Controversies in the management of Graves' disease in children.

Graves' disease (GD) is the most prevalent cause of thyrotoxicosis in children. Because spontaneous and lasting resolution of this condition occurs in only a minority of patients, most pediatric patients with GD will need radioactive iodine treatment (131I) or thyroidectomy. Whereas the medication propylthiouracil (PTU) had been used in the past, only methimazole (MMI) should be now used in children, as PTU is associated with an unacceptable risk of liver failure. However, MMI may be associated minor and major side effects, which may be minimized using lower doses. An area of controversy involves the optimal duration of antithyroid drug (ATD) therapy. For some children, the prolonged use of antithyroid drugs is a valid approach, but for most, this will not increase the chance of remission. When 131I is administered, dosages should be greater than 150 uCi/gm of thyroid tissue, with higher dosages needed for larger glands. Considering that there will be low-level whole body radiation exposure associated with 131I, this treatment is viewed as controversial by some and should be avoided in young children. When surgery is performed, near-total or total thyroidectomy is the recommended procedure. Complications for thyroidectomy in children are considerably higher than in adults. Thus, an experienced thyroid surgeon is needed when children have surgery. Overall, when different treatment options for GD are considered, the benefits, risks and viewpoints of the family need to be considered and discussed in full.

Putative melatonin receptors in a human biological clock.

In vitro autoradiography with 125I-labeled melatonin was used to examine melatonin binding sites in human hypothalamus. Specific 125I-labeled melatonin binding was localized to the suprachiasmatic nuclei, the site of a putative biological clock, and was not apparent in other hypothalamic regions. Specific 125I-labeled melatonin binding was consistently found in the suprachiasmatic nuclei of hypothalami from adults and fetuses. Densitometric analysis of competition experiments with varying concentrations of melatonin showed monophasic competition curves, with comparable half-maximal inhibition values for the suprachiasmatic nuclei of adults (150 picomolar) and fetuses (110 picomolar). Micromolar concentrations of the melatonin agonist 6-chloromelatonin completely inhibited specific 125I-labeled melatonin binding, whereas the same concentrations of serotonin and norepinephrine caused only a partial reduction in specific binding. The results suggest that putative melatonin receptors are located in a human biological clock.

Balancing the benefits and harms of thyroid cancer surveillance in survivors of Childhood, adolescent and young adult cancer: Recommendations from the international Late Effects of Childhood Cancer Guideline Harmonization Group in collaboration with the PanCareSurFup Consortium.

Radiation exposure to the thyroid gland during treatment of childhood, adolescent and young adult cancer (CAYAC) may cause differentiated thyroid cancer (DTC). Surveillance recommendations for DTC vary considerably, causing uncertainty about optimum screening practices. The International Late Effects of Childhood Cancer Guideline Harmonization Group, in collaboration with the PanCareSurFup Consortium, developed consensus recommendations for thyroid cancer surveillance in CAYAC survivors. These recommendations were developed by an international multidisciplinary panel that included 33 experts in relevant medical specialties who used a consistent and transparent process. Recommendations were graded according to the strength of underlying evidence and potential benefit gained by early detection and appropriate management. Of the two available surveillance strategies, thyroid ultrasound and neck palpation, neither was shown to be superior. Consequently, a decision aid was formulated to guide the health care provider in counseling the survivor. The recommendations highlight the need for shared decision making regarding whether to undergo surveillance for DTC and in the choice of surveillance modality.

Tissue-specific expression of GTPas RalA and RalB during embryogenesis and regulation by epithelial-mesenchymal interaction.

RalA and RalB are members of the Ras-like GTP-binding protein family. To investigate if Ral GTPases play a role in development, temporal and spatial patterns of RalA and RalB gene expression were examined during embryogenesis. RalA and RalB had tissue-specific pattern of expression in developing organs. Both GTPases were expressed in endodermal derivatives in the digestive tract, kidney, mesonephros, gonad, adrenal gland, trachea, and blood vessels. RalA and RalB were also expressed in the ectodermal derivatives in the brain, spinal cord, ganglia, eye, skin, hair, whiskers, and teeth. RalA and RalB were also expressed in some mesodermal tissues including the lung mesenchyme and myocardium. Tissue recombination experiments showed that of Ral expression was increased by epithelial-mesenchymal interaction in the lung, indicating that Ral expression is functionally regulated. These data identify that RalA and RalB as inducible GTPases in the developing embryo.

RFL9 encodes an A2b-adenosine receptor.

We recently reported the cloning of a cDNA (designated RFL9) that encodes a novel A2-adenosine receptor subtype. We now fully characterize the pharmacological properties of RFL9 in stably transfected CHO cells by examining cAMP responses to drug treatments. The pharmacological profile of cAMP responses in RFL9-transfected cells was similar to that expected for A2b-adenosine receptors and distinct from that of CHO cells transfected with the A2a-adenosine receptor. When RFL9-transfected cells were compared with VA 13 fibroblasts, the human cell line in which endogenous A2b-adenosine receptors were originally characterized, the dose-response curves of cAMP responses to drug treatments were highly correlated. Northern blot analysis of RNA prepared from VA 13 fibroblasts revealed specific hybridizing transcripts when probed for RFL9, but no hybridizing signal for A2a-adenosine receptor mRNA. Using degenerate oligonucleotide primers designed to detect adenosine receptors by the polymerase chain reaction, only one cDNA fragment homologous to the rat A2b-adenosine receptor was isolated from VA 13 cells. These results strongly suggest that RFL9 encodes the proposed A2b-adenosine receptor subtype. The identification of the cDNA for an A2b-adenosine receptor will allow more rigorous characterization of its anatomical distribution and functional properties.

Molecular cloning and characterization of a rat A1-adenosine receptor that is widely expressed in brain and spinal cord.

An A1-adenosine receptor has been cloned from a rat brain cDNA library using a probe generated by the polymerase chain reaction. The cDNA encodes a protein of 327 amino acids which is 91% identical to a recently cloned dog A1-adenosine receptor (RDC7). Expression of the rat cDNA in COS-6M and NIH 3T3 cells resulted in ligand binding and functional activity characteristics of an A1-adenosine receptor that is coupled to inhibition of adenylyl cyclase. Examination of the distribution of A1-adenosine receptor mRNA by Northern blot analysis showed that it is highly expressed in brain, spinal cord, testis, and white adipose tissue. In situ hybridization studies revealed an extensive hybridization pattern in the central nervous system, with high levels in cerebral cortex, hippocampus, cerebellum, thalamus, brainstem, and spinal cord. The cloned A1-adenosine receptor may thus mediate many of the modulatory actions of adenosine in neural and endocrine systems.

Molecular cloning and expression of the cDNA for a novel A2-adenosine receptor subtype.

A novel adenosine receptor subtype has been cloned from a rat brain cDNA library using a probe generated by the polymerase chain reaction. The cDNA, designated RFL9, encodes a protein of 332 amino acids. The structure of RFL9 is most similar to that of the recently cloned rat A2-adenosine receptor, with a sequence identity of 73% within the presumed seven transmembrane domains. Expression of RFL9 in COS-6M cells resulted in ligand binding and functional activity characteristics of an adenosine receptor that is coupled positively to adenylyl cyclase. Examination of the tissue distribution of RFL9 mRNA by Northern blot analysis showed a restricted distribution with highest levels expressed in large intestine, cecum, and urinary bladder; this pattern was distinct from that of either the A1- or A2-adenosine receptor mRNAs. In situ hybridization studies of RFL9 mRNA showed no specific hybridization pattern in brain, but a hybridization signal was readily observed in the hypophyseal pars tuberalis. Thus, RFL9 encodes a novel A2-adenosine receptor subtype.

Localization and characterization of adenosine receptor expression in rat testis.

Adenosine has long been suspected to play an important role in regulating male reproduction. To determine the sites of adenosine action in testis, the distribution of a family of recently cloned adenosine receptors was examined in rats. Northern blot analysis and in situ hybridization studies revealed high levels of testicular A3 adenosine receptor messenger RNA and lower levels of A1 adenosine receptor messenger RNA. Neither A2a nor A2b adenosine receptor gene expression could be detected. In situ hybridization and comparative polymerase chain reaction studies showed high level A3 receptor gene expression in germ cells (spermatocytes and spermatids), whereas high levels of A1 receptor gene expression was seen in Sertoli cells. To test for the presence of functional A1 and A3 receptors, the regulation of adenylyl cyclase by adenosine analogs was examined in seminiferous tubules and spermatozoa. Treatment of seminiferous tubules with forskolin resulted in a 5-fold increase in cAMP levels. The A1/A3 receptor agonist iodo-N6-aminobenzyladenosine inhibited forskolin-stimulated cAMP levels in the presence of the A1 8-cyclopentyl-1,3-dipropylxanthine antagonist 8-cyclopentyl-1,3-dipropylxanthine, showing that functional A3 receptors were present. The A1-selective agonist N6-cyclopentyladenosine also inhibited forskolin-stimulated cAMP levels, showing that functional A1 receptors were present. Treatment of spermatozoa with forskolin resulted in a 2-fold increase in cAMP levels. However, neither iodo-N6-aminobenzyladenosine nor N6-cyclopentyladenosine altered basal or forskolin-stimulated cAMP levels in sperm. These data show that functional inhibitory adenosine receptors are widely distributed in testis, with different patterns of expression.

A1 adenosine receptor activation increases adipocyte leptin secretion.

A1 adenosine receptors (A1ARs) are heavily expressed in adipocytes and influence fat cell metabolism. Because increasing evidence suggests a role for leptin in mediating appetite and fat cell metabolism, we tested whether ALARs regulate leptin production. Rats were treated with the A1AR agonist N6-cyclopentyladenosine (CPA), and changes in circulating levels of leptin and leptin gene expression were examined. Serum leptin levels rose 2- to 10-fold, with peak increases seen 8-16 h after injection of CPA (P < 0.05). In contrast, CPA did not alter steady state levels of adipose tissue leptin mRNA. To assess the influence of endogenous adenosine on circulating leptin levels, rats were also injected with dipyridamole (DPY), an adenosine reuptake blocker. DPY induced 80% increases in serum levels at 8 h after injections (P < 0.05). Supporting the idea that stimulation of leptin production is A1AR mediated, pretreatment with the A1AR antagonist 8-cyclopentyl-1,3-dipropylxanthine completely blocked increases in leptin levels after DPY treatment. To complement in vivo studies, the effect of A1AR activation on leptin secretion was also studied in epididymal fat pad cultures. In cultures, CPA treatment increased leptin secretion by 37% (P < 0.05). Collectively, these data show that the adenosinergic system can increase leptin secretion by directly activating A1ARs in fat tissue.

Solubilization and purification of melatonin receptors from lizard brain.

Melatonin receptors in lizard brain were identified and characterized using 125I-labeled melatonin ([125I]MEL) after solubilization with the detergent digitonin. Saturation studies of solubilized material revealed a high affinity binding site, with an apparent equilibrium dissociation constant of 181 +/- 45 pM. Binding was reversible and inhibited by melatonin and closely related analogs, but not by serotonin or norepinephrine. Treatment of solubilized material with the non-hydrolyzable GTP analog, guanosine 5'-(3-O-thiotriphosphate) (GTP-gamma-S), significantly reduced receptor affinity. Gel filtration chromatography of solubilized melatonin receptors revealed a high affinity, large (Mr 400,000) peak of specific binding. Pretreatment with GTP-gamma-S before solubilization resulted in elution of a lower affinity, smaller (Mr 150,000) peak of specific binding. To purify solubilized receptors, a novel affinity chromatography resin was developed by coupling 6-hydroxymelatonin with Epoxy-activated Sepharose 6B. Using this resin, melatonin receptors were purified approximately 10,000-fold. Purified material retained the pharmacologic specificity of melatonin receptors. These results show that melatonin receptors that bind ligand after detergent treatment can be solubilized and substantially purified by affinity chromatography.

Melatonin receptors in chick brain: characterization and localization.

Melatonin receptors in chick brain were characterized by RRA and localized by in vitro autoradiography, using [125I]melatonin [( 125I]MEL), a biologically active melatonin analog. In membranes from whole brain, radioreceptor studies revealed a high affinity [125I]MEL binding site with an equilibrium dissociation constant of 47.2 +/- 11.5 (mean +/- SEM) pM and a density of 37.8 +/- 8.5 fmol/mg protein. Binding was reversible and competitively inhibited by melatonin and closely related melatonin analogs, but not be norepinephrine or serotonin. In vitro autoradiographic studies of brain revealed a widespread distribution of melatonin receptors. Specific I-MEL binding was observed in retinorecipient and integrative nuclei of the visual system including the avian homolog of the mammalian suprachiasmatic nuclei. Auditory relay nuclei and limbic structures associated with arousal and vocalization were also specifically labeled. This widespread distribution of putative melatonin receptors in chick brain is in marked contrast to the very restricted distribution of melatonin receptors in mammalian brain and suggests that avian sensory systems are affected by melatonin.

Djungarian hamsters exhibit reproductive responses to changes in daylength at extreme photoperiods.

In seasonally breeding species, an animal's photoperiodic history (the daylength or photoperiod previously experienced) influences the reproductive response to new photoperiods. However, this has only been examined over a relatively narrow range of photoperiods. We assessed whether Djungarian hamsters (Phodopus sungorus) respond to daylength changes at extremely long and extremely short photoperiods. To determine the extent and temporal sequence of reproductive responses to photoperiod changes, ultrasonography was employed to determine serial changes in testis weight in individual animals; serial changes in body weight and pelage color were also assessed. Male animals (60-day-old) shifted from 6 h of light/day (6L) to 10L showed partial testicular recrudescence and darkening of the coat. In control animals remaining in 6L, testes remained small, and coat color continued to lighten. Animals shifted from 20L to 16L showed partial and transient testicular regression, whereas testes remained large in control animals remaining in 20L. There were no significant differences in body weight between control and experimental animals in either study. These findings indicate that Djungarian hamsters respond reproductively to changes in photoperiod at extreme daylengths, but the magnitude of the response appears to be dependent on the absolute daylength.

Tertiary hyperparathyroidism during high phosphate therapy of familial hypophosphatemic rickets.

We report the development of severe tertiary hyperparathyroidism in three girls treated for familial hypophosphatemic rickets and characterize parathyroid function in vivo and in vitro. All patients had been previously treated with relatively large doses of inorganic phosphorus (125 mm/day) and ergocalciferol or calcitriol for several years and had radiographic evidence of long-standing hyperparathyroidism. Even in the presence of extremely elevated PTH levels, oral phosphate lowered serum calcium levels in vivo and further stimulated PTH secretion. Profound multiglandular parathyroid hyperplasia was found in each patient at surgery. Examination of the secretory characteristics of the excised parathyroid tissue revealed that either relatively high calcium concentrations were generally needed to suppress PTH secretion or PTH secretion was not suppressible. Caution is recommended when relatively large doses of phosphate are used to treat familial hypophosphatemic rickets.

Calcium-dependent release of N-terminal fragments and intact immunoreactive parathyroid hormone by human pathological parathyroid tissue in vitro.

The regulation of PTH secretion by extracellular calcium was studied in parathyroid tissue obtained from patients with hyperparathyroidism (adenoma or hyperplasia) using both an amino (N)-terminal RIA as well as an immunoradiometric assay (intact assay) specific for the intact hormone. The parathyroid glands separated into three major groups when examined in terms of absolute amounts of PTH secreted, degree of suppressibility, and set-point for calcium (the concentration of calcium causing half-maximal inhibition of PTH release). In cell preparations from group A (two different adenomas, two hyperplastic glands from a patient with renal failure, and a hyperplastic gland from a patient with hypophosphatemic rickets), both assays showed comparable PTH release (agreeing within 2-fold), similar degrees of suppressibility and similar, if not identical, set-points. In group B (two adenomas and one hyperplastic gland from a patient with renal failure), PTH secretion, as measured in the N-terminal assay, was 3- to 6-fold more than that measured in the intact assay. The set-points and maximal degrees of suppressibility were, however, still comparable. In group C [two adenomas and one gland from a patient with hypophosphatemic rickets (the sister of the patient in group A)], no suppressibility was observed when PTH release was measured using the intact assay (i.e. less than 50% suppression of PTH release at 2-3 mM Ca2+). In one of these three glands, the N-terminal assay was used in addition to the intact assay, and no suppressibility was present with either assay. Thus, in general, the effects of extracellular Ca2+ on PTH secretion from pathological parathyroid tissue were similar when assessed with both an N-terminal and an intact assay, at least with respect to setpoint and maximal suppressibility. In a few cases, maximal PTH release was greater when measured with the N-terminal assay, consistent with substantial release of N-terminal fragments in addition to intact PTH. In addition, nonsuppressible glands were not uncommon when PTH release was measured by the intact assay, confirming previous studies with less specific assays.

Adenosine A1 receptors are located predominantly on axons in the rat hippocampal formation.

The nucleoside adenosine exerts potent biological effects via specific receptors, including the inhibitory A1 adenosine receptor (A1AR). In the hippocampus A1ARs play an important role in regulating neuronal activity. However, the cellular sites of hippocampal A1ARs are undefined. Using in situ hybridization, receptor autoradiography, and single- and double-label immunocytochemistry techniques, we have characterized the cellular sites of A1AR expression in the rat hippocampus. In situ hybridization and receptor autoradiography studies revealed strikingly different patterns of labeling. In situ hybridization studies revealed heaviest labeling of cell bodies in the granular layer of the dentate gyrus and the pyramidal layers of Ammon's horn. In contrast, using [3H]DPCPX, we observed heavy specific labeling over the neuropil in the dentate hilus stratum moleculare, stratum lacunosum-moleculare, stratum radiatum, and stratum oriens, and little labeling over cell bodies. Using single-label immunocytochemistry, A1AR immunoreactivity was found to be heaviest over fibers in regions corresponding with heavy [3H]DPCPX labeling. Double-label florescent confocal microscopy was then used to determine the identity of labeled fibers. A1AR immunoreactivity was found to co-localize with SMI-31 that labels axons, but not with MAP2a,b that labels cell bodies and dendrites, or with synaptophysin that labels synapses. These data identify axons as the predominant site of A1AR expression in hippocampus. Activation of A1ARs may be a powerful mechanism by which adenosine alters axonal transmission to inhibit neurotransmitter release.

A1 adenosine receptors mediate hypoglycemia-induced neuronal injury.

The cellular mechanisms that lead to neuronal death following glucose deprivation are not known, although it is recognized that hypoglycemia can lead to perturbations in intracellular calcium ([Ca2+]i) levels. Recently, activation of A1 adenosine receptors (A1AR) has been shown to alter [Ca2+]i and promote neuronal death. Thus, we examined if A1AR activation contributes to hypoglycemia-induced neuronal injury using rat cortical neurons. First, we observed that hypoglycemia was associated with large increases in neuronal adenosine release. Next, decreased neuronal viability was seen with progressive reduction in glucose concentration (25, 6, 3, 0.75 and 0 mM). Using the calcium-sensitive dye, Fluo-3, we observed both acute and long-term changes in relative [Ca2+]i during hypoglycemic conditions. Demonstrating a role for adenosine in this process, both the loss in neuronal viability and the early changes in [Ca2+]i were reversed by treatment with A1AR antagonists (8-cyclopentyl, 1,3-dipropylxanthine; 9-chloro-2-(2-furyl)(1,2,4)-triazolo(1,5-c)quinazolin-5-amine; and N-cyclopentyl-9-methyladenine). We also found that hypoglycemia induced the expression of the pro-apoptotic enzyme, caspase-3, and that A1AR antagonism reversed hypoglycemia-induced caspase-3 activity. Collectively, these data show that hypoglycemia induces A1ARs activation leading to alterations in [Ca2+]i, which plays a prominent role in leading to hypoglycemia-induced neuronal death.

Hypoglycemia pathogenesis in children with dumping syndrome.

Three children with severe hypoglycemic reactions secondary to dumping syndrome were studied to discern the mechanism by which hypoglycemia occurred. Symptoms in patient 1 developed after fundoplication, generalized autonomic dysfunction occurred in patient 2, and dumping syndrome developed in patient 3 after malplacement of a feeding gastrostomy tube. Average blood glucose levels studied during and after two to seven meals in each child were 375 +/- 97 mg/dL (mean +/- SD) 30 minutes postprandially and 35 +/- 10 mg/dL greater than 120 minutes later. Swings in glucose values were proportional to the volume of meals. Insulin and glucagon levels were followed during a single meal challenge test in each patient; the average glucose concentration increased to 356 +/- 59 mg/dL 30 minutes postprandially and decreased to 32 +/- 11 mg/dL at 150 +/- 30 minutes. Hormonal analyses indicated (1) inappropriate early release of glucagon (300 pg/mL at 15 minutes) in patient 1, (2) exuberant early release of insulin (maximum 190 +/- 15 microU/mL) resulting in rapid decrease in glucose concentration in all patients, (3) development and/or persistence of hypoglycemia after the decline in circulating insulin to undetectable levels, and (4) inadequate glucagon response to hypoglycemia resulting in sustained hypoglycemia. These data indicate that gross disturbances of the insulin-glucagon axis attend childhood dumping syndrome.

Maternal communication of circadian phase to the developing mammal.

In rodents, an entrainable circadian clock begins oscillating prenatally in the hypothalamic suprachiasmatic nuclei (SCN). The maternal circadian system coordinates (entrains) the timing of the developing clock to the prevailing light-dark cycle during both late fetal and early neonatal life. This maternal communication of circadian phase ensures that the developing animal is coordinated to the outside world until maturation of the normal pathway of light-dark entrainment in adults, the retinohypothalamic pathway, permits direct photic entrainment through the neonatal eye. The mechanism of maternal communication of circadian phase remains to be determined, but the necessity of the maternal SCN in this communication has been demonstrated. Indirect lines of evidence suggest that a similar scheme occurs for the development of circadian rhythms in humans.

D1 receptors mediate dopamine action in the fetal suprachiasmatic nuclei: studies of mice with targeted deletion of the D1 dopamine receptor gene.

Studies in rodents suggest the presence of a dopaminergic system that influences the function of a biological clock in the hypothalamic suprachiasmatic nuclei (SCN). To provide insights into mechanisms of dopamine action in the SCN, we studied transgenic mice that had either one allele (+¿-) or both alleles (-/-) of the D1 dopamine receptor gene deleted, along with normal (+/+) littermates. As expected, receptor labelling autoradiography studies using [125I]SCH 23982 showed a complete absence of D1 dopamine receptor binding sites in the SCN of -/- animals. When pregnant mice from +¿- x +¿- matings were injected with the D1 receptor agonist SKF 38393, or the dopamine reuptake blocker GBR 12909 at day 19 of gestation, c-fos mRNA expression was observed in the SCN of +/+ fetuses. In contrast, c-fos mRNA induction was not seen in -/- or +¿- litter mates. Injection of cocaine into pregnant dams also resulted in robust SCN c-fos mRNA expression in +/+ mice. Increases in SCN c-fos mRNA expression were also seen in +¿- and -/- mice suggesting that cocaine action in the SCN involves both D1 receptor-dependent and -independent mechanisms. Collectively, our studies of transgenic mice deficient in D1 receptors support the presence of a functional dopaminergic system in the fetal SCN. We also identify D1 receptors as the prominent transducer of dopamine action in the fetal SCN.