Improvement of Storage Medium for Cultured Human Retinal Pigment Epithelial Cells Using Factorial Design.

Storage of human retinal pigment epithelium (hRPE) can contribute to the advancement of cell-based RPE replacement therapies. The present study aimed to improve the quality of stored hRPE cultures by identifying storage medium additives that, alone or in combination, contribute to enhancing cell viability while preserving morphology and phenotype. hRPE cells were cultured in the presence of the silk protein sericin until pigmentation. Cells were then stored for 10 days in storage medium plus sericin and either one of 46 different additives. Individual effects of each additive on cell viability were assessed using epifluorescence microscopy. Factorial design identified promising additive combinations by extrapolating their individual effects. Supplementing the storage medium with sericin combined with adenosine, L-ascorbic acid and allopurinol resulted in the highest cell viability (98.6 ± 0.5%) after storage for three days, as measured by epifluorescence microscopy. Flow cytometry validated the findings. Proteomics identified 61 upregulated and 65 downregulated proteins in this storage group compared to the unstored control. Transmission electron microscopy demonstrated the presence of melanosomes after storage in the optimized medium. We conclude that the combination of adenosine, L-ascorbic acid, allopurinol and sericin in minimal essential medium preserves RPE pigmentation while maintaining cell viability during storage.

Hypophosphatemic osteomalacia: an unusual clinical presentation of multiple myeloma.

An unusual case of a 75-year-old man is presented who had multiple stress fractures due to adult onset hypophosphatemic osteomalacia, which was the result of Fanconi syndrome, with light chain cast proximal tubulopathy due to multiple myeloma. A 75-year-old man presented with diffuse pain and muscle weakness. He had multiple stress fractures, low serum phosphate, decreased renal tubular reabsorption of phosphate, and normal PTH and FGF23, indicating adult onset hypophosphatemic osteomalacia. Phosphate supplements with calcitriol resulted in clinical recovery and healing of stress fractures. Because of proteinuria, a renal biopsy was performed that revealed Fanconi syndrome with light chain cast proximal tubulopathy and light kappa chains were found in serum and urine. A bone biopsy confirmed the diagnosis of multiple myeloma, and treatment with chemotherapy resulted in cytological and clinical recovery.

Moxifloxacin is not anti-inflammatory in experimental pneumococcal pneumonia.

OBJECTIVES: Anti-inflammatory functions of antibiotics may counteract deleterious hyperinflammation in pneumonia. Moxifloxacin reportedly exhibits immunomodulatory properties, but experimental evidence in pneumonia is lacking. Therefore, we investigated moxifloxacin in comparison with ampicillin regarding pneumonia-associated pulmonary and systemic inflammation and lung injury. METHODS: Ex vivo infected human lung tissue and mice with pneumococcal pneumonia were examined regarding local inflammatory response and bacterial growth. In vivo, clinical course of the disease, leucocyte dynamics, pulmonary vascular permeability, lung pathology and systemic inflammation were investigated. In addition, transcellular electrical resistance of thrombin-stimulated endothelial cell monolayers was quantified. RESULTS: Moxifloxacin reduced cytokine production in TNF-α-stimulated, but not in pneumococci-infected, human lung tissue. In vivo, moxifloxacin treatment resulted in reduced bacterial load as compared with ampicillin, whereas inflammatory parameters and lung pathology were not different. Moxifloxacin-treated mice developed less pulmonary vascular permeability during pneumonia, but neither combination therapy with moxifloxacin and ampicillin in vivo nor examination of endothelial monolayer integrity in vitro supported direct barrier-stabilizing effects of moxifloxacin. CONCLUSIONS: The current experimental data do not support the hypothesis that moxifloxacin exhibits potent anti-inflammatory properties in pneumococcal pneumonia.

Behavioral demonstration of a somatosensory neuroprosthesis.

Tactile sensation is critical for effective object manipulation, but current prosthetic upper limbs make no provision for delivering somesthetic feedback to the user. For individuals who require use of prosthetic limbs, this lack of feedback transforms a mundane task into one that requires extreme concentration and effort. Although vibrotactile motors and sensory substitution devices can be used to convey gross sensations, a direct neural interface is required to provide detailed and intuitive sensory feedback. In light of this, we describe the implementation of a somatosensory prosthesis with which we elicit, through intracortical microstimulation (ICMS), percepts whose magnitude is graded according to the force exerted on the prosthetic finger. Specifically, the prosthesis consists of a sensorized finger, the force output of which is converted into a regime of ICMS delivered to primary somatosensory cortex through chronically implanted multi-electrode arrays. We show that the performance of animals (Rhesus macaques) on a tactile task is equivalent whether stimuli are delivered to the native finger or to the prosthetic finger.

High-dose tirofiban pretreatment reduces the need for bail-out study medication in patients with ST-segment elevation myocardial infarction: results of a subgroup analysis of the On-TIME 2 trial.

OBJECTIVE: This study investigated the outcome of patients who received bail-out study medication and evaluated whether high-dose tirofiban (HDT) pretreatment may reduce the need for bail-out study medication. DESIGN: A prespecified analysis of the multicentre, double-blind, placebo controlled, randomised On-TIME 2 trial. Bail-out use of study medication was predefined and part of the combined clinical end point. PATIENTS: 984 patients excluded from many coronary intervention hospitals in different countries were randomly assigned to HDT or placebo. In the subgroup who received blinded bail-out treatment, patients pretreated with placebo who received bail-out HDT were compared with those pretreated with HDT who received bail-out placebo. Interventions Routine prehospital initiation of HDT versus bail-out use of HDT. MAIN OUTCOME MEASURES: Electrocardiographic and clinical outcome. RESULTS: Blinded bail-out study medication was used in 24% (237/980) of patients, with a higher rate in patients pretreated with placebo: 29% (140/492) versus 20% (97/488), p=0.002. Bail-out versus no bail-out use of study medication was associated with more residual ST deviation (5.5±7.2 vs 3.7±4.8 mm, p=0.005), and worse clinical outcome (major adverse cardiac events (MACE) at 30 days 12.2% vs 5.6%, p<0.001), mainly due to poor outcome in patients who received HDT bail-out. In patients pretreated with HDT who received placebo bail-out study medication, residual ST deviation and clinical outcome did not differ significantly compared with patients who did not receive bail-out medication (4.0±4.6 vs 3.7± 4.8 mm, p=0.703, MACE 7.2% vs 5.6%, p=0.535). CONCLUSIONS: Routine prehospital treatment with HDT significantly reduced the use of blinded bail-out study medication. The need for bail-out therapy was associated with a less favourable outcome. This analysis suggests that routine pretreatment is superior to provisional use of HDT in patients with ST-segment elevation myocardial infarction.

Miglustat therapy in juvenile Sandhoff disease.

GM(2)-gangliosidosis is a rare and heterogeneous inherited metabolic disorder caused by autosomal recessive mutations in genes encoding the lysosomal enzyme ß-hexosaminidase, resulting in the accumulation of ganglioside GM(2) in various tissues, particularly the central nervous system. It is characterized by progressive neurological deterioration that mainly affects motor and spinocerebellar function. Several forms of GM(2)-gangliosidosis exist, including the Sandhoff variant. Currently there is no treatment for these conditions, except for palliative care. Miglustat (Zavesca) is a reversible inhibitor of glucosylceramide synthase, which catalyses the first committed step in the synthesis of glucose-based glycolipids. Miglustat has pharmacokinetic properties that allow it to cross the blood-brain barrier, and preclinical data suggest that it may benefit neuronopathic lysosomal storage diseases. Here we present a case report of a Norwegian patient with Sandhoff disease treated with miglustat at our centre in Norway. The patient initially presented with ataxia and dysarthria at 2-3 years of age, which progressed slowly during childhood. At age 14, he experienced episodes of depression and apathy, leading to weight loss. He was diagnosed with Sandhoff disease at age 16. Following 2.5 years of treatment with miglustat, his body weight was stabilized and disease progression appeared to have slowed, as evidenced by the lack of progressive brain atrophy. His depressive symptoms were managed using electroconvulsive treatment (ECT), which improved general functioning. These findings suggest that miglustat may provide beneficial effects in patients with juvenile Sandhoff disease, and that ECT may alleviate depressive symptoms.

Marked reduction of early stent thrombosis with pre-hospital initiation of high-dose Tirofiban in ST-segment elevation myocardial infarction.

BACKGROUND: No randomized comparisons are yet available evaluating the effect of pre-hospital high dose tirofiban on the incidence of early stent thrombosis after primary percutaneous coronary intervention (PCI). OBJECTIVES: The aim of this analysis was to evaluate whether routine pre-hospital administration of high-dose tirofiban in ST-segment elevation myocardial infarction (STEMI) decreases the incidence of early stent thrombosis after primary PCI. PATIENTS/METHODS: The Ongoing Tirofiban in Myocardial Evaluation (On-TIME) 2 trial was a prospective multicenter study of consecutive STEMI patients referred for primary PCI in which patients were randomized to pre-hospital no high-dose tirofiban/placebo. We examined the incidence of Academic Research Consortium definite and probable early stent thrombosis and determined predictors and outcome of early stent thrombosis. RESULTS: Primary PCI was performed in 1203 out of 1398 patients (86.1%). In 1073 patients (89.2%) a coronary stent was placed. Early stent thrombosis occurred in 39 patients (3.6%). Pre-hospital initiation of high-dose tirofiban significantly reduced early stent thrombosis (2.1% vs. 5.2%, P = 0.006) and was associated with a lower incidence of urgent repeat PCI (1.9% vs. 5.2%, P = 0.005). Early stent thrombosis, as well as pre-hospital initiation of high-dose tirofiban, was independently associated with 30-day mortality. CONCLUSIONS: Pre-hospital initiation of high-dose tirofiban reduces the 30-day incidence of stent thrombosis in STEMI patients treated with primary PCI and stenting. Early stent thrombosis and pre-hospital initiation of high-dose tirofiban were independent predictors of 30-day mortality.

Fatty acid composition of plasma lipids in patients with pancreatic, lung and oesophageal cancer in comparison with healthy subjects.

BACKGROUND & AIMS: Despite the general notion of impaired nutritional status in cancer patients, studies on fatty acid status in cancer patients are limited. The aim of the present study was to investigate whether plasma n-3 fatty acids concentrations are reduced in patients with different tumour types. METHODS: We measured fatty acid composition in plasma phospholipids (PLs) and cholesteryl esters (CEs) in 71 newly diagnosed, untreated cancer patients of three tumour types: oesophageal or cardia cancer (n = 35), non-small cell lung cancer (n = 22) and pancreatic cancer (n = 15) and in 45 healthy subjects. RESULTS: In pancreatic cancer, plasma n-3 fatty acids showed a substantial reduction in both plasma PLs and CES. Although n-3 fatty acids in lung cancer also tended to be reduced, this difference failed to reach statistical significance. n-3 Fatty acid levels were especially reduced in pancreatic cancer patients without diabetes mellitus, and in lung cancer patients with weight loss. In oesophageal cancer, n-3 fatty acid concentrations were comparable to those in healthy subjects. CONCLUSION: We conclude that plasma n-3 fatty acid levels were reduced in pancreatic cancer, tended to be reduced in lung cancer, but were not altered in oesophageal cancer. Further studies are needed to assess the mechanisms underlying the observed changes in n-3 fatty acid concentrations.

Circuit and protocol for hypoxic hyperthermic isolated limb perfusion to treat malignant melanoma.

In the treatment of cancer, isolated limb perfusion (ILP) allows what would be a lethal systemic dose of cytotoxic drugs to be administered directly to a tumour site of an extremity. Unfortunately, ILP is a complex, expensive, time-consuming treatment that requires general anaesthesia, vascular surgery and expertise with extracorporeal circuits that may not be available outside a cardiac centre. By streamlining the traditional ILP protocols and eliminating the oxygenator from the circuit, an equally safe and effective technique of hypoxic hyperthermic isolated limb perfusion has been developed.

Dimensions of sexuality in the perimenopausal transition: a model for practice.

A biopsychosocial-cultural model of the sexuality of women during the perimenopausal transition includes dimensions related to physiology, psychosocial issues, and culture. It is an amalgam of biomedical and psychosocial models, yet has the added focus on culture. This holistic approach to sexual health is recommended by researchers and clinicians engaged in the study of midlife women. Clinicians can use this model to guide assessment and interventions, examining all of the dimensions of sexuality during the perimenopausal transition.

Myosin-X, a novel myosin with pleckstrin homology domains, associates with regions of dynamic actin.

Myosin-X is the founding member of a novel class of unconventional myosins characterized by a tail domain containing multiple pleckstrin homology domains. We report here the full-length cDNA sequences of human and bovine myosin-X as well as the first characterization of this protein's distribution and biochemical properties. The 235 kDa myosin-X contains a head domain with <45% protein sequence identity to other myosins, three IQ motifs, and a predicted stalk of coiled coil. Like several other unconventional myosins and a plant kinesin, myosin-X contains both a myosin tail homology 4 (MyTH4) domain and a FERM (band 4.1/ezrin/radixin/moesin) domain. The unique tail domain also includes three pleckstrin homology domains, which have been implicated in phosphatidylinositol phospholipid signaling, and three PEST sites, which may allow cleavage of the myosin tail. Most intriguingly, myosin-X in cultured cells is present at the edges of lamellipodia, membrane ruffles, and the tips of filopodial actin bundles. The tail domain structure, biochemical features, and localization of myosin-X suggest that this novel unconventional myosin plays a role in regions of dynamic actin.

Pharmacokinetics of intravenous ATP in cancer patients.

OBJECTIVE: To characterise the pharmacokinetics of adenosine 5'-triphosphate (ATP) in patients with lung cancer after i.v. administration of different ATP dosages. METHODS: Twenty-eight patients received a total of 176 i.v. ATP courses of 30 h. Fifty-two infusions were given as low-dose infusions of 25-40 microg kg(-1) min(-1), 47 as middle-dose infusions of 45-60 microg kg(-1) min(-1) and 77 as high-dose infusions of 65-75 microg kg(-1) min(-1) ATP. Kinetic data of ATP concentrations in erythrocytes were available from 124 ATP courses. Results are expressed as mean +/- SEM. RESULTS: Most ATP courses in cancer patients were without side effects (64%), and side effects occurring in the remaining courses were mild and transient, resolving within minutes after decreasing the infusion rate. Baseline ATP concentration in erythrocytes was 1,554 +/- 51 micromol l(-1). ATP plateau levels at 24 h were significantly increased by 53 +/- 3, 56 +/- 3 and 69 +/- 2% after low-dose, middle-dose and high-dose ATP infusions, respectively. At the same time, significant increases in plasma uric acid concentrations were observed: 0.06 +/- 0.01, 0.11 +/- 0.01 and 0.16 +/- 0.01 mmol l(-1), respectively. The mean half-time for disappearance of ATP from erythrocytes, measured in five patients, was 5.9 +/- 0.5 h. CONCLUSIONS: During constant i.v. infusion of ATP in lung cancer patients, ATP is taken up by erythrocytes and reaches dose-dependent plateau levels 50-70% above basal concentrations at approximately 24 h.

Effects of ATP infusion on glucose turnover and gluconeogenesis in patients with advanced non-small-cell lung cancer.

Cancer cachexia is associated with elevated lipolysis, proteolysis and gluconeogenesis. ATP infusion has been found to significantly inhibit loss of body weight, fat mass and fat-free mass in patients with advanced lung cancer. The present study was aimed at exploring the effects of ATP on whole-body glucose turnover, alanine turnover and gluconeogenesis from alanine. Twelve patients with advanced non-small-cell lung cancer (NSCLC) were studied 1 week before and during 22-24 h of continuous ATP infusion. After an overnight fast, turnover rates of glucose and alanine, and gluconeogenesis from alanine, were determined using primed constant infusions of ¿6, 6-(2)H(2)glucose and ¿3-(13)Calanine. Thirteen NSCLC patients and eleven healthy subjects were studied as control groups without ATP infusion. During high-dose ATP infusion (75 microg.min(-1).kg(-1)), glucose turnover was 0.62+/-0.07 mmol.h(-1).kg(-1), compared with 0. 44+/-0.13 mmol.h(-1).kg(-1) at baseline (P=0.04). For gluconeogenesis a similar, but non-significant, trend was observed ¿baseline, 0.30+/-0.16 mmol.h(-1).kg(-1); during ATP, 0.37+/-0.13 mmol.h(-1).kg(-1) (P=0.08). At lower ATP doses (37-50 microg. min(-1).kg(-1)) these effects were not detected. The relative increase in glucose turnover during ATP infusion compared with baseline showed a significant correlation with the ATP dose (r=0.58, P=0.02). No change in alanine turnover was observed at any ATP dose. The results of this study indicate an increase in glucose turnover during high-dose ATP infusion compared with baseline levels. During high-dose ATP infusion, glucose turnover was similar to that during low-dose ATP infusion and to that in control NSCLC patients. Between ATP infusions, however, glucose turnover in patients treated with high-dose ATP was significantly lower than that in the low-dose and control NSCLC patients (P=0.04 and P=0.03 respectively), and similar to that in healthy subjects. This would suggest that repeated high-dose ATP infusions may inhibit glucose turnover between infusion periods.

Autologous blood collection and transfusion in cats undergoing partial craniectomy.

OBJECTIVE: To describe the procedure for autologous blood donation and associated complications in cats undergoing partial craniectomy for mass removal. DESIGN: Prospective case series. ANIMALS: 15 cats with intracranial mass confirmed by computed tomographic scan, no evidence of renal failure, and PCV > or = 22%. PROCEDURE: One unit (60 ml) of blood was collected and stored 7 to 17 days before surgery and transfused during the perioperative period if needed. The PCV was measured before donation, before surgery, during surgery, and after surgery to assess effect of donation on PCV before surgery and effect of transfusion on PCV after surgery. Cats were evaluated for donation complications, iatrogenic anemia, and adverse reactions associated with administration of autologous blood. RESULTS: Complications associated with phlebotomy were not detected. Fifteen cats underwent partial craniectomy 7 to 17 days after blood donation; all had histologic confirmation of meningioma by examination of tissue obtained at surgery. Eleven cats received autologous blood transfusions. None of the cats received allogeneic blood transfusions. Transfusion reactions were not observed. Subclinical iatrogenic anemia was detected in 3 cats. Two cats were considered to have received excessive transfusion, and 3 cats received inadequate transfusion. All cats undergoing partial craniectomy were discharged from the hospital and were alive > 6 months after surgery. CONCLUSIONS AND CLINICAL RELEVANCE: Autologous blood donation before surgery was considered safe for cats undergoing partial craniectomy for resection of meningioma. The only complication observed was iatrogenic anemia. The procedure contributed to blood conservation in our hospital.

Hepatic sugar phosphate levels reflect gluconeogenesis in lung cancer: simultaneous turnover measurements and 31P magnetic resonance spectroscopy in vivo.

Stable-isotope tracers were used to assess whether levels of phosphomonoesters (PME) and phosphodiesters (PDE) in the livers of lung cancer patients, as observed by (31)P magnetic resonance (MR) spectroscopy, reflect elevated whole-body glucose turnover and gluconeogenesis from alanine. Patients with advanced non-small-cell lung cancer without liver metastases (n=24; weight loss 0-24%) and healthy control subjects (n=13) were studied after an overnight fast. (31)P MR spectra of the liver in vivo were obtained, and glucose turnover and gluconeogenesis from alanine were determined simultaneously using primed-constant infusions of [6, 6-(2)H(2)]glucose and [3-(13)C]alanine. Liver PME concentrations were 6% higher in lung cancer patients compared with controls (not significant); PME levels in patients with >/=5% weight loss were significantly higher than in patients with <5% weight loss (P<0.01). PDE levels did not differ between the groups. In lung cancer patients, whole-body glucose production was 19% higher (not significant) and gluconeogenesis from alanine was 42% higher (P<0. 05) compared with healthy subjects; turnover rates in lung cancer patients with >/=5% weight loss were significantly elevated compared with both patients with <5% weight loss and healthy subjects (P<0. 05). PME levels were significantly correlated with glucose turnover and gluconeogenesis from alanine in lung cancer patients (r=0.48 and r=0.48 respectively; P<0.05). In conclusion, elevated PME levels in lung cancer patients appear to reflect increased glucose flux and gluconeogenesis from alanine. These results are consistent with the hypothesis that elevated PME levels are due to contributions from gluconeogenic intermediates.

Eicosapentaenoic acid ethyl ester supplementation in cachectic cancer patients and healthy subjects: effects on lipolysis and lipid oxidation.

BACKGROUND & AIMS: Recent reports suggest that weight loss in cachectic cancer patients may be inhibited by supplementation of the n-3 fatty acid eicosapentaenoic acid (20:5n-3; EPA), presumably due to inhibition of lipolysis. The aim of the present double-blind, randomized trial was to assess whether short-term oral EPA ethyl ester (EE) supplementation inhibits lipolysis and lipid oxidation in weight-losing cancer patients and in healthy subjects. METHODS: Seventeen weight-losing, cancer patients of different tumor types, and 16 healthy subjects were randomized to receive EPA-EE (6 g/d) or placebo (oleic acid (OA)-EE; 6 g/d) for seven days. At baseline (day 0) and during supplementation (days 2 and 7) whole-body lipolysis and palmitic acid release were measured in the overnight fasting state using [1, 1, 2, 3, 3-(2)H(5)]glycerol and [1-(13)C]palmitic acid. Palmitate oxidation was determined by measuring(13)CO(2)enrichment in expired breath. RESULTS: No significant effects of EPA-EE on whole-body lipolysis, palmitic acid release, or palmitate oxidation were detected in cancer patients nor in healthy subjects in comparison with OA-EE. EPA-EE supplementation reduced plasma-free fatty acid and triacylglycerol concentrations significantly in healthy subjects but not in cancer patients. CONCLUSION: We conclude that supplementation of EPA-EE does not significantly inhibit lipolysis or lipid oxidation in weight-losing cancer patients or in healthy subjects during short-term supplementation when using OA-EE as a placebo supplement.

Effects of azetidine-2-carboxylic acid on treatments of hepatoma cells with single or fractionated X-ray irradiations and on thermal radiosensitization in normal and thermotolerant cells.

The amino acid analog azetidine-2-carboxylic acid (azetidine) is a potent sensitizer to both hyperthermia and ionizing radiation. Incubation of H35 hepatoma cells with 2.5 mM azetidine before or after treatments with X-rays causes a time- and sequence-dependent enhancement of cell killing. Exposure of cells to 1-1.5 mM azetidine for 96 h in combination with repeated doses of 3 Gy X-rays at 24 h intervals causes an enhanced reduction of the surviving cell population due to both radiosensitization and an additional growth inhibition. Azetidine does not prevent the induction of thermotolerance after a heat shock. This thermotolerance proportionally reduces thermal radiosensitization but does not seem to affect azetidine radiosensitization. It is suggested that thermal radiosensitization and azetidine radiosensitization operate by different mechanisms.

Incorporation and washout of orally administered n-3 fatty acid ethyl esters in different plasma lipid fractions.

The aim of the present study was to quantify the incorporation of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) into plasma lipids after oral administration of n-3 fatty acid ethyl esters, since little is known about the rate and pattern of incorporation into plasma lipid fractions. In addition, we aimed to obtain preliminary information regarding EPA half-life, which is needed to establish an optimal dosing schedule. Five healthy volunteers ingested two 8.5 g doses of n-3 fatty acid ethyl esters daily for 7 d, supplying 6.0 g EPA/d and 5.3 g DHA/d. The fatty acid compositions of plasma phospholipids (PL), cholesteryl esters (CE) and triacylglycerols (TAG) were determined during supplementation and during a washout period of 7 d. Half-lives of EPA and DHA were calculated. The proportion of EPA in PL showed a 15-fold increase after 7 d (P < 0.001), while DHA showed a smaller increase (P < 0.01). In CE, EPA also increased (P < 0.05), while DHA did not increase at all. Remarkably, incorporation of DHA into TAG was even higher than that of EPA. Half-life of EPA in PL ranged from 1.63 to 2.31 d (mean 1.97 (SE 0.15) d), whereas mean half-life of EPA in CE was 3.27 (SE 0.56) d. In three subjects, washout of EPA and DHA from TAG seemed to follow a bi-exponential pattern, with a short half-life (< 1 d) in the initial phase and a half-life of several days in the second phase. In conclusion, EPA ethyl esters are rapidly incorporated into plasma lipids, especially into PL. The relatively long half-life of EPA in plasma would permit a dosing schedule with intervals of > or = 12 h in supplementation studies.