Long-term efficacy of ultrasound-guided laser ablation for benign solid thyroid nodules. Results of a three-year multicenter prospective randomized trial.

BACKGROUND: The aim of the present trial on ultrasound (US)-guided laser ablation therapy (LAT) of solid thyroid nodules is to assess long-term clinical efficacy, side effects, and predictability of outcomes in different centers operating with the same procedure. PATIENTS: Two hundred consecutive patients were randomly assigned to a single LAT session (group 1, 101 cases) or to follow-up (group 2, 99 cases) at four thyroid referral centers. Entry criteria were: solid thyroid nodule with volume of 6-17 mL, repeat benign cytological findings, normal thyroid function, no autoimmunity, and no thyroid gland treatment. METHODS: Group 1: LAT was performed in a single session with two optical fibers, a 1064 nm Nd-YAG laser source, and an output power of 3 W. Volume and local symptom changes were evaluated 1, 6, 12, 24, and 36 months after LAT. Side effects and tolerability of treatment were registered. Group 2: Follow-up with no treatment. RESULTS: One patient was lost to follow-up in each group. Group 1: Volume decrease after LAT was -49 ± 22%, -59 ± 22%, -60 ± 24%, and -57 ± 25% at 6, 12, 24, and 36 months, respectively (P < .001 vs baseline). LAT resulted in a nodule reduction of >50% in 67.3% of cases (P < .001). Local symptoms decreased from 38 to 8% of cases (P = .002) and cosmetic signs from 72 to 16% of cases (P = .001). Baseline size, presence of goiter (P = .55), or US findings (fluid component ≤ 20% [P = .84], halo [P = .46], vascularization [P = .98], and calcifications [P = .06]) were not predictive factors of a volume decrease > 50%. The procedure was well tolerated in most (92%) cases. No changes in thyroid function or autoimmunity were observed. In group 2, nodule volume increased at 36 months (25 ± 42%; P = .04). The efficacy and tolerability of the procedure were similar in different centers. CONCLUSIONS: A single LAT treatment of solid nodules results in significant and persistent volume reduction and local symptom improvement, in the absence of thyroid function changes.

Beta-adrenergic agonism does not impair the GH response to acylated ghrelin in humans.

BACKGROUND: Acylated ghrelin (AG) is a physiological GH secretion amplifier, in part stimulating GHRH neurones and antagonizing somatostatin activity. In humans, AG is one of the most potent pharmacological stimuli of GH secretion and, unlike GHRH, is refractory to the inhibitory effect of glucose, free fatty acids (FFA) and somatostatin. Somatotroph secretion is also profoundly modulated by the adrenergic system. Indeed, beta-adrenergic agonists abolish spontaneous and GHRH-stimulated GH secretion. Based on these data, the aim of the present study was to investigate the effects of beta adrenergic agonism on the GH response to AG. SUBJECTS AND MEASUREMENTS: Six young healthy male volunteers underwent: (a) acute AG intravenous (iv) administration (1.0 microg/kg); (b) salbutamol infusion (SLB; 0.06 microg/kg/min iv); (c) AG + SLB; and (d) saline infusion. In all sessions GH levels were assayed every 15 min from time -30 to +210 min. RESULTS: SLB induced a significant (P < 0.05) inhibition of spontaneous GH secretion that persisted up to 75 min after SLB withdrawal. AG induced a marked increase (P < 0.01) in GH that was not modified by SLB. CONCLUSIONS: The GH-releasing effect of AG is refractory to the inhibitory effect of SLB-induced beta-adrenergic receptor activation. Although further studies are needed to confirm these results during the lifespan and particularly during prolonged exposure to beta agonists, the present data clearly suggest that, among GH stimulatory tests, AG administration might be the most suitable in clinical conditions of chronic treatment with beta-2 agonists, such as in asthmatic disease.

The continuous infusion of acylated ghrelin enhances growth hormone secretion and worsens glucose metabolism in humans.

CONTEXT: Acylated ghrelin (AG) has been discovered as a natural ligand of the GH secretagogue receptor type 1a and is now recognized as an important orexigenic factor. Besides stimulation of GH secretion and appetite, it exerts other central and peripheral actions including modulation of insulin secretion, glucose and lipid metabolism. OBJECTIVE: To define the effects of the continuous iv infusion of AG in humans with particular attention to metabolic parameters. MATERIALS AND METHODS: We studied the effects of 16- h (from 21:00 to 13:00 h) infusion of AG (0.5 microg/kg/h) or saline in 8 young volunteers who were provided with isocaloric balanced meals. GH, cortisol, insulin, glucose, free fatty acid (FFA), and ghrelin levels were assayed every 20 min. RESULTS: AG infusion increased circulating total ghrelin to a steady state that was maintained over 16 h infusion of the peptide. With respect to saline, AG infusion significantly modified GH, cortisol, insulin, and glucose profiles and decreased FFA area under the curve (p<0.01). AG increased GH pulse frequency and approximate entropy (p<0.05). AG enhanced the glucose response to both dinner (p<0.02) and breakfast (p<0.03). AG infusion blunted the early insulin response to dinner (p<0.03) but enhanced the second-phase insulin response to dinner and breakfast (p<0.05). CONCLUSIONS: The continuous exposure to AG in humans enhances somatotroph secretion but also worsens glucose metabolism, although it inhibits lipolysis. These findings in normal young volunteers are consistent with data from studies in animals and suggest that acylated ghrelin is likely to play a negative role in glucose metabolism.

Sleep abnormalities in type 2 diabetes may be associated with glycemic control.

Sleep disturbances may be associated with impaired glucose metabolism. The aim of this study was to evaluate sleep duration and quality in relation to glycemic control in patients with type 2 diabetes. In a cross-sectional study, sleep duration and quality were assessed in 47 middle-aged patients with type 2 diabetes treated with oral agents and without sleep disturbing complications and 23 healthy control subjects similar by age, sex, body mass index, occupation and schooling. Sleep was recorded by wrist-actigraphy for three consecutive days under free-living conditions. Univariate analysis showed lower sleep maintenance (P = 0.002) and sleep efficiency (P = 0.005), and higher fragmentation index (P < 0.0001), total activity score (P = 0.05) and moving time (P < 0.0001) in patients with type 2 diabetes. After adjusting for age, gender and schooling, fragmentation index and moving time remained significantly higher in the patients with diabetes (P < 0.05, both). HbA1c correlated inversely with sleep efficiency (r = -0.29; P = 0.047) and positively with moving time (r = 0.31; P = 0.031). These findings suggest that type 2 diabetes is associated with sleep disruptions even in the absence of complications or obesity. The relevance of sleep abnormalities to metabolic control and possible strategies to improve sleep quality in type 2 diabetes deserve further investigation.

Cortistatin-8, a synthetic cortistatin-derived ghrelin receptor ligand, does not modify the endocrine responses to acylated ghrelin or hexarelin in humans.

Cortistatin (CST), a neuropeptide with high structural homology with somatostatin (SST), binds all SST receptor (SST-R) subtypes but, unlike SST, also shows high binding affinity to ghrelin receptor (GHS-R1a). CST exerts the same endocrine activities of SST in humans, suggesting that the activation of the SST-R might mask the potential interaction with ghrelin system. CST-8, a synthetic CST-analogue devoid of any binding affinity to SST-R but capable to bind the GHS-R1a, has been reported able to exert antagonistic effects on ghrelin actions either in vitro or in vivo in animals. We studied the effects of CST-8 (2.0 microg/kg i.v. as a bolus or 2.0 microg/kg/h i.v. as infusion) on both spontaneous and ghrelin- or hexarelin- (1.0 microg/kg i.v. as bolus) stimulated GH, PRL, ACTH and cortisol secretion in 6 normal volunteers. During saline, no change occurred in GH and PRL levels while a spontaneous ACTH and cortisol decrease was observed. As expected, both ghrelin and hexarelin stimulated GH, PRL, ACTH and cortisol secretion (p<0.05). CST-8, administered either as bolus or as continuous infusion, did not modify both spontaneous and ghrelin- or hexarelin-stimulated GH, PRL, ACTH and cortisol secretion. In conclusion, CST-8 seems devoid of any modulatory action on either spontaneous or ghrelin-stimulated somatotroph, lactotroph and corticotroph secretion in humans in vivo. These negative results do not per se exclude that, even at these doses, CST-8 might have some neuroendocrine effects after prolonged treatment or that, at higher doses, may be able to effectively antagonize ghrelin action in humans. However, these data strongly suggest that CST-8 is not a promising candidate as GHS-R1a antagonist for human studies to explore the functional interaction between ghrelin and cortistatin systems.

d-Lys-GHRP-6 does not modify the endocrine response to acylated ghrelin or hexarelin in humans.

Acylated ghrelin exerts numerous endocrine and non-endocrine activities via the GH Secretagogue receptor type 1a (GHS-R1a). D-Lys-GHRP-6 has been widely studied in vitro and in vivo in animal studies as GHS-R1a antagonist; its action in humans has, however, never been tested so far. Aim of our study was to verify the antagonistic action of D-Lys-GHRP-6 on the endocrine responses to acylated ghrelin and hexarelin, a peptidyl synthetic GHS, in humans. The effects of different doses of D-Lys-GHRP-6 (2.0microg/kg iv as bolus or 2.0microg/kg/h iv as infusion) on both spontaneous and acylated ghrelin- or hexarelin (1.0microg/kg iv as bolus) -stimulated GH, PRL, ACTH and cortisol levels were studied in six normal volunteers (age [mean+/-SEM]: 25.4+/-1.2yr; BMI: 22.3+/-1.0kg/m(2)). The effects of D-Lys-GHRP-6 (2.0microg/kg iv as bolus+4.0microg/kg/h iv) on the GH response to 0.25microg/kg iv as bolus acylated ghrelin was also studied. During saline, spontaneous ACTH and cortisol decrease was observed while non changes occurred in GH and PRL levels. Acylated ghrelin and hexarelin stimulated (p<0.05) GH, PRL, ACTH and cortisol secretions. D-Lys-GHRP-6 administered either as bolus or a continuous infusion did not modify both spontaneous and acylated ghrelin- or hexarelin-stimulated GH, PRL, ACTH and cortisol secretion. D-Lys-GHRP-6 did not modify even the GH response to 0.25microg/kg iv acylated ghrelin. In conclusion, D-Lys-GHRP-6 does not affect the neuroendocrine response to both ghrelin and hexarelin. These findings question D-Lys-GHRP-6 as an effective GHS-R1a antagonist for human studies.

Ghrelin: from somatotrope secretion to new perspectives in the regulation of peripheral metabolic functions.

Ghrelin, a peptide predominantly produced by the stomach, has been discovered as natural ligand of the GH secretagogue (GHS) receptor type 1a (GHS-R1a), suggesting the existence a new endogenous modulator of somatotrope secretion. Subsequently, ghrelin turned out to exert pleiotropic actions, consistent with the widespread distribution of ghrelin and GHS-R expression in central and peripheral tissues. Despite that the binding to GHS-R1a requires ghrelin to be acylated in serine 3, some ghrelin actions are independent of such acylation; thus suggesting the possibility of the existence of other GHS-R subtypes. Ghrelin secretion (70% in its unacylated form) is mainly under metabolic control being modulated by glucose, insulin and feeding. On the other hand, ghrelin influences energy metabolism acting both as a central orexigenic factor and directly on the endocrine pancreas, liver and adipose tissue. Recently, another gastric hormone derived from the same ghrelin gene has been isolated and named obestatin. Obestatin in rats resulted in reduced food intake, jejunal contraction and body weight gain, via specific distinct receptors. Thus, all these data indicate that we are exploring a very complex system deeply involved in the modulation of metabolic functions, whose understanding will probably increase our knowledge about diabetes mellitus and the metabolic syndrome.

Ghrelin: endocrine, metabolic and cardiovascular actions.

Ghrelin is a peptide predominantly produced by the stomach, although expressed by many other tissues, including the pancreas and the cardiovascular system. Its secretion is negatively associated to body mass index and undergoes fluctuations during the day. It is stimulated by energy restriction and acetylcholine, while it is reduced by gastrectomy, food intake, glucose, insulin and SRIF. Ghrelin is a natural ligand of the GH secretagogue (GHS) receptor 1a (GHS-R1a), known being specific for synthetic GHS. GHS-R1a expression and binding studies showed GHS-R in the hypothalamus-pituitary area but also in other brain areas as well as in peripheral, endocrine and non-endocrine tissues, including the pancreas and the cardiovascular system. Besides its potent GH-releasing effect, ghrelin: 1) stimulates lactotroph and corticotroph secretion while it negatively modulates the gonadal axis; 2) exerts central actions including orexigenic effect coupled with control of energy expenditure; 3) influences either the exocrine or the endocrine pancreatic function and the glucose and lipid metabolism; 4) controls gastric motility and acid secretion; 5) exerts cardiovascular actions; 6) modulates cell proliferation. Ser3-acylation of ghrelin is essential for binding the GHS-R1a and for its endocrine actions. However, both non-acylated and acylated ghrelin are bound by GHS-R subtypes at both the pancreatic and the cardiovascular level. Non-acylated ghrelin is not an inactive peptide; it exerts some non-endocrine actions such as cardiovascular activities, modulation of cell proliferation and even some metabolic action such as modulation of insulin secretion, glucose and lipid metabolism. Notably, some GHS-R subtypes are not ghrelin receptors; cardiovascular receptors specific for peptidyl GHS have been found and it has been shown they mediate some specific actions that are not shared by ghrelin.

Cortistatin-17 and -14 exert the same endocrine activities as somatostatin in humans.

Cortistatin (CST) is a neuropeptide, which binds with high affinity all somatostatin (SS) receptor subtypes and shows high structural homology with SS itself. A receptor specific for CST only, i.e., not recognized by SS, has been recently described in agreement with data reporting that not all CST actions are shared by SS. Interestingly, CST but not SS also binds ghrelin receptor (GHS-R1a) in vitro, suggesting a potential interplay between CST and ghrelin system. The aim of this study was to investigate in humans the endocrine and metabolic activities of human CST-17 in comparison with rat CST-14 that has previously been shown to exert the same endocrine actions of SS in healthy volunteers. To this aim, in six healthy male volunteers (age [median, 3rd-97th centiles]: 28.5; 23.6-34.3 years; Body Mass Index: 23.5; 21.0-25.1 kg/m(2)), we studied the effects of human CST-17 (2.0 microg/kg/h iv over 120 min), rat CST-14 (2.0 microg/kg/h iv over 120 min) and SS-14 (2.0 microg/kg/h iv over 120 min) on: (a) spontaneous GH, ACTH, PRL, cortisol, insulin and glucose levels; (b) the GH responses to GHRH (1.0 microg/kg iv at 0 min); (c) the GH, PRL, ACTH, cortisol, insulin and glucose responses to ghrelin (1.0 microg/kg iv at 0 min). CST-17 inhibited (p < 0.01) basal GH secretion to the same extent of CST-14 and SS-14. Spontaneous PRL, ACTH and cortisol secretion were not significantly modified by CST-17, CST-14 or SS-14. CST-17 as well as CST-14 and SS-14 also inhibited (p < 0.05) spontaneous insulin secretion to a similar extent. None of these peptides modified glucose levels. The GH response to GHRH was inhibited to the same extent by CST-17 (p < 0.01), CST-14 (p < 0.01) and SS-14 (p < 0.05 ). The ghrelin-induced GH response was higher than that elicited by GHRH (p < 0.01) and inhibited by CST-17 (p < 0.05) as well as by CST-14 (p < 0.05) and SS-14 (p < 0.01). The PRL, ACTH and cortisol responses to ghrelin were unaffected by CST-17, CST-14 or SS-14. On the other hand, the inhibitory effect of ghrelin on insulin levels was abolished by CST-17, CST-14 or SS-14 (p < 0.05) that, in turn, did not modify the ghrelin-induced increase in glucose levels. In conclusion, this study demonstrates that human CST-17 and rat CST-14 exert the same endocrine activities of SS in humans. The endocrine actions of human and rat CST therefore are likely to reflect activation of classical SS receptors.

Suppression of interleukin-6 production in endotoxin tolerance in a mouse glioma cell line: reversal by phorbol ester.

Tolerance to endotoxin (lipopolysaccharide, LPS) was shown to be mediated by an inhibition of cytokine production. We have studied the effect of 3-day pretreatment with LPS on production of IL-6 in response to a subsequent challenge with LPS in a mouse glioma. The results indicated that in this model, a complete blockage of IL-6 production is induced by LPS pretreatment. This is associated with a decrease of LPS-induced IL-6 mRNA levels. LPS-induced IL-6 production can be restored by PMA, as it was previously observed in vivo, suggesting that down-regulation of IL-6 response in LPS tolerance occurs at the transcriptional level, probably by down-regulating protein kinase C or some other PMA-activable signaling system. IL-6 production is also down-regulated by 3-day preincubation with IL-6 and, to a lesser extent, with IL-1 or TNF, indicating that IL-6 can down-regulate its own production.

Differential sensitivity of in vivo TNF and IL-6 production to modulation by anti-inflammatory drugs in mice.

The effect of dexamethasone and two non-steroidal anti-inflammatory agents ibuprofen and indomethacin on the production of serum interleukin 6(IL-6) and tumor necrosis factor (TNF) levels in mice treated with endotoxin (2.5 micrograms/mouse, i.p.) was investigated. Pretreatment of mice with dexamethasone (0.3-30.0 mg/kg, i.p., 30 min before endotoxin) completely blocked TNF production but did not affect that of IL-6. Conversely, pretreatment with indomethacin (5 mg/kg, i.p.) or ibuprofen (30 mg/kg, i.p.) potentiated the production of both IL-6 (+ 80% with INDO; + 100% with IBU) and TNF (+ 500% with INDO; + 50% with IBU). In the case of IL-6, the two anti-inflammatory drugs were able per se to induce significant levels of this cytokine even in the absence of LPS. These data indicate that IL-6 and TNF production are differently susceptible to glucocorticoids, and that prostaglandins can physiologically provide a negative feedback regulation of IL-6 and TNF synthesis.

The unique interaction with immunity of FCE 24517, an antitumor drug with a novel mode of action.

The present study was designed to characterize in mice the effects on the immune system of the antineoplastic agent FCE 24517, a benzoic acid mustard derivative of distamycin A with specificity for AT-rich base pair sequences of beta-DNA. At antitumorally therapeutic doses, single injections of FCE 24517 caused profound leukopenia and a decrease of spleen cell numbers. However, primary and secondary antibody production to the T-dependent antigen sheep red blood cells were markedly increased by FCE 24517 given before or together with antigen. Antibody production to the T-independent antigen type III pneumococcal polysaccharide was not appreciably affected by FCE 24517. The delayed type hypersensitivity response to sheep erythrocytes was only marginally decreased, whereas proliferation of spleen cells to mitogens was markedly depressed. The ability of natural killer cells and macrophages to mediate cytotoxicity was not affected by FCE 24517 treatment. The ability of carrier-primed cells from drug-treated mice to cooperate for anti-trinitrophenyl antibody production increased twofold over that of vehicle-injected controls, suggesting an increase in T-helper cell activity. Serum cytokine levels were studied in mice injected with bacterial lipopolysaccharide used as a model stimulus. Peak levels of TNF and IL-6 were not modified by FCE 24517, but at later times higher amounts of cytokines were found in drug-treated mice compared with the control, suggesting a longer exposure of immunocompetent cells to these factors. Two compounds (FCE 24561 and CC-1065), also capable of binding AT-rich sequences in the minor groove of beta-DNA and containing an alkylating moiety, had immunomodulatory activity similar to FCE 24517 in increasing primary anti-sheep erythrocytes response. It is concluded that FCE 24517 exhibits a unique interaction with the immune system, markedly different from that of alkylating agents and may be representative of a novel group of antiproliferative-immunomodulating agents.

Impairment of cytokine production in mice fed a vitamin D3-deficient diet.

C57Bl/6 female mice fed a Vitamin D (VIT-D)-deficient diet had serum levels of 25-hydroxyvitamin D decreasing with the time of diet exposure (3 and 8 weeks). Cytokine production (IL-6, TNF and IL-1) by peritoneal macrophages cultured in vitro with a standard stimulus, LPS, evaluated in the supernatants as biological activity, was significantly reduced in VIT-D-deficient animals. The defect in monokine production was partial and was evident at suboptimal LPS concentrations and incubation times. I-A antigen expression, induced in macrophages by in vitro exposure to IFN-gamma, was not modified in VIT-D-deficient mice, but IFN-gamma-inducible macrophage cytotoxicity to tumour target cells was significantly decreased in VIT-D-deficient animals. Moreover, basal and Poly I:C-induced NK activity was not modified by VIT-D deficiency. Thus, macrophage functions, such as cytokine production and tumour cytotoxicity induction, are down-modulated in vitro by VIT-D deprivation. To give more support to the relevance of VIT-D availability for cytokine production, TNF and IL-6 have been evaluated in the sera of control and VIT-D-deficient mice given LPS as a model stimulus. Serum peak levels of both cytokines were at least halved in VIT-D-deprived mice. Thus, VIT-D deficiency may represent a model of partial defect of monokine production.

Interleukin-6 gene expression and production induced in human monocytes by membrane proteoglycans from Klebsiella pneumoniae.

The present study was designed to investigate the effect of membrane proteoglycans (MPG) from Klebsiella pneumoniae on IL-6 production by human peripheral blood monocytes. Exposure in vitro to MPG induced release of IL-6 activity from human monocytes, as assessed by the 7TD1 hybridoma assay. MPG-induced hybridoma growth factor activity was blocked by anti-IL-6 antibodies. MPG induced expression in human monocytes of IL-6 mRNA transcripts as assessed by Northern blot analysis. Induction of IL-6 in mononuclear phagocytes may play a role in the immunomodulatory activity of MPG.