Growth hormone-releasing peptide-2 stimulates GH secretion in GH-deficient patients with mutated GH-releasing hormone receptor.

GH-releasing peptides (GHRPs) are synthetic peptides that bind to specific receptors and thereby stimulate the secretion of pituitary GH. In vivo it is uncertain whether these peptides act directly on somatotroph cells or indirectly via release of GHRH from the hypothalamus. In this study we compared the pituitary hormone response to GHRP-2 in 11 individuals with isolated GH deficiency (GHD) due to a homozygous mutation of the GHRH receptor (GHRH-R) gene and in 8 normal unrelated controls. Basal serum GH levels were lower in the GHD group compared with controls [0.11 +/- 0.11 (range, <0.04 to 0.38) vs. 0.59 +/- 0.76 microg/L (range, 0.04-2.12 microg/L); P = 0.052]. After GHRP-2 administration there was a 4.5-fold increase in serum GH relative to baseline values in the GHD group (0.49 +/- 0.41 vs. 0.11 +/- 0.11 microg/L; P = 0.002), which was significantly less than the 79-fold increase in the control group (46.8 +/- 17.6 vs. 0.59 +/- 0.76 microg/L; P = 0.008). Basal and post-GHRP-2 serum levels of ACTH, cortisol, and PRL were similar in both groups. Basal levels of serum TSH were significantly higher in the GHD group than in the control group (3.23 +/- 2.21 vs. 1.37 +/- 0.34 microIU/mL; P = 0.003). TSH levels in both groups did not change after GHRP-2 administration. These results suggest that an intact GHRH signaling system is not an absolute requirement for GHRP-2 action on GH secretion and that GHRP-2 has a GHRH-independent effect on pituitary somatotroph cells.

Study of the multiple endocrine neoplasia type 1, growth hormone-releasing hormone receptor, Gs alpha, and Gi2 alpha genes in isolated familial acromegaly.

Familial acromegaly may occur as an isolated pituitary disorder or as a feature of hereditary syndromes, such as multiple endocrine neoplasia type 1 (MEN1) or the Carney complex. Herein, we characterized a newly identified kindred with isolated acromegaly and searched for germline mutation in genes that have been associated with endocrine tumors [i.e. MEN1, Gs alpha (GNAS1), and Gi2 alpha (GNAI2)], as well as the GHRH receptor (GHRH-R) gene. Genomic DNA was used to amplify exons 2-10 of MEN1, followed by dideoxy fingerprinting mutation analysis and direct sequencing. The GHRH-R gene was analyzed via direct sequencing of PCR-amplified fragments representing the coding exons and intron-exon junctions. To exclude mutation at hot spot areas of GNAS1 and GNAI2, exons 8 and 9 of GNAS1 and exons 5 and 6 of GNAI2 were amplified and screened for mutation via denaturing gradient gel electrophoresis. No mutations were detected in any of the four genes. The present data extend prior reports of the absence of mutation in MEN1, GHRH-R, and GNAS1 and describe the first family with isolated acromegaly in which germline mutation in GNAI2 has been searched.

Familial growth hormone deficiency with mutated GHRH receptor gene: clinical and hormonal findings in homozygous and heterozygous individuals from Itabaianinha.

OBJECTIVE: To characterize clinically and hormonally the syndrome of autosomal recessive familial growth hormone deficiency (FGHD) recently identified in Itabaianinha, Sergipe, Brazil, caused by a novel mutation (mt) that inactivates the growth hormone-releasing hormone receptor (GHRH-R) gene. DESIGN: Clinical and hormonal evaluations were performed in 21 FGHD individuals (mt/mt group) aged 8 to 63 years, 13 heterozygotes for the GHRH-R mutation (wt/mt group) and 5 homozygotes for the wild type (wt) allele (wt/wt group), identified by genotyping of peripheral blood leukocyte DNA. METHODS: Clinical and hormonal characterization included physical examination and measurement of GH, IGF-I, IGF binding protein-3 (IGFBP-3), cortisol, prolactin, LH, FSH, and free thyroxine (FT4). RESULTS: Clinical features were consistent with isolated growth hormone deficiency. Height was significantly reduced in the mt/mt group compared with the wt/mt group (mean height standard deviation score (SDS) +/- s.d.: -7.35+/-1.37 vs -1.84+/-1.44 respectively, P<0. 0001), and the wt/wt group (-1.85 +/- 0.81, P=0.0007). The height of the 13 wt/mt subjects did not differ from the 5wt/wt individuals. Serum GH, IGF-I, IGF-I SDS, IGFBP-3 and IGFBP-3 SDS were all significantly lower in the mt/mt group than in the wt/mt and wt/wt groups. Two affected children treated with GH for 1 year showed a normal growth response. Serum IGF-I and IGF-I SDS were lower in wt/mt compared with wt/wt group, but did not reach statistical significance. IGF-I and IGF-I SDS correlated inversely with age in wt/mt group. CONCLUSIONS: FGHD due to an autosomal recessive GHRH-R gene mutation leads to marked dwarfism, phenotypically and hormonally indistinguishable from other forms of isolated GH deficiency. Heterozygotes for the GHRH-R mutation appear to have a partial defect in the GH/IGF axis, with no apparent height impairment.

Calcitonin deficiency in early stages of chronic autoimmune thyroiditis.

OBJECTIVES: Although calcitonin (Ct) deficiency has been described in chronic autoimmune thyroiditis (CAT) it is unclear at what stage in the disease it develops. We have analysed the Ct secretory responses of patients in two different evolutionary stages of CAT, namely the goitrous and atrophic phases. DESIGN: We studied the Ct response to combined calcium (2 mg/kg) and pentagastrin (0.5 microgram/kg) intravenous infusion in 27 patients with CAT and 30 normal adult controls. The cases were divided into two groups. The first comprised eleven women with CAT and goitrous subclinical hypothyroidism (GH), aged 28.6 +/- 10.1 years--at diagnosis they had increased thyroid autoantibody titres and cytological features compatible with stages 1 and 2 of Hashimoto's thyroiditis. The second comprised 16 females with CAT and an atrophic thyroid confirmed by ultrasound scan, aged 38.0 +/- 9.2 years--these patients were severely hypothyroid at diagnosis and were termed AH (atrophic hypothyroidism). Both groups (GH and AH) received replacement doses of thyroxine sufficient to restore euthyroidism for at least six months before the stimulation tests. Control group (C) consisted of 20 healthy women (A), aged 30.0 +/- 9.6 years, and 10 healthy men (B), aged 34.7 +/- 8.0 years. Serum Ct was measured by IRMA. The Ct secretory response was related to thyroid size and cytological data, when available. RESULTS: Basal Ct concentrations in groups GH (0.08 ng/l, median) and AH (0.07 ng/l, median) were significantly lower than those of female controls (0.58 ng/l, median). Stimulated Ct peak values in groups GH (0.08 ng/l, median) and AH (0.19 ng/l, median) were significantly lower than those of female controls (13.61 ng/l, median). Also, both basal (2.72 ng/l, median) and stimulated Ct levels (35.73 ng/l, median) in male controls were significantly higher than in female controls given already. A positive correlation between the Ct secretory reserve and thyroid dimensions, evaluated by ultrasound scan, was found only in patients with thyroid atrophy (AH; rs = 0.61, P < 0.05). CONCLUSIONS: We have found low basal and stimulated calcitonin values in patients with chronic autoimmune thyroiditis and thyroid enlargement, which represents an early phase of chronic autoimmune thyroiditis. Our data have also confirmed previous findings of deficient calcitonin secretion in advanced stages of chronic autoimmune thyroiditis in which thyroid atrophy is usually found. These findings may be associated with C-cell destruction following progressive, nonspecific follicular cell damage caused by lymphocytic infiltration and fibrosis of the gland.

A homozygous mutation in the luteinizing hormone receptor causes partial Leydig cell hypoplasia: correlation between receptor activity and phenotype.

Leydig cell hypoplasia (LCH) is characterized by a decreased response of the Leydig cells to LH. As a result, patients with this syndrome display aberrant male development ranging from complete pseudohermaphroditism to males with micropenis but with otherwise normal sex characteristics. We have evaluated three brothers with a mild form of LCH. Analysis of their LH receptor (LHR) gene revealed a homozygous missense mutation resulting in a substitution of a lysine residue for a isoleucine residue at position 625 of the receptor. In vitro analysis of this mutant LHR, LHR(I625K), in HEK293 cells indicated that the signaling efficiency was significantly impaired, which explains the partial phenotype. We have compared this mutant LHR to two other mutant LHRs, LHR(A593P) and LHR(S616Y), identified in a complete and partial LCH patient, respectively. Although the ligand-binding affinity for all three mutant receptors was normal, the hormonal response of LHR(A593P) was completely absent and that of LHR(S616Y) and LHR(I625K) was severely impaired. Low cell surface expression explained the reduced response of LHR(S616Y), while for LHR(I625K) this diminished response was due to a combination of low cell surface expression and decreased coupling efficiency. For LHR(A593P), the absence of a reduced response resulted from both poor cell surface expression and a complete deficiency in coupling. Our experiments further show a clear correlation between the severity of the clinical phenotype of patients and overall receptor signal capacity, which is a combination of cell surface expression and coupling efficiency.

Lactoferrin in thyroid lesions: immunoreactivity in fine needle aspiration biopsy samples.

OBJECTIVE: Lactoferrin is an iron-binding protein that has been used for distinguishing normal from neoplastic conditions in many different tissues. In order to improve evaluation of thyroid lesions, we studied the lactoferrin immunoreaction in cytologic smears obtained by fine needle aspiration and in biopsy samples from primary neoplasms and from adenomatous goiter. STUDY DESIGN: A retrospective study on fine needle aspiration cytology samples and corresponding available biopsies from thyroid lesions in patients examined at São Paulo County Hospital between 1982 and 1992, performed in order to evaluate lactoferrin immunoreactivity in morphologically well characterized samples from neoplastic and nonneoplastic lesions. Immunoperoxidase procedures were performed using monospecific polyclonal rabbit antihuman lactoferrin as a primary antibody and biotinylated goat antirabbit IgG as a secondary antibody. Amplification was performed with the avidin-biotin-peroxidase complex, and the color sign of the positive reactions was developed using a diaminobenzidine solution. RESULTS: Lactoferrin was not detected in cytologic smears from goiters, whereas only one biopsy was slightly positive (1/21, or 4.76%). One smear from adenoma showed low positive staining (1/19, or 5.26%), which was present in 4 of 13 biopsies (30.77%) from adenoma. Papillary carcinomas were positive in 19 of 33 smears (57.58%) and in 100% of biopsies, whereas 31.25% (5/16) of follicular carcinoma smears were positive for lactoferrin, detected in all the biopsy samples. CONCLUSION: Lactoferrin immunoreactivity was strongly associated with neoplastic proliferation and may be used as a useful auxiliary marker to distinguish malignant from benign thyroid lesions in cytologic smears and biopsy samples.

Molecular and immunohistochemical analysis of P53 in phaeochromocytoma.

We searched for mutations of the p53 gene in 25 phaeochromocytomas using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis of the entire conserved region of the gene, encompassing exons 4-8; expression of the p53 protein was assessed by immunohistochemistry. No mutations were found, while a polymorphism in codon 72 was observed. Immunohistochemistry revealed nuclear p53 overexpression in one tumour sample. We conclude that mutations of the 'hotspot' region of the p53 gene do not seem to play a role in the pathogenesis of phaeochromocytoma.

Neuron-specific enolase is a useful marker of neuroendocrine origin in pheochromocytoma cell culture

Neuron-specific enolase (NSE) has been used as a marker for neuroendocrine tumors either in immunocytochemical studies or in serum measurements. ln this paper NSE levels were determined in cultured pheochromocytoma cells to test whether it is also a useful marker ín cell culture of tumors derived from neuroendocrine system. Cultured pheochromocytoma cells came from a primary explant and were grown in RPMI supplemented with 20 per cent fetal calf serum, 100 mug/mL ampicillin and 100 mug/mL streptomycin. NSE was measured in culture medium and cell homogenates. Samples from different pheochromocytoma cultures were analyzed and compared to normal cultured fibroblast cells derived from human skin. NSE was measured by a commercially available radioimmunoassay kit. NSE levels were higher in cell homogenates as compared to those in culture medium, reaching levels as high as 6-fold in the former in TE cell line (26.46 ng/mL and 4.39 ng/mL respectively. Serial NSE measurements in culture medium from TE cell line evidenced decreasing values in subsequential subcultures (from 9.24 ng/mL during primary explant to 1.7 ng/ml. in the 10th subculture). In cultured normal fibroblasts, NSE levels in cultured media were definitely lower than those obtained from pheochromocytoma cultures. These preliminary data suggest that NSE may be a useful marker of neuroendocrine derived tumors, such as pheochromocytoma, in culture. Thus, the simplicity and availability of NSE radioimmunoassay provides an alternative to catecholamine measurement to better characterize pheochromocytoma cell lines in culture, with the advantage of faster results at lower costs.

Low cord blood levels of catecholamine from a newborn of a pheochromocytoma patient.

Association of pheochromocytoma and pregnancy is rare and usually related to high maternal and fetal mortality rates. Maternal effects of the tumor have been studied extensively and the clinical outcome has markedly improved during the last decade. However, the role of excess catecholamines on fetal development has been discussed very little. We report here a case of pheochromocytoma during pregnancy. In which catecholamine levels from the cord blood were low despite simultaneous elevated maternal values (1.93 and 29.46 nmol/l norepinephrine, respectively), possibly owing to the high activity of the catecholamine degradative enzymes monoamine oxidase and COMT at the placental level. We suggest that in pregnancies complicated by pheochromocytoma, fetal well-being may be related mainly to good control of maternal blood pressure instead of the amount of catecholamines in the fetal circulation, because the placenta performs a protective role through an effective process of hormone inactivation.

Immunohistochemistry of medullary thyroid carcinoma and C-cell hyperplasia by an affinity-purified anti-human calcitonin antiserum.

BACKGROUND: The diagnosis of medullary thyroid carcinoma (MTC) depends on the calcitonin immunohistochemistry. Familial MTC is associated with C-cell hyperplasia (CCH), whereas sporadic MTC is not. A specific and sensitive calcitonin immunohistochemistry is necessary for the diagnosis of MTC and CCH. METHODS: An affinity-purified anti-calcitonin antiserum (APxCT) was used for immunohistochemistry of the thyroids of 15 patients with MTC. The thyroids of five patients with familial MTC were studied in detail, with each gland sectioned in 48 areas. RESULTS: Between three and ten independent MTC were found in each thyroid, and CCH was found in all five patients (24.2%, varying from 8.4-56.3% of the 48 areas from each thyroid). MTC and CCH were localized mainly in the middle third and in the central axis of the thyroid lobes. They often were found together in the same area (in a total of 21 areas for the five thyroids sectioned in 48 areas) but ten areas with MTC did not have CCH, and 37 areas with CCH did not have MTC. In ten thyroids partially studied, CCH was indicated in three patients thought to have sporadic MTC. In two thyroids, with follicular and papillary carcinoma, a higher density of C-cells was found around the tumors, but disease was not characterized as CCH. CONCLUSIONS: APxCT antiserum increased the immunohistochemical specificity and sensitivity. The distinction of the familial from the sporadic MTC requires a careful and extensive search of CCH. C-cells in high density may be found around follicular cell carcinomas, being a potential source of diagnostic error.

Long-term treatment of central precocious puberty with a long-acting analogue of luteinizing hormone release hormone (D-Tryp6-GnRH) in monthly injections. Its possible use in normal puberty.

The gonadotropin-releasing-hormone-like agonist D-Tryp6-GnRH (GnRHa) has been shown to induce reversible suppression of gonadotropins and gonadal steroids in patients with central precocious puberty. We examined the effect of a long-acting preparation of GnRHa in biodegradable microcapsules. D-Tryptophane6-GnRH, administered intramuscularly at 1 month intervals, for 12 consecutive months, on growth and skeletal maturation in 3 girls and 4 boys with neurogenic or idiopathic precocious puberty. Suppression of gonadotropin release after GnRH stimulation and gonadal steroids was maintained in all subjects. Growth velocity fell from a mean rate (+/- SEM) or 8.60 +/- 0.75 cm/year before treatment to 5.81 +/- 0.60 cm/year (p < 0.005) after 1 year. Bone age advanced a mean of 8.0 +/- 0.45 months during treatment, suggesting an increase in predicted height from the ratio delta bone age/delta chronological age. Two subjects, one of them with compensated Bartter's syndrome with normal hypothalamic pituitary-gonadal-axis, received the analogue to delay pubertal growth with the hope to improve final height. In the first one, the growth velocity fell from 9.9 cm/year to 8 cm/year and delta bone age/delta chronological age decreased from 1.28 to 1.0 and in the other subject, the growth velocity fell from 12 cm/year to 6.0 cm/year in the last year of treatment and delta bone age/delta chronological age fell from 3.2 to 0.75, indicating an improvement in predicted height.

[Serum growth hormone levels in Hunter's syndrome]. / Níveis séricos de hormônio de crescimento na síndrome de Hunter.

Serum growth hormone (GH) levels were measured in 3 brothers with Hunter syndrome. The secretion of GH was studied by means of insulin (ITT), glucose (GTT), lysine-vasopressin (LVP), and L-Dopa administrations. Mean basal GH levels during the 4 tests were high (x = 14.2 ng/ml) in all cases. In the ITT and LVP tests, GH responses correlated positively with the patients' ages. Contrarily, after L-Dopa administration, GH elevations were normal in the two younger and absent in the oldest case. During GTT, GH levels were suppressed in all cases as expected. Basal cortisol and prolactin serum levels during the tests were normal. In order to clarify these data, GH levels were then determined during 120 min. (20-20 min.) under basal conditions. The means (+/- SD) of GH were 178 +/- 0.15; 4.42 +/- 2.47; and 2.30 +/- 0.71, for cases 1, 2 and 3, respectively (normal values 0-5 ng/ml). Basal somatomedin-C levels were in low-normal ranges. As patients were not undernourished and albumin levels were normal, a slight dysfunction of hypothalamic-pituitary-GH-somatomedin-C axis might occurred in these cases. The hypothesis here offered is that a primary sub-production of somatomedin-C, mainly by liver and kidneys, could be present in Hunter syndrome. This situation would lead to normal-high GH serum levels, as seen in the present cases. GH serum measurements in Hunter syndrome were not documented previously.

[Early detection of medullary thyroid carcinoma in multiple endocrine neoplasia type II]. / Detecção precoce do carcinoma medular de tireóide na neoplasia endócrina múltipla tipo II.

The early diagnosis of medullary thyroid carcinoma was made in two among six examined siblings belonging to two sibships that were offsprings of multiple endocrine neoplasia type II parents. The calcitonin secretory reserve was determined by a combined test using Ca++ (2 mg/kg) and pentagastrin (0.5 mcg/kg), intravenously. Two abnormal tests made on different days supported the diagnosis. Basal calcitonin levels were moderately high (90-500 pg/ml; NL = 15-85 pg/ml) and peak levels were also abnormal (480-1500 pg/ml; NL less than 320 pg/ml), in both cases. Total thyroidectomy associated to prophylactic resection of lymph nodes from central neck region were performed in both. A small nodule (3-5 mm) was found in each lobe in both cases. Pathological and immunocytochemical data supported the diagnosis of medullary thyroid carcinoma. C-cell hyperplasia was present in the peritumoral zones. Pheochromocytoma and definite hyperparathyroidism were not detected in these cases. Two years after surgery, basal and stimulated serum calcitonin levels remained normal. Carcinoembryonic antigen levels were and continue to be normal, in both. These seem to be the first cases published in this country in which this early diagnosis was made.

Serum levels of total testosterone and sex hormone binding globulin in hypothyroid patients and normal subjects treated with incremental doses of L-T4 or L-T3.

Plasma testosterone (T) and sex hormone binding globulin (SHBG) were assayed in normal controls (N = 9) and hypothyroid patients (N = 17) receiving increasing doses of L-T4 (0.2 mg, 0.4 mg for 30 days), followed first by 30 days without medication and then by 30 days each of 0.05 mg L-T3 and 0.2 mg L-T3. Normal male controls showed a significant increase in plasma T only at high doses of either L-T4 (0.4 mg) or L-T3 (0.2 mg). A small but significant increase in plasma T levels was observed in normal female subjects at 0.4 mg of T4. In both men and women, plasma SHBG increased in a dose-dependent manner with L-T4 or L-T3 and correlated positively and significantly with serum thyroid hormone levels. Hypothyroid male subjects had significantly lower levels of plasma T (mean +/- SD) of 279 +/- 131 ng/dl as compared with normal males (431 +/- 118 ng/dl), which reached the normal range only at a relatively high dose of either L-T4 (0.4 mg) or L-T3 (0.2 mg). No significant changes in plasma T were seen in the hypothyroid female group. Basal plasma SHBG levels were significantly lower in both hypothyroid men and women and increased towards normal levels during L-T4 and L-T3 therapy, although the response to thyroid hormones was significantly lower than that of normal controls. These results indicate that thyroid hormone therapy increases plasma SHBG levels in both normal and hypothyroid patients and that this increase precedes the expected elevation of plasma T in males.

Estudo clinico, citogenetico, histologico e da regulacao hormonal em dois casos de hermafroditismo verdadeiro. / Clinical, cytogenetic, hystological and hormonal regulation studies in 2 cases of true hermaphroditism

Foi realizado estudo clinico, citogenetico histologico e da secrecao hormonal em dois casos de hermafroditismo verdadeiro (HV).O primeiro paciente apresentava cariotipo XX e o segundo XX/XY, sendo os dois antigeno HY positivo. O exame histologico revelou ovotestis no primeiro caso com presenca de espermatogonias ate agora nao descritas no HV. O segundo paciente apresentava ovario histologicamente normal e testiculo com areas de hialinizacao tubular. A avaliacao hormonal no paciente 1 revelou grandes flutuacoes dos niveis de LH, FSH, estradiol e progesterona. Os dois pacientes apresentavam baixa resposta testicular ao estimulo com gonadotrofina corionica e liberacao de LH apos uso de estrogeno semelhante a observada no ser feminino.O citrato de clomifeno induziu resposta ovulatoria no caso testado, podendo ser utilizado na identificacao da presenca de ovotestis em caso de suspeita de HV