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This paper was presented at the 4th Micro and Nano Flows Conference (MNF2014), which was held at University College, London, UK. The conference was organised by Brunel University and supported by the Italian Union of Thermofluiddynamics, IPEM, the Process Intensification Network, the Institution of Mechanical Engineers, the Heat Transfer Society, HEXAG - the Heat Exchange Action Group, and the Energy Institute, ASME Press, LCN London Centre for Nanotechnology, UCL University College London, UCL Engineering, the International NanoScience Community, www.nanopaprika.eu. Traditionally, in vitro investigations on biology and physiology of cells rely on averaging the responses eliciting from heterogeneous cell populations, thus being unsuitable for assessing individual cell behaviors in response to external stimulations. In the last years, great interest has thus been focused on single cell analysis and screening, which represents a promising tool aiming at pursuing the direct and deterministic control over cause-effect relationships guiding cell behavior. In this regard, a high-throughput microfluidic platform for trapping and culturing adherent single cells was presented. A single cell trapping mechanism was implemented based on dynamic variation of fluidic resistances. A round-shaped culture chamber (Φ=250μm, h=25μm) was conceived presenting two connections with a main fluidic path: (i) an upper wide opening, and (ii) a bottom trapping junction which modulates the hydraulic resistance. Several layouts of the chamber were designed and computationally validated for the optimization of the single cell trapping efficacy. The optimized chamber layouts were integrated in a polydimethylsiloxane (PDMS) microfluidic platform presenting two main functionalities: (i) 288 chambers for trapping single cells, and (ii) a chaoticmixer based serial dilution generator for delivering both soluble factors and non-diffusive molecules under spatio-temporally controlled chemical patterns. The devices were experimentally validated and allowed for trapping individual U87-MG (human glioblastoma-astrocytoma epithelial-like) cells and culturing them up to 3 days.

Previous studies demonstrated that GHRP-6 has modest GH-releasing activity in primary pituitary cell monolayer cultures. However, the effects of this peptide have always been tested on cells very sensitive to GHRH. We have previously reported that GHRH is unable to stimulate GH secretion in the GH1 rat tumor cell line. The aim of the study was to assess for the first time the effect on GH secretion of the GHRP-6 analog, hexarelin, in the GH1 cells; moreover, we investigated the potential involvement of GHRH in the effects of hexarelin in the GH1 rat cell line. The GHRP-6 analog hexarelin (0.01-1 microM) significantly stimulated GH release in both normal and GH1 rat cells. The greatest GH-releasing effect of hexarelin was observed with the 1 microM dose both in GH1 (155+/-25% vs. control wells) and in normal rat pituitary cells (185+/-23% vs. control wells). GHRH significantly stimulated GH secretion in normal rat somatotrophs (3-fold increase). In this latter cell model, GHRH and hexarelin were demonstrated to have additive stimulatory effects on GH secretion. Conversely, GHRH did not affect hexarelin-stimulated GH release in GH1 cells at any of the doses used. Finally, 8Br-cAMP significantly stimulated GH secretion in both normal rat and GH1 cells. These results provide in vitro evidence that non-GHRH-mediated pathways for GHRP action exist. Moreover, the observation that cells not sensitive to GHRH can be significantly stimulated by hexarelin strongly suggests that GHRPs and GHRH have two distinct sites and modes of action at the pituitary level.

Eptastigmine is a long-lasting acetyl-cholinesterase inhibitor, currently being developed for the symptomatic treatment of Alzheimer's disease. In the present study, we investigated the relationship between pharmacokinetics and pharmacodynamics of eptastigmine in young healthy volunteers. Eight male subjects received single oral doses of 10, 20, and 30 mg of eptastigmine and placebo according to a double-blind, randomized, crossover design. Blood was collected before and 0.5, 1, 1.5, 2, 3, 4, 6, and 24 hours after drug administration. Cholinesterase activity was measured using a potentiometric method in both plasma (butyryl-cholinesterase) and in red blood cells (acetyl-cholinesterase). Eptastigmine plasma levels were measured by a very sensitive high-performance liquid chromatography method (limit of quantitation 0.2 ng/mL). Eptastigmine plasma concentrations increased proportionally with the dose (mean +/- SEM AUC0-24 was 0.74 +/- 0.58, 3.61 +/- 1.15, and 6.25 +/- 1.51 ng.h/mL with 10, 20, and 30 mg, respectively) and were undetectable at 24 hours. The inhibition of acetyl-cholinesterase was dose-dependent (peak inhibition was 15 +/- 2%, 30 +/- 4%, and 36 +/- 6% with 10, 20, and 30 mg, respectively) and long-lasting, with a residual inhibition of 8 to 11% at 24 hours. Acetyl-cholinesterase inhibition and drug plasma levels were related over time with a counterclockwise hysteresis curve, suggesting the formation of active metabolites and/or a slow association to and dissociation from the enzyme in red blood cells. Butyryl-cholinesterase inhibition was weak and not dose-dependent (peak inhibition was 12 +/- 4%, 13 +/- 3%, and 12 +/- 2% with 10, 20, and 30 mg, respectively). The drug was well tolerated by all subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

Aim of our study was to investigate the effect of clonidine and galanin (alone or in combination) on growth hormone (GH) secretion in normal subjects and in adult patients with increased somatostatin tone due to chronic daily immunosuppressive glucocorticoid treatment. We studied 7 adult patients undergoing long-term (no less than 6 months) immunosuppressive glucocorticoid treatment for non endocrine diseases (4F, 3M; age 49.7 +/- 6.3 years). Six normal adult nonobese subjects (3F, 3M; age 34 +/- 2.7 years) served as controls. All subjects underwent the following three tests in random order: 1) iv infusion of clonidine, 150 micrograms in 10 mL of saline, from time 0 to 10 min; 2) iv infusion of synthetic porcine galanin, 500 micrograms in 100 mL of saline from -15 to 30 min; 3) iv infusion of clonidine from 0 to 10 min combined with synthetic porcine galanin iv infusion from -15 to 30 min. Blood samples for GH assay were taken at -15, 0, 15, 30, 45, 60, 90, 120 min. No significant differences in GH absolute values were observed at any time between the three different tests within each group of subjects. Normal subjects showed significantly (p0.05) higher GH peaks and GH absolute values from 15 to 90 min after galanin alone, clonidine alone and clonidine+galanin with respect to the glucocorticoid-treated patients. The absence of any either synergistic or at least additive effect on GH secretion of galanin and clonidine in conditions of both normal and increased somatostatin tone suggests that also in man, as well as in the rat, the action of galanin on the GH axis may be mediated through alpha-adrenergic pathways.

One of the most prominent metabolic effects of the systemic administration of the synthetic neuropeptide galanin in man is the increase in growth hormone (GH) secretion. This stimulating action of galanin is thought to occur directly at the hypothalamic level through the release of GHRH. Recently, it has been shown that also dopaminergic drugs may elicit GH secretion through an increase in hypothalamic GHRH secretion. The aim of this study was to investigate if the action of galanin on GHRH and consequently on GH release may be mediated via dopaminergic pathways evaluating the effects of a potent central dopaminergic receptor blocker, metoclopramide (MCP), on the galanin-induced growth hormone (GH) secretion in normal subjects. We studied seven young non obese healthy subjects (three females and four males). GH secretion was evaluated after 45 min iv infusion of porcine galanin (0.5 mg in 100 ml of saline) from 0 to 45 min combined with a 60 min iv infusion of a) saline (100 ml) or b) MCP (10 mg in 100 ml of saline) from -15 to 45 min. In all the seven subjects, during galanin infusion, GH values increased with respect to baseline with peaks occurring between 30 and 60 min after the beginning of galanin infusion. Peak GH values ranged between 3.5 and 15.4 micrograms/l (mean 10.4 +/- 1.6 micrograms/l). During MCP infusion no significant differences in the GH response to galanin with respect to saline were observed both when absolute GH levels and GH AUC were examined.(ABSTRACT TRUNCATED AT 250 WORDS)

The aim of the present study was to characterize in a large series (N = 12) of cultured somatotrope adenomas the in vitro effects of the neuropeptide galanin on growth hormone (GH) secretion. This was contrasted with two peptides known to be GH secretagogues (GH-releasing hormone [GHRH] and thyrotropin-releasing hormone [TRH]) and a peptide with a known GH-inhibitory effect (the somatostatin analog octreotide). Groups of three wells were incubated for 4 hours with growth medium alone (control incubation), galanin, GHRH(1–29)NH 2 , TRH, or octreotide. Galanin and octreotide were applied at concentrations of 0.1, 1, and 10 μmol/L, and GHRH and TRH at concentrations of 0.01, 0.1, and 1 μmol/L. Galanin was able to inhibit GH release in nine of 12 cultured somatotrope adenoma cells. This inhibitory effect was clearly dose-dependent in five adenomas. Overall, the mean GH nadir after galanin was −36.1% in nine responder adenoma cultures versus control wells. Octreotide inhibited GH release in five of eight cultured somatotrope adenoma cells. The mean GH nadir after octreotide was −32.7% in five responder adenoma cultures compared with control wells. GHRH and TRH were able to stimulate GH release, respectively, in seven of 11 and in six of seven cultured somatotrope adenoma cells. The mean GH peaks after either GHRH or TRH in responder adenoma cultures were, respectively, +71.5% and +143.7% compared with levels in the control wells. In conclusion, the consistency and potency of the in vitro GH-inhibitory effect of galanin in a large series of somatotrope adenomas are at least similar to those of the most effective available GH-lowering agent, the somatostatin analog octreotide.

The aim of our study was to investigate the effect of hexarelin, a novel GH-releasing peptide-6 analog, and GH-releasing hormone (GHRH) (alone or in combination) on GH secretion in adult patients with increased somatostatin tone due to chronic glucocorticoid excess. We studied seven adult patients undergoing long-term (no less than 6 months) immunosuppressive glucocorticoid treatment for non-endocrine diseases (six females and one male, age range 42–68 years) and one subject (female, age 31 years) with endogenous hypercortisolism due to adrenal adenoma. Six normal subjects (four females and two males) matched for sex and age with the patients and not undergoing any therapy served as controls. All the subjects underwent the following three tests in random order: (1) human GHRH (1–29)NH2 (100 μg in 1 ml saline) injected as an i.v. bolus at 0 min, (2) hexarelin (100 μg in 1 ml saline) injected as an i.v. bolus at 0 min and (3) hexarelin (100 μg in 1 ml of saline) plus GHRH (100 μg in 1 ml saline) injected as an i.v. bolus at 0 min. After GHRH alone the patients with glucocorticoid excess showed a blunted GH response as compared with normal subjects (median delta GH: 0·9, range 0–5·6 μg/l vs 7·1, range 0·3–14·9 μg/l). No significant differences were observed in the steroid-treated group with respect to normal subjects after hexarelin alone (median delta GH: 15·5, range 1·9–45·2 μg/l vs 17·9, range 5·5–53·9 μg/l). The GH responses after the combined stimuli were significantly decreased in the patients with glucocorticoid excess as compared with normal subjects (median delta GH: 43·5, range 21–56·9 μg/l vs 73·7, range 19·6-116·8 μg/l). The two stimuli acted in a synergistic fashion in both groups of subjects. It may be hypothesized that hexarelin influences the GH inhibitory effect of glucocorticoids in humans, acting at the hypothalamic somatostatin level. Journal of Endocrinology (1995) 146, 227–232

The aim of our study was to evaluate the effect of the iv administration of two different beta 2- receptors agonists, salbutamol and broxaterol, on the growth hormone (GH) response to maximal exercise in 11 patients (8 males and 3 females; age range 18-65 yr; mean +/- SE age 56 +/- 13 yr; BMI 26.2 +/- 1.4 kg/m2) with chronic asthmatic bronchitis. All the subjects underwent four cycloergometric exercise tests (incremental workload until maximal predicted heart rate). At baseline, at maximal exercise, at the end of the recovery period and 60 min after the end of each exercise, blood samples were drawn for the assay of GH, glucose, insulin, lactates, norepinephrine and epinephrine. Two exercises were performed without treatment while the remaining two were performed 60 min after the administration of 400 micrograms of either salbutamol or broxaterol (both diluted in 10 ml of saline) according to a randomized double blind cross-over design. Both exercise tests performed without treatment caused a significant (p0.05) and similar GH peak with respect to baseline values (from 0.3 +/- 0.1 micrograms/L to 2.8 +/- 1.3 micrograms/L, mean of the two exercise tests). Salbutamol pretreatment blunted the GH response to exercise which caused a no more significant serum GH peak over the baseline levels (from 0.6 +/- 0.2 micrograms/L to 1.4 +/- 0.6 micrograms/L,). Moreover, broxaterol completely abolished the GH response to exercise (baseline level 0.6 +/- 0.2 micrograms/L; peak levels 0.4 +/- 0.1 micrograms/L). The serum GH peak after exercise + broxaterol was significantly (p0.05) lower as compared to exercise + salbutamol. In conclusion, we have demonstrated for the first time that beta 2 stimulation blunts the physiological GH response to maximal exercise in adult human subjects. It can be suggested that changes in brain neurotransmitters, possibly an increase in the alpha-adrenergic tone, are likely to be involved in this endocrine effects of exercise.

The aim of our study was to elucidate the physiological role of the neuropeptide galanin in the regulation of anterior pituitary function in human subjects. Six healthy men (age range 26-35 yr, body mass index range 20-24 kg/m2) underwent in random order 1) an intravenous bolus injection of growth hormone-releasing hormone (GHRH)-(1-29)-NH2 (100 micrograms) + thyrotropin-releasing hormone (TRH, 200 micrograms) + luteinizing hormone-releasing hormone (LHRH, 100 micrograms) + corticotropin-releasing hormone (CRH, 100 micrograms), and 2) intravenous saline (100 ml) at time 0 plus either human galanin (500 micrograms) in saline (100 ml) or saline (100 ml) from -15 to +30 min. Human galanin determined a significant increase in serum GH (GH peak: 11.3 +/- 2.2 micrograms/l) from both baseline and placebo levels. No significant differences were observed between GH values after galanin and those after GHRH alone (24.3 +/- 5.2 micrograms/l). Human galanin significantly enhanced the GH response to GHRH (peak 49.5 +/- 10 micrograms/l) with respect to either GHRH or galanin alone. Human galanin caused a slight decrease in baseline serum adrenocorticotropic hormone (ACTH; 16.3 +/- 2.4 pg/ml) and cortisol levels (8 +/- 1.5 micrograms/dl). Galanin also determined a slight reduction in both the ACTH (peak 27 +/- 8 pg/ml) and cortisol (peak 13.8 +/- 1.3 micrograms/dl) responses to CRH. Baseline and releasing hormone-stimulated secretions of prolactin, thyroid-stimulating hormone, LH, and follicle-stimulating hormone were not altered by galanin. Our data suggest a physiological role for the neuropeptide galanin in the regulation of GH secretion in humans.(ABSTRACT TRUNCATED AT 250 WORDS)

The growth hormone (GH) releasing effect of GH-releasing hormone (GHRH) and galanin, a 29-amino acid peptide widely distributed in mammalian CNS, was investigated in cultured rat pituitary tumor cells (GH 1 ) as compaerd to normal rat somatotrophs. GHRH stimulated dose-dependently GH secretion in normal somatotrophs but did not affect GH secretion in GH 1 cells. Galanin (1–10 μM) stimulated GH release in a concentration-dependent manner, but with lower potency as compared to GHRH, in normal rat pituitaries but was inhibitory in rat GH 1 cells. The results of this study indicate that while galanin has the ability to stimulate GH release from dispersed pituitary cells of normal rats it has potent direct inhibitory effects on GH release from tumor rat cells.

Galanin elicits growth hormone (GH) secretion in normal man but may cause a paradoxical fall of GH in acromegaly. The aim of our study was to investigate the effects of long-term treatment with bromocriptine on the galanin-induced GH decrease in acromegalic subjects. Six acromegalic patients (5F, 1M) chronically treated with bromocriptine underwent in randomized order: (i) iv infusion of 100 ml saline from 0 to 45 min and (ii) iv infusion of synthetic porcine galanin (0.5 mg in 100 ml saline) from 0 to 45 min. In acromegalic patients, GH values fell from baseline (10.5 +/- 2.7 micrograms/l) to a mean nadir of 6.9 +/- 2.2 micrograms/l after galanin infusion (57.8 +/- 9.4% vs basal levels). Saline infusion did not cause any change in circulating GH levels. The mean change in GH values with respect to baseline after galanin in these subjects significantly differed from that observed after saline from time 15 to 90 min. Serum prolactin levels were not significantly affected by galanin. Our results confirm that the dose of galanin capable of increasing plasma GH levels in normal subjects can decrease GH values in patients with acromegaly. Moreover, our data show that this paradoxical GH decrease induced by galanin can also be observed in patients chronically treated with bromocriptine. Therefore, the paradoxical decreasing effect of galanin on plasma GH levels in acromegaly seems not to be mediated via dopaminergic pathways.

The aim of our study was to analyze the effects of sex and age on the GH response to galanin infusion in healthy subjects. We have studied 12 young (age,40 yr) nonobese healthy volunteers [6 females: age, 31.0 +/- 2.5 yr; body mass index (BMI), 21.6 +/- 0.9 kg/m2; 6 males: age, 29.2 +/- 1.4 yr; BMI, 23.3 +/- 0.4 kg/m2] and 11 old (age,65 yr) healthy subjects (5 females: age, 83.8 +/- 3.8 yr; BMI, 23.4 +/- 1.4 kg/m2; 6 males: age, 79.7 +/- 4.6 yr; BMI, 23.3 +/- 0.2 kg/m2). All subjects received an infusion of synthetic porcine galanin (500 micrograms, iv) in 100 mL saline from 0-45 min. Blood samples for GH measurement were drawn at -15, 0, 15, 30, 45, 60, 90, and 120 min. The GH peaks after galanin treatment in young females (11.9 +/- 2.9 micrograms/L) were significantly (P0.05) higher than those in the young males (5.1 +/- 1.8 micrograms/L). Old males showed significantly higher peak GH levels after galanin treatment (8.6 +/- 3.1 micrograms/L) than old females (2.4 +/- 0.6 micrograms/L). The GH peaks and areas under the curve after galanin treatment were significantly (P0.05) higher in young than in old females. On the contrary, no significant differences were observed after galanin treatment in young and old males. The magnitude of galanin-induced GH secretion significantly correlated with estradiol levels in young women. Our data seem to suggest that circulating estrogen levels play a crucial permissive role in galanin-induced GH secretion in humans.

In patients with acromegaly, circulating growht hormone (GH) levels and GH responses to GH-releasing hormone (GHRH) are decreased by long-term administration of pharmacological doses of glucocorticoids. The aim of our study was to investigate the acute effects of intravenous (IV) infusion of hydrocortisone combined either with saline or arginine infusion on circulating GH levels in acromegaly. We studied five adult patients with acromegaly, two men and three women aged 54.6 ± 4 years having a body mass index of 25.9 ± 1.2 kg/m 2 . On two randomized occasions, patients underwent a bolus IV injection of 100 mg hydrocortisone succinate at time 0 followed by a 120-minute IV infusion of 250 mg hydrocortisone in 250 mL saline, combined with a 90-minute (from −15 to 75 minutes) IV infusion of (1) 60 g arginine hydrochloride in 200 mL saline, or (2) 200 mL saline. In all of the acromegalic patients during the infusion of hydrocortisone alone, serum GH levels clearly decreased (nadir range, 26.4% to 68.1%) with respect to GH levels before hydrocortisone administration (mean of time −15 and 0, basal level), with a nadir between 90 and 180 minutes after the beginning of the infusion. After arginine pretreatment, GH levels were significantly enhanced compared with levels attained with hydrocortisone saline, and they were also significantly increased (peak, 167.5% ± 27.7%) with respect to basal levels. Our data show that arginine blocks the inhibitory effect of acute and sustained hypercortisolism on circulating GH levels in acromegaly. It also seems likely that in both acromegalic patients and normal subjects, acute increases in serum cortisol levels may cause an enhancement of hypothalamic somatostatin secretion, which in turn may be responsible for the glucocorticoid-mediated GH inhibition.

Thrombohemorrhagic complications are a major cause of morbidity and mortality in patients with essential thrombocythemia (ET) and polycythemia vera (PV), The pathogenesis of these complications is not completely clarified, Several studies have described abnormalities of red blood cells and platelets in these patients. However, no studies are available on changes in the polymorphonuclear leukocytes (PMNs), which can play an important role in the activation of the hemostatic System. In patients with ET (n = 37) and PV (n = 34), a series of PMN activation parameters (PMN membrane CD11b and leukocyte alkaline phosphatase [LAP] antigen expression, cellular elastase content, plasma elastase, and myeloperoxidase levels) was evaluated simultaneously with the levels of plasma markers of endothelial damage (thrombomodulin and von Willebrand factor antigen) and hypercoagulation (thrombin-antithrombin complex, prothrombin fragment 1 + 2, and D-dimer). The results show the occurrence of PMN activation in both groups of patients compared with a control group of healthy subjects. An increase in CD11b and LAP expression by PMN membrane was observed, together with a significant increase in cellular elastase content, plasma elastase, and myeloperoxidase levels. In addition, patients had high plasma levels of endothelial and hypercoagulation markers compared with controls, For the first time, these data show that in ET and PV, 2 hematologic conditions that place patients at increased risk for thrombosis, an in vivo leukocyte activation occurs and is associated with laboratory signs of endothelium and coagulation system activation. (C) 2000 by The American Society of Hematology. Thrombohemorrhagic complications are a major cause of morbidity and mortality in patients with essential thrombocythemia (ET) and polycythemia vera (PV). The pathogenesis of these complications is not completely clarified. Several studies have described abnormalities of red blood cells and platelets in these patients. However, no studies are available on changes in the polymorphonuclear leukocytes (PMNs), which can play an important role in the activation of the hemostatic system. In patients with ET (n = 37) and PV (n = 34), a series of PMN activation parameters (PMN membrane CD11b and leuko-cyte alkaline phosphatase [LAP] antigen expression, cellular elastase content, plasma elastase, and myeloperoxidase levels) was evaluated simultaneously with the levels of plasma markers of endothelial damage (thrombomodulin and von Willebrand factor antigen) and hypercoagulation (thrombin-antithrombin complex, prothrombin fragment 1 + 2, and D-dimer). The results show the occurrence of PMN activation in both groups of patients compared with a control group of healthy subjects. An increase in CD11b and LAP expression by PMN membrane was observed, together with a significant increase in cellular elastase content, plasma elastase, and myeloperoxidase levels. In addition, patients had high plasma levels of endothelial and hypercoagulation markers compared with controls. For the first time, these data show that in ET and PV, 2 hematologic conditions that place patients at increased risk for thrombosis, an in vivo leukocyte activation occurs and is associated with laboratory signs of endothelium and coagula-tion system activation. © 2000 by The American Society of Hematology.