Acute toxicities in proton therapy for head and neck cancer - A matched analysis of the DAHANCA 35 feasibility study.

Background and purpose: As preparation for a national randomized study comparing proton radiotherapy to photon radiotherapy, DAHANCA 35, we performed a non-randomized pilot study to investigate patient selection, logistics, planning, and treatment delivery. With the present study, as a comprehensive safety analysis, we want to compare toxicity during and up to two months after therapy to a historically matched group of patients treated with photon radiotherapy. Materials and methods: 62 patients treated with protons were matched to 124 patients who received photon treatment outside a protocol. Available data were retrieved from the DAHANCA database. Patients were matched on treatment centre, concurrent chemotherapy, tumour site, stage, p16 status for oropharynx cancers. Selection of patients for proton therapy was based on comparative treatment plans with a NTCP reduction for dysphagia and xerostomia at six months. Results: Baseline characteristics between groups were well balanced, except for the type of drug used concurrently; more photon patients received Carboplatin (21.2 % vs 5.8 %, p = 0.01). Proton therapy was associated with significantly less weight loss at the end of treatment, mean weight loss of 3 % for protons and 5 % for photons (p < 0.001). There were more grade 3 skin reactions and grade 3 mucositis after proton treatment compared with photons at the end of treatment, Risk Ratio (RR) 1.9 (95 % CI: 1.01-3.5, p = 0.04) and RR 1.5 (95 % CI: 1.3-1.7, p < 0.001), respectively. All differences resolved at follow up two months after treatment. There were no significant differences between groups on opioid use, use of feeding tubes, or hospitalization during the observation period. Conclusion: Proton treatment resulted in excess objective mucositis and dermatitis, which was transient and did not seem to negatively influence weight or treatment compliance and intensity. Selection bias was likely especially since NTCP models were used for selection of proton treatment and photon treated patients were matched manually. We are currently including patients in a randomized controlled trial.

Fractional exhaled nitric oxide as a potential biomarker for radiation pneumonitis in patients with non-small cell lung cancer: A pilot study.

INTRODUCTION: The aim of the study was to investigate repetitive fractional exhaled nitric oxide (FeNO) measurements during high-dose radiation therapy (HDRT) and to evaluate the use of FeNO to predict symptomatic radiation pneumonitis (RP) in patients being treated for non-small cell lung cancer (NSCLC). MATERIALS AND METHODS: A total of 50 patients with NSCLC referred for HDRT were enrolled. FeNO was measured at baseline, weekly during HDRT, one month- and every third month after HDRT for a one-year follow-up period. The mean FeNO(visit 0-6) was calculated using the arithmetic mean of the baseline and weekly measurements during HDRT. Patients with grade ≥ 2 of RP according to the Common Terminology Criteria for Adverse Events (CTCAE) were considered symptomatic. RESULTS: A total of 42 patients completed HDRT and weekly FeNO measurements. Grade ≥ 2 of RP was diagnosed in 24 (57%) patients. The mean FeNO(visit 0-6) ±â€¯standard deviation in patients with and without RP was 15.0 ±â€¯7.1 ppb (95%CI: 12.0-18.0) and 10.3 ±â€¯3.4 ppb (95%CI: 8.6-11.9) respectively with significant differences between the groups (p = 0.0169, 95%CI: 2.3-2.6). The leave-one-out cross-validated cut-off value of the mean FeNO(visit 0-6) ≥ 14.8 ppb was predictive of grade ≥ 2 RP with a specificity of 71% and a positive predictive value of 78%. CONCLUSIONS: The mean FeNO(visit 0-6) in patients with symptomatic RP after HDRT for NSCLC was significantly higher than in patients without RP and may serve as a potential biomarker for RP.

Effects of RYGB on energy expenditure, appetite and glycaemic control: a randomized controlled clinical trial.

OBJECTIVES: Increased energy expenditure (EE) has been proposed as an important mechanism for weight loss following Roux-en-Y gastric bypass (RYGB). However, this has never been investigated in a controlled setting independent of changes in energy balance. Similarly, only few studies have investigated the effect of RYGB on glycaemic control per se. Here, we investigated the effect of RYGB on EE, appetite, glycaemic control and specific signalling molecules compared with a control group in comparable negative energy balance. SUBJECTS/METHODS: Obese normal glucose-tolerant participants were randomized to receive RYGB after 8 (n=14) or 12 weeks (n=14). The protocol included a visit at week 0 and three visits (weeks 7, 11 and 78) where 24-h EE, appetite and blood parameters were assessed. Participants followed a low-calorie diet from weeks 0-11, with those operated at week 12 serving as a control group for those operated at week 8. RESULTS: Compared with controls, RYGB-operated participants had lower body composition-adjusted 24-h EE and basal EE 3 weeks postoperatively (both P<0.05) but EE parameters at week 78 were not different from preoperative values (week 7). Surgery changed the postprandial response of glucagon-like peptide-1 (GLP-1), peptide YY3-36 (PYY), ghrelin, cholecystokinin, fibroblast growth factor-19 and bile acids (all P<0.05). Particularly, increases in GLP-1, PYY and decreases in ghrelin were associated with decreased appetite. None of HOMA-IR (homeostasis model assessment-estimated insulin resistance), Matsuda index, the insulinogenic index, the disposition index and fasting hepatic insulin clearance were different between the groups, but RYGB operated had lower fasting glucose (P<0.05) and the postprandial glucose profile was shifted to the left (P<0.01). CONCLUSIONS: Our data do not support that EE is increased after RYGB. More likely, RYGB promotes weight loss by reducing appetite, partly mediated by changes in gastrointestinal hormone secretion. Furthermore, we found that the early changes in glycaemic control after RYGB is to a large extent mediated by caloric restriction.

Maternal fish oil supplementation during lactation is associated with reduced height at 13 years of age and higher blood pressure in boys only.

Dietary long-chain n-3 PUFA (n-3 LCPUFA) in infancy may have long-term effects on lifestyle disease risk. The present follow-up study investigated whether maternal fish oil (FO) supplementation during lactation affected growth and blood pressure in adolescents and whether the effects differed between boys and girls. Mother-infant pairs (n 103) completed a randomised controlled trial with FO (1·5 g/d n-3 LCPUFA) or olive oil (OO) supplements during the first 4 months of lactation; forty-seven mother-infant pairs with high fish intake were followed-up for 4 months as the reference group. We also followed-up 100 children with assessment of growth, blood pressure, diet by FFQ and physical activity by 7-d accelerometry at 13·5 (sd 0·4) years of age. Dried whole-blood fatty acid composition was analysed in a subgroup (n 49). At 13 years of age, whole-blood n-3 LCPUFA, diet, physical activity and body composition did not differ between the three groups. The children from the FO group were 3·4 (95 % CI 0·2, 6·6) cm shorter (P=0·035) than those from the OO group, and tended to have less advanced puberty (P=0·068), which explained the difference in height. There was a sex-specific effect on diastolic blood pressure (P sex×group=0·020), which was driven by a 3·9 (95 % CI 0·2, 7·5) mmHg higher diastolic blood pressure in the FO compared with the OO group among boys only (P=0·041). Our results indicate that early n-3 LCPUFA intake may reduce height in early adolescence due to a delay in pubertal maturation and increase blood pressure specifically in boys, thereby tending to counteract existing sex differences.

No effect of high-dose atorvastatin on leukotriene B4 formation from neutrophils in patients treated with coronary bypass surgery: a randomized placebo-controlled double-blinded trial with a crossover design.

Inflammation plays a pivotal role in the pathophysiology of cardiovascular disease, (CVD) and leukotrienes may play a role in atherogenesis. Statins reduce mortality from CVD by reducing LDL cholesterol and potentially by other (pleiotropic) mechanisms. The aim of this study was to investigate if atorvastatin exerts an anti-inflammatory effect by reducing leukotriene B4 (LTB4) formation from stimulated neutrophils in patients treated with coronary artery bypass grafting. The study was a randomized, placebo-controlled, double-blinded crossover study. Patients (n=80) were allocated to 80 mg atorvastatin or placebo for 6 weeks before crossing over to the opposite treatment for another 6 weeks. There was no significant correlation between baseline LDL cholesterol levels on formation of LTB4, and atorvastatin had no effect on LTB4 formation. Hence, this study does not support any effect of atorvastatin on LTB4 formation as part of the explanation for its beneficial effect on CVD.

Development of neuromodulation treatments in a large animal model--do neurosurgeons dream of electric pigs?

The Göttingen minipig has been established as a translational research animal for neurological and neurosurgical disorders. This animal has a large gyrencephalic brain suited for examination at sufficient resolution with conventional clinical scanning modalities. The large brain, further, allows use of standard neurosurgical techniques and can accommodate clinical neuromodulatory devises such as deep brain stimulation (DBS) electrodes and encapsulated cell biodelivery devices making the animal ideal for basic scientific studies on neuromodulation mechanisms and preclinical tests of new neuromodulation technology for human use. The use of the Göttingen minipig is economical and does not have the concerns of the public associated with the experimental use of primates, cats, and dogs, thus providing a cost-effective research model for translation of rodent data before clinical trials are initiated.

Neuromodulation in a minipig MPTP model of Parkinson disease.

Large animal neuroscience enables the use of conventional clinical brain imagers and the direct use and testing of surgical procedures and equipment from the human clinic. The greater complexity of the large animal brain additionally enables a more direct translation to human brain function in health and disease. Economical, ethical, scientific and practical issues may on the other hand hamper large animal neuroscience. Large animal neuroscience should therefore either be performed in order to examine large animal species dependent problems or to complement promising small animal basic studies by constituting an intermediate research system, bridging small animal CNS research to the human CNS. We have, accordingly, during the last ten years used the Gottingen minipig to examine neuromodulatory treatment modalities such as stem cell transplantation and deep brain stimulation directed towards Parkinson disease. This has been accomplished by the development of a MPTP-based large animal model of Parkinson disease in the Gottingen minipig and the development of stereotaxic and surgical approaches needed to manipulate the Gottingen minipig CNS. The instituted changes in the CNS can be evaluated in the live animal by brain imaging (PET and MR), cystometry, gait analysis, neurological evaluation and by post mortem examination based on histology and stereological analysis.

Intramammary infusion of leptin decreases proliferation of mammary epithelial cells in prepubertal heifers.

High energy intake and excessive body fatness impair mammogenesis in prepubertal ruminants. High energy intake and excessive fatness also increase serum leptin. Our objective was to determine if an infusion of leptin decreases proliferation of mammary epithelial cells of prepubertal heifers in vivo. Ovine leptin at 100 microg/ quarter per d with or without 10 microg of insulin-like growth factor (IGF)-I was infused via the teat canal into mammary glands of prepubertal dairy heifers; contralateral quarters were used as controls. After 7 d of treatment, bromodeoxyuridine was infused intravenously and heifers were slaughtered approximately 2 h later. Tissue from 3 regions of the mammary parenchyma was collected and immunostained for bromodeoxyuridine (BrdU), proliferating cell nuclear antigen (Ki-67), and caspase-3. Leptin decreased the number of mammary epithelial cells in the S-phase of the cell cycle by 48% in IGF-I-treated quarters and by 19% in saline-treated quarters. Leptin did not alter the number of mammary epithelial cells within the cell cycle, as indicated by Ki-67 labeling. Caspase-3 immunostaining within the mammary parenchyma was very low in these heifers, but leptin significantly increased labeling in saline-treated quarters. Leptin enhanced SOCS-3 expression in IGF-I-treated quarters but did not alter SOCS-1 or SOCS-5 expression. We conclude that a high concentration of leptin in the bovine mammary gland reduces proliferation of mammary epithelial cells. The reduced proliferation is accompanied by an increase in SOCS-3 expression, suggesting a possible mechanism for leptin inhibition of IGF-I action. Whether leptin might be a physiological regulator of mammogenesis remains to be determined.

Effects of feeding prepubertal heifers a high-energy diet for three, six, or twelve weeks on mammary growth and composition.

The experimental objective was to determine the effects of feeding prepubertal dairy heifers a high-energy diet for 3, 6, or 12 wk on mammary growth and composition. Holstein heifers (age = 11 wk; body weight = 107 +/- 1 kg) were assigned to 1 of 4 treatments (n = 16/ treatment). The treatment period lasted 12 wk and treatments were H0 (low-energy diet fed for 12 wk, with no weeks on the high-energy diet); H3 (low-energy diet fed for 9 wk, followed by the high-energy diet for 3 wk); H6 (low-energy diet fed for 6 wk, followed by the high-energy diet for 6 wk); and H12 (high-energy diet for all 12 wk). The low- and high-energy diets were formulated to achieve 0.6 and 1.2 kg of average daily gain, respectively. Heifers were slaughtered at 23 wk of age and mammary tissue was collected. A longer duration of feeding the high-energy diet increased total mass of the mammary gland, extraparenchymal fat, and intraparenchymal fat, but did not alter the mass of fat-free parenchymal tissue. When adjusted for carcass weight to reflect differences in physical maturity, the mass of fat-free parenchymal tissue decreased in a linear fashion with a longer duration on the high-energy diet. Total masses of mammary parenchymal DNA and RNA were not different. However, after adjustment for carcass weight, the masses of DNA and RNA decreased as heifers were fed the high-energy diet for a longer duration. The percentages of epithelium, stroma, and lumen, the number of epithelial structures, and the developmental scores of mammary parenchymal tissue were not different among treatments. However, the percentage of proliferating epithelial cells in the terminal ductal units, as indicated by Ki-67 labeling, decreased as heifers were fed the high-energy diet for a longer duration. We concluded that feeding prepubertal heifers a high-energy diet for a longer duration resulted in a linear decrease in both the percentage of mammary epithelial cells that were proliferating and in the mass of fat-free mammary parenchyma per unit of carcass. High-energy feeding hastens puberty and, in this study, decreased mammary epithelial cell proliferation in areas of active ductal expansion. These data are consistent with the idea that feeding heifers a high-energy diet will reduce mammary parenchymal mass at puberty.

Short communication: intramammary infusion of IGF-I increases bromodeoxyuridine labeling in mammary epithelial cells of prepubertal heifers.

When dairy heifers are fed to gain more than 900 g of body weight/d, they have less mammary parenchymal DNA at puberty but more insulin-like growth factor-I (IGF-I) in serum. This negative relationship between serum IGF-I concentration and mammary epithelial cell proliferation is in disagreement with the extensively reported role of IGF-I as a stimulator of mammary epithelial cell proliferation. Despite the large body of evidence suggesting that an increase in IGF-I concentration should lead to an increase in mammary epithelial cell proliferation of prepubertal heifers, it had not been previously tested. Our objective was to determine if intramammary infusions of IGF-I would stimulate mammogenesis in prepubertal heifers in vivo. After 7 d of treatment, bromodeoxyuridine was infused intravenously and heifers were slaughtered 3 h later. Samples from 3 regions of the mammary parenchyma were collected, fixed, sliced, and incubated with bromodeoxyuridine monoclonal antibody to identify cells in the S-phase of the cell cycle. Intramammary infusion of IGF-I increased the percentage of epithelial cells in the S-phase by 52% (6.4 vs. 4.2%, +/- 0.3%). Proliferation was similar in all 3 parenchymal regions, and the response to IGF-I was similar in each region. We conclude that local IGF-I increases proliferation of mammary parenchymal epithelial cells in prepubertal heifers.

Effect of increasing energy and protein intake on mammary development in heifer calves.

The objective of this study was to determine if increased energy and protein intake from 2 to 14 wk of age would affect mammary development in heifer calves. At 2 wk of age, Holstein heifer calves were assigned to 1 of 4 treatments in a 2 x 2 factorial arrangement with 2 levels of protein and energy intake (moderate, M; high, H) in period 1 (2 to 8 wk of age) and 2 levels of protein and energy intake (low, L; high, H) in period 2 (8 to 14 wk of age), so that mean initial body weights were approximately equal for all 4 treatments (ML, MH, HL, and HH). The M diet in period 1 consisted of a standard milk replacer (21.3% CP, 21.3% fat) fed at 1.1% of BW on a DM basis and a 16.5% CP grain mix fed at restricted intake to promote 400 g of daily gain, whereas the L diet in period 2 consisted only of the grain mix. The H diet in period 1 consisted of a high-protein milk replacer (30.3% CP, 15.9% fat) fed at 2.0% of body weight on a DM basis and a 21.3% CP grain mix available ad libitum. In period 2, the H diet consisted of just the 21.3% grain mix. Calves were gradually weaned from milk replacer by 7 wk and slaughtered at 8 (n = 11) or 14 wk of age (n = 41). Parenchyma from the distal region, midgland, and proximal region relative to the teat from one half of the udder was collected, fixed, and embedded in paraffin. The other half of the gland was used to determine parenchymal mass, protein, fat, DNA, RNA, and extraparenchymal mass. Total parenchymal tissue, parenchymal DNA, parenchymal RNA, and concentrations of DNA and RNA were higher for calves on the H diet during period 1, but were not affected by diet during period 2. Parenchymal fat percentage was increased by the H diet during period 2. The H diet increased extraparenchymal fat during both periods. The area of parenchyma occupied by epithelium was not affected by treatment, but at the end of period 2, the percentage of proliferating epithelial cells as indicated by Ki67, an marker of cell proliferation, expression was greater for calves on the M diet in period 1 compared with calves on the H diet in period 1. Diets did not influence parenchymal protein percentage or the ratio of RNA to DNA. Higher energy and protein intake from 2 to 8 wk of age increased parenchymal mass and parenchymal DNA and RNA in mammary glands of heifer calves without increasing deposition of parenchymal fat. Diet also influenced histological development of mammary parenchyma and subsequent proliferation of ductal epithelial cells. Implications of these effects for future milk production potential are unknown.

Routine X-ray control of upper central venous lines: Is it necessary?

BACKGROUND: Insertion of central venous catheters (CVCs) is a procedure associated with a varying risk of complications, depending on the setting and the skill of the clinician who undertakes the procedure. The aim of this study was to monitor the complication rate of CVC insertion and evaluate the value of routine chest X-ray control. METHODS: Anesthesiologists at eight hospitals filled in a questionnaire immediately after insertion of a CVC. The post-procedural clinical evaluation, including expected complications, was compared to actual radiological findings. Chest X-ray was ordered by the anesthesiologist, and described by staff radiologists. RESULTS: The clinicians had from 2 months to 30 years of experience as anesthesiologists, and trainees inserted 34% and specialists 66% of the catheters, using landmark techniques. Over a period of 2 months, 473 CVC-insertion procedures were included in the investigation. Two patients (0.4%) had a pneumothorax: one was among the 11 cases in which the clinician suspected complications after the procedure, and another was found in a high-risk patient 13 h after CVC insertion. Both patients were treated successfully with chest tubes. The favorite approach was right vena jugularis interna with 324 (69%) catheters; of these patients one had a pneumothorax, catheter-tip placement was correctly predicted in 317 (97%), and no catheters were repositioned. CONCLUSION: In the hands of trained clinicians, insertion of CVCs is a safe procedure. We found no value of routine X-ray control and omission of routine chest X-ray must be considered.

Evidence for a local effect of leptin in bovine mammary gland.

On average, high-energy diets promoting body growth rates above 1 kg/d before puberty impair mammary development by 15 to 20% in cattle. We hypothesized that leptin, a protein produced by adipocytes, mediates the inhibitory effect of high-energy diets on mammary development. Therefore, our objectives were to determine the effect of leptin on mammary epithelial cell proliferation, and the distribution of mRNA for two leptin receptor isoforms in prepubertal bovine mammary glands and other peripheral tissues. Addition of leptin to culture media containing either 5 ng/ml of insulin-like growth factor-I (IGF-I) or 1% fetal bovine serum decreased DNA synthesis of a bovine mammary epithelial cell line (MAC-T) in a dose-dependent manner. The minimal doses of leptin that decreased IGF-I- and fetal bovine serum-stimulated cell proliferation were 64 and 1 ng/ml, respectively. In addition, we determined that MAC-T cells and isolated bovine mammary epithelial cells express the long form of leptin receptor (Ob-Rb) mRNA. Ob-Rb mRNA was detected in all bovine tissues examined. In contrast with reports on other species, mRNA expression of the short form of leptin receptor (Ob-Ra) was detected only in bovine liver, pituitary body, and spleen. These results support the concept that leptin mediates the inhibitory effect of high-energy diets on mammary development.

Maxi K+ channels co-localised with CFTR in the apical membrane of an exocrine gland acinus: possible involvement in secretion.

The primary secretion formed in various exocrine glands has a [K+] 2-5 times that of plasma. In this study we measured the transepithelial flux of 36Cl-, 22Na+ and 42K+ across the frog skin and applied the single-channel patch-clamp technique to the apical membrane of frog skin gland acini to investigate the pathway taken by K+ secreted by the glands. Transepithelial K+ secretion was active and was driven by a larger force than the secretion of Na+. When driving Na+ through the epithelium by clamping the transepithelial potential to 100 mV (apical solution reference), blockers of cellular secretion (apical 5-nitro-2-(3-phenylpropylamino)benzoate or basolateral quinine or furosemide) decreased K+ secretion but left Na+ secretion unaffected. We conclude that K+ follows a transcellular pathway across the epithelium. Patch-clamp analysis of the apical membrane of microdissected gland acini revealed a population of voltage- and calcium-activated K+ channels of the maxi K+ type. In cell-attached patches these channels were activated by membrane potential depolarisation or exposure to prostaglandin E2 and had a permeability of 3.6 +/- 0.3 x 10(-13) cm3 s-1, giving a calculated conductance of 170 pS with 125 mM K+ on both sides of the membrane. In inside-out patches the channels were activated by increasing intracellular [Ca2+] from 10(-7) to 10(-6) M and were blocked by Ba2+ added to the cytoplasmic side. Exposure of inside-out patches containing the maxi K+ channel to ATP on the inside activated cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels, confirming that both channels are co-localised to the apical membrane. We interpret these findings in terms of a model where transepithelial NaCl secretion can be supported in part by an apical K+ conductance.

Effect of alpha1-adrenergic stimulation of Cl- secretion and signal transduction in exocrine glands (Rana esculenta).

In the present work, the effect of stimulation of alpha-adrenergic receptors on Cl- secretion via exocrine frog skin glands was investigated. The alpha-adrenergic stimulation was performed by addition of the adrenergic agonist noradrenaline in the presence of the beta-adrenergic antagonist propranolol. In the presence of propranolol, noradrenaline had no effect on the cellular cAMP content. The Cl- secretion was measured as the amiloride-insensitive short circuit current (ISC). Addition of noradrenaline induced a biphasic increase in the ISC. The increase in ISC coincided with an increase in the net 36Cl- secretion. The noradrenaline-induced increase in ISC was dose-dependent with an EC50 of 13 +/- 0.3 microM. Epifluorescence microscopic measurements of isolated, fura-2-loaded frog skin gland acini were used to characterize the intracellular calcium ([Ca2+]i) response. Application of noradrenaline induced a biphasic [Ca2+]i response, which was dose-dependent with an EC50 of 11 +/- 6 microM. The Ca2+ plateau unlike the peak-response was sensitive to removal of Ca2+ from the extracellular medium. The noradrenaline-induced increase in the Cl- secretion as well as in [Ca2+]i was sensitive to the alpha1-adrenergic antagonist prazosine. Ryanodine and caffeine had no effect on [Ca2+]i indicating that the release was independent of ryanodine-sensitive Ca2+ stores. Noradrenaline mediated a significant increase in the cellular inositol 1,4,5-trisphosphate (IP3) content suggesting that the signal transduction pathway leading to the noradrenaline-induced increase in Ca2+ involved IP3 and a release of Ca2+ from IP3-sensitive stores.

Trichosanthin interacts with and enters cells via LDL receptor family members.

The type-I ribosome-inactivating protein trichosanthin displays selective cytotoxicity, suggesting specific mechanisms for entry into cells. Here we show that trichosanthin binds specifically to the endocytic receptors LRP and megalin, and that binding as well as uptake into cells is inhibited by the receptor-associated protein (RAP). The results suggest that the known abortifacient and renotoxic actions of trichosanthin are caused by LRP-mediated uptake in trophoblasts and megalin-mediated uptake in proximal tubule epithelial cells, respectively.

Effect of carbachol and prostaglandin E2 on chloride secretion and signal transduction in the exocrine glands of frog skin (Rana esculenta).

Carbachol (CCH) increased the short-circuit current across frog skin glands in a biphasic manner, which coincided with an increase in the transepithelial Cl- net flux. CCH also induced a biphasic increase in [Ca2+]i. Both these responses were mediated via muscarinic receptors. The plateau phase of the CCH-induced Cl- secretion was modestly inhibited by indomethacin and unaffected by tetrodotoxin or tetrodotoxin plus indomethacin, indicating that CCH can increase Cl- secretion directly via receptors on the secretory cells. Prostaglandin E2 (PGE2) increased secretion and cAMP production, indicating expression of EP2 and/or EP4 receptors. PGE2 failed to increase [Ca2+]i ruling out involvement of EP1 receptors. The secretory response to CCH was potentiated by prestimulation with PGE2, and it was investigated whether this potentiation is caused by interaction at the level of the messengers involved. Stimulation by CCH plus PGE2 failed to stimulate cAMP production further than PGE2 alone. Addition of PGE2 during the CCH-elevated [Ca2+]i plateau phase in most cases reduced the level of [Ca2+]i. These data show that the synergy between CCH and PGE2 is not based on interactions at the intracellular messenger level.

Increased mortality associated with growth hormone treatment in critically ill adults.

BACKGROUND: The administration of growth hormone can attenuate the catabolic response to injury, surgery, and sepsis. However, the effect of high doses of growth hormone on the length of stay in intensive care and in the hospital, the duration of mechanical ventilation, and the outcome in critically ill adults who are hospitalized for long periods is not known. METHODS: We carried out two prospective, multicenter, double-blind, randomized, placebo-controlled trials in parallel involving 247 Finnish patients and 285 patients in other European countries who had been in an intensive care unit for 5 to 7 days and who were expected to require intensive care for at least 10 days. The patients had had cardiac surgery, abdominal surgery, multiple trauma, or acute respiratory failure. The patients received either growth hormone (mean [+/-SD] daily dose, 0.10 +/- 0.02 mg per kilogram of body weight) or placebo until discharge from intensive care or for a maximum of 21 days. RESULTS: The in-hospital mortality rate was higher in the patients who received growth hormone than in those who did not (P<0.001 for both studies). In the Finnish study, the mortality rate was 39 percent in the growth hormone group, as compared with 20 percent in the placebo group. The respective rates in the multinational study were 44 percent and 18 percent. The relative risk of death for patients receiving growth hormone was 1.9 (95 percent confidence interval, 1.3 to 2.9) in the Finnish study and 2.4 (95 percent confidence interval, 1.6 to 3.5) in the multinational study. Among the survivors, the length of stay in intensive care and in the hospital and the duration of mechanical ventilation were prolonged in the growth hormone group. CONCLUSIONS: In patients with prolonged critical illness, high doses of growth hormone are associated with increased morbidity and mortality.

alpha-synuclein binds to Tau and stimulates the protein kinase A-catalyzed tau phosphorylation of serine residues 262 and 356.

alpha-Synuclein has been implicated in the pathogenesis of several neurodegenerative disorders based on the direct linking of missense mutations in alpha-synuclein to autosomal dominant Parkinson's disease and its presence in Lewy-like lesions. To gain insight into alpha-synuclein functions, we have investigated whether it binds neuronal proteins and modulates their functional state. The microtubule-associated protein tau was identified as a ligand by alpha-synuclein affinity chromatography of human brain cytosol. Direct binding assays using (125)I-labeled human tau40 demonstrated a reversible binding with a IC(50) about 50 pM. The interacting domains were localized to the C terminus of alpha-synuclein and the microtubule binding region of tau as determined by protein fragmentation and the use of recombinant peptides. High concentrations of tubulin inhibited the binding between tau and alpha-synuclein. Functionally, alpha-synuclein stimulated the protein kinase A-catalyzed phosphorylation of tau serine residues 262 and 356 as determined using a phospho-epitope-specific antibody. We propose that alpha-synuclein modulates the phosphorylation of soluble axonal tau and thereby indirectly affects the stability of axonal microtubules.

The carboxy-terminal domain of the receptor-associated protein binds to the Vps10p domain of sortilin.

Binding of the receptor-associated protein (RAP) to the newly identified putative sorting receptor, sortilin, was analyzed by surface plasmon resonance analysis of recombinant RAP and sortilin domains and compared with binding to megalin and low density lipoprotein receptor-related protein (LRP). The data show that the RAP-binding site in sortilin is localized in the cysteine-rich lumenal part homologous to yeast vacuolar protein-sorting 10 protein (Vps10p), and the sortilin-binding site in RAP is localized in the carboxy-terminal domain III of the three homologous domains in RAP. Whereas sortilin bound only RAP domain III, megalin and LRP bound all RAP domains with the functional affinity order: domain III >domain I > domain II.