive physical training. The influence of these factors has been documented in a variety of sports, but there is limited information on rhythmic gymnasts, who have entirely different training and performance requirements.

The study was conducted during the 13th European Championships in Patras, Greece, and included 255 female rhythmic gymnasts, aged 11–23 yr. The study included measurement of height and weight, assessment of breast and pubic hair development, estimation of body fat and skeletal maturation, and registration of menarcheal age and parental height.

Gymnasts were taller than average height for age, with mean height above and mean weight below the 50th percentile. Actual height sd score was positively correlated to weight sd score (P < 0.001), number of competitions (P = 0.01), and body mass index (BMI; P < 0.001). Predicted adult height sd score was positively correlated to weight sd score (P < 0.001) and negatively to body fat (P = 0.004).

There was a delay in skeletal maturation of 1.3 yr (P < 0.001). Pubertal development was following bone age rather than chronological age. The mean age of menarche was significantly delayed from that of their mothers and sisters (P = 0.008 and P = 0.05, respectively), was positively correlated to the intensity of training and to the difference between chronological age and bone age (P < 0.001 and P = 0.002, respectively), and was negatively correlated to body fat (P < 0.001).

In the elite female rhythmic gymnasts, psychological and somatic efforts have profound effects on growth and sexual development. Despite these aberrations, adult height is not expected to be affected.
Increased Vitreous Concentrations of Human Hepatocyte Growth Factor in Proliferative Diabetic Retinopathy1
Authors: Nishimura Masato , Ikeda Tsunehiko , Ushiyama Masaji , Nanbu Akira , Kinoshita Shigeru , Yoshimura Manabu
Published: January 2014

Human hepatocyte GF (hHGF) has strong neoangiogenesis activity. The present study was designed to investigate the possible involvement of hHGF in neovascularization in proliferative diabetic retinopathy (PDR) by measuring vitreous hHGF concentrations. The mean vitreous hHGF concentration was higher in subjects with PDR (5.70 ± 0.68 ng/mL, n = 33) than in nondiabetic control subjects (1.50 ± 0.20 ng/mL, n = 18, P < 0.01), nondiabetic subjects with proliferative vitreoretinopathy (3.31 ± 0.57 ng//mL, n = 10, P < 0.05), or diabetic subjects without PDR (1.29 ± 0.28 ng/mL, n = 8, P < 0.01). PDR subjects with neovascularization of iris, which suggests advanced retinal ischemia, showed a higher mean vitreous hHGF concentration than those without iridal neovascularization [7.33 ± 1.16 ng/mL (n = 14) vs. 4.49 ± 0.72 ng/mL (n = 19), P < 0.05]. The mean vitreous hHGF concentration was higher in PDR subjects with retinal neovascularization at the optic disc than in those with neovascularization elsewhere [7.3 ± 1.1 ng/mL (n = 15) vs. 4.4 ± 0.7 ng/mL (n = 18), P < 0.05]. Our results indicate that vitreous hHGF may play a role in retinal neovascularization in PDR.
A Limited Repertoire of Mutations of the Luteinizing Hormone (LH) Receptor Gene in Familial and Sporadic Patients with Male LH-Independent Precocious Puberty1
Authors: Kremer H. , Martens J. W. M. , van Reen M. , Verhoef-Post M. , Wit J. M. , Otten B. J. , Drop S. L. S. , Delemarre-van de Waal H. A. , Pombo-Arias M. , De Luca F. , Potau N. , Buckler J. M. H. , Jansen M. , Parks J. S. , Latif H. A. , Moll G. W. , Epping W. , Saggese G. , Mariman E. C. M. , Themmen A. P. N. , Brunner H. G.
Published: January 2014

Herein, we report mutation analysis of the LH receptor gene in 17 males with LH-independent precocious puberty, of which 8 were familial and 9 had a negative family history. A total of 7 different mutations (all previously reported) were detected in 12 patients. Among 10 European familial male-limited precocious puberty (FMPP) patients who had a LH receptor gene mutation, none had the Asp578Gly mutation, which is responsible for the vast majority of cases in the U.S. The restricted number of activating mutations of the LH receptor observed in this and other studies of FMPP strongly suggests that an activating phenotype is associated with very specific sites in the receptor protein. Clinical follow-up of the 5 patients who did not have LH receptor mutations shows that such cases most likely do not have true FMPP. LH receptor mutation analysis provides a sensitive tool for distinguishing true FMPP from other causes of early-onset LH-independent puberty in males.
Changes in Bone Turnover in Young Women Consuming Different Levels of Dietary Protein1
Authors: Kerstetter Jane E. , Mitnick MaryAnn E. , Gundberg Caren M. , Caseria Donna M. , Ellison Alice F. , Carpenter Thomas O. , Insogna Karl L.
Published: January 2014

Although high protein diets are known to increase urinary calcium excretion and induce negative calcium balance, the impact of dietary protein on bone turnover and fractures is controversial. We therefore evaluated the effect of dietary protein on markers of bone turnover in 16 healthy young women. The experiment consisted of 2 weeks of a well balanced diet containing moderate amounts of calcium, sodium, and protein followed by 4 days of an experimental diet containing one of three levels of protein (low, medium, or high). On day 4, serum and urinary calcium, serum PTH, 1,25-dihydroxyvitamin D, serum osteocalcin, bone-specific alkaline phosphatase, and urinary N-telopeptide excretion were measured. Urinary calcium excretion was significantly higher on the high than on the low protein diet. Secondary hyperparathyroidism occurred on the low protein diet. Urinary N-telopeptide excretion was significantly greater during the high protein than during the low protein intake (48.2 ± 7.2 vs. 32.7 ± 5.3 nM bone collagen equivalents/mM creatinine; P < 0.05). There was no increase in osteocalcin or bone-specific alkaline phosphatase when comparing the low to the high diet, suggesting that bone resorption was increased without a compensatory increase in bone formation. Our data suggest that at high levels of dietary protein, at least a portion of the increase in urinary calcium reflects increased bone resorption.
Association of Collagen Type 1 α1 Gene Polymorphism with Bone Density in Early Childhood1
Authors: Sainz Jesus , Van Tornout Jan M. , Sayre James , Kaufman Francine , Gilsanz Vicente
Published: January 2014

Osteoporosis is a disease characterized by the development of nontraumatic fractures, most commonly in the vertebrae of elderly women. Approximately 500,000 elderly women in the United States are newly diagnosed with vertebral fractures every year, as the compressive strength of the vertebra, mainly determined by the density of cancellous bone and its cross-sectional area, declines with age. A recent study in women suggested that a polymorphism in the Sp1 binding site of the collagen type I gene (COLIA1) was related to decreased vertebral bone mass and vertebral fractures. Determining the phenotypic trait(s) responsible for this relationship and whether this association is manifested in childhood would further define the structural basis for decreased bone mass and help identify children “at risk” for fractures later in life. We therefore studied the COLIA1 gene polymorphism and measurements of the size and the density of vertebral bone in 109 healthy, prepubertal girls. On average, 22 girls with the Ss genotype and one girl with the ss genotype had 6.7% and 49.4% lower cancellous bone density in the vertebrae than girls with the SS genotype. In contrast, there was no association between the size of the vertebrae and the COLIA1 genotypes.
Two Novel Cysteine Substitutions (C1263R and C1995S) of Thyroglobulin Cause a Defect in Intracellular Transport of Thyroglobulin in Patients with Congenital Goiter and the Variant Type of Adenomatous Goiter1
Authors: Hishinuma Akira , Takamatsu Junta , Ohyama Yoshihide , Yokozawa Tamotsu , Kanno Yumiko , Kuma Kanji , Yoshida Shigeru , Matsuura Nobuo , Ieiri Tamio
Published: January 2014

We analyzed the thyroglobulin (Tg) gene of 2 unrelated patients with congenital goiter and the Tg gene of 2 siblings with the variant type of adenomatous goiter. The clinical characteristics of the patients with congenital goiter and the variant type of adenomatous goiter were very similar, except for serum Tg levels, which were less than 15 pmol/L in the patients with congenital goiter, but 117–181 pmol/L in the patients with the variant type of adenomatous goiter (normal, 15–50 pmol/L). The tissue content of Tg in the thyroid glands of all 4 patients was reduced at 0.9–3.8% of total protein (normal, 19–40%). The missense mutation C1263R was detected in the 2 unrelated patients with congenital goiter; the pedigree study showed an autosomal recessive pattern of inheritance. In the 2 siblings with the variant type of adenomatous goiter, the missense mutation C1995S was homozygously detected. In the Tg complementary DNA of 110 normal subjects, the allelic frequencies of the C1263R and C1995S mutations were each less than 0.5%. Also in the normal subjects were detected 35 nucleotide polymorphisms, the insertion of 3 nucleotides, and 1 alternative splicing, each of which was not associated with any specific thyroid disease. From these data, the molecular mechanism of the C1263R and C1995S mutations was elucidated. We first analyzed the carbohydrate residues of C1263R Tg and C1995S Tg. Sensitivity to treatment by endoglycosidase H suggests that C1263R Tg and C1995S Tg were retained in the endoplasmic reticulum (ER). Also, the presence of endoglycosidase H-resistant Tg as well as endoglycosidase H-sensitive Tg in the patients with the variant type of adenomatous goiter suggests that a fraction of C1995S Tg was transported to the Golgi and associated with the mildly increased serum Tg levels. Native PAGE and Western blot analysis with anti-Tg antibody showed that C1263R Tg and C1995S Tg form high mol wt aggregates in the ER.

Our results suggest that missense mutations that replace cysteine with either arginine or serine cause an abnormal three-dimensional structure of Tg. Such misfolded Tg polypeptides are retained in the ER as high mol wt aggregates.
A Novel 9-Base Pair Duplication in RET Exon 8 in Familial Medullary Thyroid Carcinoma1
Authors: Pigny Pascal , Bauters Catherine , Wemeau Jean-Louis , Lecomte Houcke Martine , Crepin Michel , Caron Philippe , Giraud Sophie , Calender Alain , Buisine Marie-Pierre , Kerckaert Jean-Pierre , Porchet Nicole
Published: January 2014

Familial medullary thyroid carcinoma (FMTC) and multiple endocrine neoplasia type 2A syndromes are dominantly inherited diseases caused by activating germline mutations of the RET protooncogene. The majority of these patients carry a germline point mutation affecting one of five cysteine residues encoded by exon 10 (codon 609, 611, 618, or 620) or 11 (codon 634). In a few FMTC families, point mutations involving noncysteine codons in exon 13 (codons 768, 790, and 791), 14 (codon 804), or 15 (codon 891) have been reported. Hirschsprung’s disease is a nonneoplastic disorder associated with RET mutations leading to a loss of function effect. Mutations are identified in 50% of the familial cases and are scattered along the gene. We now report the study of a FMTC family with four affected members and a history of fatal neonatal intestinal obstruction in the sister of the proband. Genetic analysis demonstrated the absence of an usual FMTC mutation and the presence of a germline 9-bp duplication in RET exon 8 in the heterozygous state in all patients with MTC. This new mutation creates an additional cysteine residue in the extracellular cysteine-rich domain of RET. Further studies are warranted to confirm whether this new mutation is causing MTC only or could be associated with Hirschsprung’s disease.
The Vitamin D Receptor (VDR) Start Codon Polymorphism in Primary Hyperparathyroidism and Parathyroid VDR Messenger Ribonucleic Acid Levels1
Authors: Correa Pamela , Rastad Jonas , Schwarz Peter , Westin Gunnar , Kindmark Andreas , Lundgren Ewa , Åkerström Göran , Carling Tobias
Published: January 2014

Vitamin D regulates parathyroid cell proliferation and secretion of PTH. Increased prevalence of the polymorphic vitamin D receptor (VDR) alleles b, a, and T has been reported in sporadic primary hyperparathyroidism (PHPT), suggesting that these genetic variants may predispose to the disease. Recently, another polymorphism in the VDR gene was related to bone mineral density, and this VDR-FokI polymorphism causes different lengths of the VDR, implying possible functional consequences. The VDR-FokI polymorphism was studied in 182 postmenopausal women with sporadic PHPT and in matched controls. No significant differences in distribution of the VDR-FokI genotypes could be detected between the groups, although there was a tendency toward overrepresentation of the F allele in the PHPT patients (P = 0.05). There were no significant associations with age, serum calcium, serum PTH, bone mineral density, or parathyroid tumor weight. The VDR genotypes were unrelated to VDR and PTH messenger ribonucleic acid levels in the parathyroid adenomas of 42 PHPT patients. In 23 PHPT patients, the Ca2+-PTH set-points were determined in vivo and were unrelated to the VDR alleles. We suggest that the VDR-FokI polymorphism has at most a minor pathogenic importance in the development of PHPT.
Jointly Amplified Basal and Pulsatile Growth Hormone (GH) Secretion and Increased Process Irregularity in Women with Anorexia Nervosa: Indirect Evidence for Disruption of Feedback Regulation within the GH-Insulin-Like Growth Factor I Axis1
Authors: Støving René K. , Veldhuis Johannes D. , Flyvbjerg Allan , Vinten Jørgen , Hangaard Jørgen , Koldkjær Ole G. , Kristiansen Jytte , Hagen Claus
Published: January 2014

Anorexia nervosa (AN) is associated with multiple endocrine alterations. In the majority of AN patients, basal and GHRH-stimulated serum GH levels are increased. The metabolic effects of GH are known to be related to its pulsatile secretory pattern. The present study was performed to examine GH pulsatility in AN using the techniques of deconvolution analysis and approximate entropy, which quantify secretory activity and serial irregularity of underlying hormone release not reflected in peak occurrence or amplitudes. To this end, 24-h GH profiles were obtained by continuous blood sampling aliquoted at 20-min intervals in 8 nonfasting patients with AN [body mass index (BMI), 14.2 ± 0.8 kg/m2; mean ± sem) and in 11 age-matched healthy women (BMI, 20.3 ± 0.5 kg/m2).

The deconvolution-estimated half-life of GH was not altered in the AN patients. The pituitary GH secretory burst frequency, burst mass, and burst duration were each significantly increased in women with AN compared to those in normal weight women. A 4-fold increase in daily pulsatile GH secretion was accompanied by a 20-fold increase in basal (nonpulsatile) GH secretion. There were significant negative correlations between BMI and the basal as well as pulsatile GH secretion rates. Moreover, AN patients exhibited significantly greater GH approximate entropy scores than the controls, denoting marked irregularity of the GH release process. In contrast to previous reports in healthy fasting subjects, cortisol levels in AN patients were positively correlated to GH secretion rates. Leptin levels were significantly inversely correlated to the pulsatile, but not the basal, GH secretion rate.

The present data demonstrate augmented basal as well as pulsatile GH secretion with disruption of the orderliness of the GH release process in AN. Accordingly, GH secretion in AN probably reflects altered neuroendocrine feedback regulation, e.g. associated with increased hypothalamic GHRH discharge superimposed on reduced hypothalamic somatostatinergic tone.
Cardiovascular Disease in the Diabetic Woman
Authors: Kaseta Jennifer R. , Skafar Debra F. , Ram Jeffrey L. , Jacober Scott J. , Sowers James R.
Published: January 2014

The incidence of cardiovascular disease (CVD) with age is increasing in the United States, and elderly women constitute a disproportional component of the aging population. Elderly women also have a relatively high incidence of diabetes, which contributes to this relatively high CVD risk. Although CVD is less common in premenopausal women than in men, this difference begins to disappear after the onset of menopause, presumably related to decreased levels of female sex hormones (estrogen and/or progesterone). Diabetes mellitus removes the normal premenopausal gender-related differences in the prevalence of CVD by mechanisms that are not clearly defined, including metabolic and hemodynamic factors associated with diabetes. Dyslipidemia in diabetes mellitus consists of low high density lipoprotein cholesterol, elevated triglyceride levels, and a small, dense, more atherogenic low density lipoprotein particle (i.e. oxidized). Dyslipidemia interacts with associated hemodynamic (i.e. hypertension) and metabolic abnormalities (i.e. increased platelet aggregation and plasminogen activator inhibitor-1 levels) to promote CVD risks in diabetic women. Recent controlled trials underscore the critical importance of aggressively treating CVD risk factors, especially dyslipidemia, in women with diabetes.
Serum Antibodies against Megalin (GP330) in Patients with Autoimmune Thyroiditis1
Authors: Marinò Michele , Chiovato Luca , Friedlander Joel A. , Latrofa Francesco , Pinchera Aldo , McCluskey Robert T.
Published: January 2014

Megalin (gp330) is a multiligand receptor found on the apical surface of selected epithelial cells, including thyroid cells. We recently showed that megalin is a high-affinity receptor for thyroglobulin. Megalin is capable of inducing autoantibodies, as shown in the rat model, Heymann nephritis. Based on this consideration and on the knowledge that autoantibodies against several thyroid antigens develop in patients with autoimmune thyroid diseases, we searched for antimegalin antibodies in 78 patients with autoimmune and nonautoimmune thyroid diseases.

We developed an assay, based on flow cytometry, to measure binding of serum IgGs to L2 cells, a rat carcinoma cell line that expresses abundant megalin. After incubation of L2 cells with serum samples and then with fluorescein isothiocynate-conjugated antihuman IgG Fc-specific antibody, the mean fluorescence intensity (MFI) was determined. Using results obtained in sera from 32 normal subjects, we established a cutoff value for MFI (50.62), above which, tests were considered positive. Significantly elevated values were found in 18 patients, including 13 of 26 patients with autoimmune thyroiditis (50.0%) and in 2 of 19 patients with Graves’ disease (10.5%). Furthermore, 2 of 19 patients with nontoxic goiter (10.5%) and 1 of 14 patients with differentiated thyroid cancer (7.14%) had MFI values greater than 50.62, associated with the presence of circulating antithyroid autoantibodies. As a control cell line, we used Chinese hamster ovary cells, which do not express megalin. We found that, among the 18 patients with positive tests for binding to L2 cells, only 1 patient with nontoxic goiter had significant binding of serum IgGs to Chinese hamster ovary cells.

Binding of serum IgGs to L2 cells was significantly reduced by coincubation with purified megalin in 15 of 18 positive patients (83.33%) and by a rabbit antimegalin antibody in 11 patients (61.11%). Further and more conclusive evidence that positive tests (MFI > 50.62) for binding to L2 cells were attributable to serum antimegalin antibodies was demonstrated by immunoprecipitation experiments. After incubation of serum samples with L2 cell extracts, incubation with antihuman IgG Fc-specific agarose beads resulted in immunoprecipitation of megalin in all the 18 positive patients, but not in normal subjects, as assessed by Western blotting using a monoclonal antibody against megalin. Furthermore, the intensity of the band corresponding to megalin precipitated by serum IgGs in the above 18 patients was significantly correlated with the L2 binding MFI.

This is the first clear-cut demonstration of antibodies against megalin in humans. Further studies are needed to determine whether antimegalin antibodies have pathogenic significance or diagnostic value in autoimmune thyroid diseases.
Expression of Calcium-Sensing Receptor and Characterization of Intracellular Signaling in Human Pituitary Adenomas1
Authors: Romoli Roberto , Lania Andrea , Mantovani Giovanna , Corbetta Sabrina , Persani Luca , Spada Anna
Published: January 2014

Extracellular Ca2+-sensing receptor (CaSR) has been recently identified in rat and mouse pituitary and in AtT-20 cells. The aim of the study was to investigate the presence of CaSR in the human pituitary and its signaling pathway. Normal parathyroid biopsies, autoptic normal pituitaries, and seven nonfunctioning and six GH-secreting adenomas were studied. Southern blot analysis of the RT-PCR products from pituitary adenomas indicated that the PCR fragments obtained were products of specific amplification of CaSR messenger ribonucleic acid. Sequence analysis showed nucleotide identity of these products with the available human parathyroid CaSR. By immunoblotting analysis CaSR, was detected in normal and adenomatous pituitary tissues. In all tumors studied, extracellular Ca2+ (2.5 mmol/L) induced a significant increase in intracellular Ca2+, mainly due to Ca2+ mobilization (from 82.7 ± 11 to 148 ± 36 nmol/L; P < 0.001). Similar results were obtained with the CaSR activators gadolinium and neomycin. Moreover, CaSR activators significantly increased cAMP levels; this effect was not mimicked by other agents able to increase intracellular Ca2+, such as TRH. CaSR agonists did not increase resting GH secretion in any GH-secreting adenomas, but amplified the GH response to GHRH. In this study we first demonstrate CaSR expression in the human pituitary and provides evidence for an additional mechanism by which calcium might regulate pituitary cell function.
A Role for Activin A and Betacellulin in Human Fetal Pancreatic Cell Differentiation and Growth1
Authors: Demeterco Carla , Beattie Gillian M. , Dib Sergio Atala , Lopez Ana D. , Hayek Alberto
Published: January 2014

Activin A (Act.A), a member of the transforming growth factorβ family of secreted proteins, has been implicated in the regulation of growth and differentiation of various cell types. Betacellulin (BTC), a member of the epidermal growth factor family, converts exocrine AR42J cells to insulin-expressing cells when combined with Act.A. We have used primary cultures of human fetal pancreatic tissue to identify the effects of Act.A and/or BTC on islet development and growth. Exposure to Act.A resulted in a 1.5-fold increase in insulin content (P < 0.005) and a 2-fold increase in the number of cells immunopositive for insulin (P < 0.005). The formation of islet-like cell clusters, containing mainly epithelial cells, during a 5-day culture, was stimulated 1.4-fold by BTC (P < 0.05). BTC alone caused a 2.6-fold increase in DNA synthesis (P < 0.005). These data suggest that Act.A induces endocrine differentiation, whereas BTC has a mitogenic effect on human undifferentiated pancreatic epithelial cells.