Follistatin-based ligand trap ACE-083 induces localized hypertrophy of skeletal muscle with functional improvement in models of neuromuscular disease.

Skeletal muscle is under inhibitory homeostatic regulation by multiple ligands of the transforming growth factor-ß (TGFß) superfamily. Follistatin is a secreted protein that promotes muscle growth and function by sequestering these ligands extracellularly. In the present study, we evaluated the potential of ACE-083 - a locally acting, follistatin-based fusion protein - as a novel therapeutic agent for focal or asymmetric myopathies. Characterization of ACE-083 in vitro revealed its high affinity for heparin and extracellular matrix while surface plasmon resonance and cell-based assays confirmed that ACE-083 binds and potently neutralizes myostatin, activin A, activin B and growth differentiation factor 11 (GDF11). Intramuscular administration of ACE-083 caused localized, dose-dependent hypertrophy of the injected muscle in wild-type mice and mouse models of Charcot-Marie-Tooth disease (CMT) and Duchenne muscular dystrophy, with no evidence of systemic muscle effects or endocrine perturbation. Importantly, ACE-083 also increased the force of isometric contraction in situ by the injected tibialis anterior muscle in wild-type mice and disease models and increased ankle dorsiflexion torque in CMT mice. Our results demonstrate the potential of ACE-083 as a therapeutic agent for patients with CMT, muscular dystrophy and other disorders with focal or asymmetric muscle atrophy or weakness.

Medición prehospitalaria del exceso de base arterial y su posible papel en la predicción del desenlace tras una parada cardiaca extrahospitalaria / Prehospital measurement of arterial base excess and its role as a possible predictor

Objetivo: La parada cardiorrespiratoria (PCR) conduce a un estado de acidosis mixtametabólica y respiratoria. Incluso tras una ventilación adecuada y la recuperación del pulso espontáneo (ROSC) la acidosis metabólica se refleja en un exceso de bases (EB).El objetivo del estudio es comprobar que el EB arterial se correlaciona con la mortalidad en el ámbito prehospitalario. Método: Se revisaron de forma retrospectiva las hojas de registro de los pacientes en PCR desde el 1 de enero de 2003 hasta 31 de diciembre de 2010. Se incluyeron 126pacientes con PCR no traumáticas en los que se obtuvo una gasometría en el curso de la reanimación cardiopulmonar (RCP). Se recogieron las siguientes variables: edad, sexo, tiempo hasta el inicio de la reanimación, causa de la PCR, ritmo inicial, duración de la reanimación, uso de trombolítico, adrenalina, bicarbonato, hipotermia terapéutica (..) (AU)

Objective: Cardiac arrest leads to a state of mixed respiratory and metabolic acidosis. Even after adequate ventilation and restoration of spontaneous circulation, metabolic acidosis as reflected by a negative base excess (BE) persists. We hypothesized that arterial BE measured in out-of-hospital cardiac arrest would be significantly associated with prehospital mortality. Methods: We retrospectively reviewed all protocol sheets of emergency medical responses to cardiac arrest in the period from January 1, 2003 to December 31, 2010. One hundred twenty-six adult non traumatic cardiac arrest patients in whom cardiopulmonary resuscitation (CPR) was attempted and an arterial blood gas sample was obtained during ongoing CPR were included for further analysis. The following data were collected: age, sex, delay, bystander or emergency medical technician CPR, cause of cardiac arrest, initial rhythm, CPR duration; use of thrombolytic therapy, epinephrine, sodium bicarbonate, and for a cooling device and blood gas sample parameters. The univariate association (..) (AU)

CTGF is overexpressed in malignant melanoma and promotes cell invasion and migration.

BACKGROUND: Malignant melanoma cells are known to have altered expression of growth factors compared with normal human melanocytes. These changes most likely favour tumour growth and progression, and influence tumour environment. The induction of transforming growth factor beta1, 2 and 3 as well as BMP4 and BMP7 expression in malignant melanoma has been reported before, whereas the expression of an important modulator of these molecules, connective tissue growth factor (CTGF), has not been investigated in melanomas until now. METHODS: Expression of CTGF was analysed in melanoma cell lines and tissue samples by qRT-PCR and immunohistochemistry. To determine the regulation of CTGF expression in malignant melanoma, specific siRNA was used. Additionally, migration, invasion and attachment assays were carried out. RESULTS: We were able to demonstrate that CTGF expression is upregulated in nine melanoma cell lines and in primary and metastatic melanoma in situ. The transcription factor HIF-1α was revealed as a positive regulator for CTGF expression. Melanoma cells, in which CTGF expression is diminished, show a strong reduction of migratory and invasive properties when compared with controls. Further, treatment of normal human epidermal melanocytes with recombinant CTGF leads to an increase of migratory and invasive behaviour of these cells. CONCLUSION: These results suggest that CTGF promotes melanoma cell invasion and migration and, therefore, has an important role in the progression of malignant melanoma.

Isolation and functional characterization of pericytes derived from hamster skeletal muscle.

AIM: At the interface of tissue and capillaries, pericytes (PC) may generate electrical signals to be conducted along the skeletal muscle vascular network, but they are functionally not well characterized. We aimed to isolate and cultivate muscle PC allowing to analyse functional properties considered important for signal generation and conduction. METHODS: Pericytes were enzymatically isolated from hamster thigh muscles and further selected during a 16-30 days' cultivation period. PC markers were studied by fluorescence activated cell scanning (FACS) and immunocytochemistry. Electrical properties of the cultured PC were investigated by patch clamp technique as well as the membrane potential sensitive dye DiBAC(4) (3). RESULTS: The cultured cells showed typical PC morphology and were positive for NG2, alpha smooth muscle actin, PDGFR-ß and the gap junction protein Cx43. Expressions of at least one single or combinations of several markers were found in 80-90% of subpopulations. A subset of the patched cells expressed channel activities consistent with a Kv1.5 channel. In vivo presence of the channels was confirmed in sections of hamster thigh muscles. Interleukin-8, a myokine known to be released from exercising muscle, increased the expression but not the activity of this channel. Pharmacologic stimulation of the channel activity by flufenamic acid induced hyperpolarization of PC alone but not of endothelial cells [human umbilical vein endothelial cells (HUVEC)] alone. However, hyperpolarization was observed in HUVEC adjacent to PC when kept in co-culture. CONCLUSION: We established a culture method for PC from skeletal muscle. A first functional characterization revealed properties which potentially enable these cells to generate hyperpolarizing signals and to communicate them to endothelial cells.

Biocatalytic approaches for the quantitative production of single stereoisomers from racemates.

Strategies for the chemoenzymatic transformation of a racemate into a single stereoisomeric product in quantitative yield have been developed. A range of industrially relevant alpha-hydroxycarboxylic acids was deracemized in a stepwise fashion via lipase-catalysed enantioselective O-acylation, followed by mandelate racemase-catalysed racemization of the remaining non-reacted substrate enantiomer. Alternatively, aliphatic alpha-hydroxycarboxylic acids were enzymatically isomerized using whole resting cells of Lactobacillus spp. Enantioselective hydrolysis of rac-sec-alkyl sulphate esters was accomplished using novel alkyl sulphatases of microbial origin. The stereochemical path of catalysis could be controlled by choice of the biocatalyst. Whereas Rhodococcus ruber DSM 44541 and Sulfolobus acidocaldarius DSM 639 act through inversion of configuration, stereo-complementary retaining sulphatase activity was detected in the marine planctomycete Rhodopirellula baltica DSM 10527.

Expression of Hugl-1 is strongly reduced in malignant melanoma.

The human gene Hugl-1 (Llgl/Lgl1) has significant homology to the Drosophila tumor suppressor gene lethal(2)giant larvae (lgl). The lgl gene codes for a cortical cytoskeleton protein, Lgl, that is involved in maintaining cell polarity and epithelial integrity. We speculate that Hugl-1 might play a role in epithelial-mesenchymal transition (EMT) and that loss of Hugl-1 expression plays a role in the development or progression of malignant melanoma. Thus, we evaluated melanoma cell lines and tissue samples of malignant melanoma for loss of Hugl-1 transcription. We found that Hugl-1 was downregulated or lost in all cell lines and in most of the tumor samples analysed, and that these losses were associated with advanced stage of the disease. Reduced Hugl-1 expression occurred as early as in primary tumors detected by both immunohistochemical and reverse transcription-polymerase chain reaction (RT-PCR) analysis. Functional assays with stable Hugl-1-transfected cell lines revealed that Hugl-1 expression increased cell adhesion and decreased cell migration. Further, downregulation of MMP2 and MMP14 (MT1-MMP) and re-expression of E-cadherin was found in the Hugl-1-expressing cell clones supporting a role of Hugl-1 in EMT. Our studies thus indicate that loss of Hugl-1 expression contributes to melanoma progression.

Body fat distribution of overweight females with a history of weight cycling.

Weight cycling may cause a redistribution of body fat to the upper body fat compartments. We investigated the distribution of subcutaneous adipose tissue (SAT) in 30 overweight women with a history of weight-cycling and age-matched controls (167 normal weight and 97 overweight subjects). Measurements of SAT were performed using an optical device, the Lipometer. The SAT topography describes the thicknesses of SAT layers at 15 anatomically well-defined body sites from neck to calf. The overweight women with a history of weight cycling had significantly thicker SAT layers on the upper body compared to the overweight controls, but even thinner SAT layers on their legs than the normal weight women. An android fat pattern was attributed to overweight females and, even more pronounced, to the weight cyclers. The majority of normal weight women showed a gynoid fat pattern. Using stepwise discriminant analysis, 89.0% of all weight cyclers and overweight controls could be classified correctly into the two groups. These findings show the importance of normal weight maintenance as a health-promoting factor.

Acute effects of nicorandil on glucose tolerance in subjects with borderline fasting blood glucose levels.

The acute effect of the anti-ischemic potassium channel opener nicorandil on glucose tolerance and post-challenge insulin levels was investigated in 11 subjects (6 males and 5 females, age 59 +/- 2 years) with borderline fasting blood glucose in a single blinded randomised study. All participants were submitted to two oral glucose tolerance tests in randomised order, once without any premedication and once 30 minutes after oral administration of 20 mg nicorandil. This single dose of nicorandil significantly increased blood glucose levels at 120 minutes (173 +/- 16 vs. 150 +/- 11 mg/dl, p < 0.05 by ANOVA) and 180 minutes (106 +/- 11 vs. 88 +/- 7 mg/dl, p < 0.05 by ANOVA) after ingestion of 75 mg of glucose. Serum insulin levels were not significantly altered. In conclusion we suggest that controlled studies in patients with coronary artery disease should be performed to investigate whether long term treatment with nicorandil increases progression rates from impaired glucose tolerance to type-II diabetes and/or from normal to impaired glucose tolerance with a possibly negative impact on the course of cardiovascular disease in comparison to conventional anti-anginal drugs.

The alpha-amino group of L-arginine mediates its antioxidant effect.

Antioxidant effects may constitute part of the possible antiatherogenic effects of the amino acid L-arginine. These antioxidant properties were further characterized in a model of lipoprotein oxidation. Oxidation of lipoproteins in unfractionated human serum was continuously monitored by a fluorescent probe. The antioxidant effects of L-arginine, N-alpha-acetyl-arginine and vitamin E in combination with L-arginine were measured after initiation of free radical generation with either copper or 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH). The half-time of the fast propagation rate for copper-induced lipoprotein oxidation increased after incubation with L-arginine in a dose-dependent manner (P < 0.01). N-alpha-acetyl-arginine did not show such effects. Vitamin E and L-arginine show different effects on copper-induced oxidation, the former increasing only lag-time, the latter increasing only propagation rate, and do not have reciprocal effects. In contrast to copper-induced oxidation, L-arginine increased the lag-time of AAPH-induced lipoprotein oxidation (P < 0.01), with no effect on the propagation rate at physiological concentrations. Again, N-alpha-acetyl-arginine did not show any antioxidant effects. Our experiments provide further evidence that mechanisms other than serving as a substrate for the NO-synthase could be involved in the antiatherosclerotic effect of L-arginine. In addition, our experiments clearly show, that the antioxidant effect of L-arginine is due to a chemical moiety different from that serving as the substrate for NO biosynthesis.

Leptin deficiency due to lipid apheresis: a possible reason for ravenous hunger and weight gain.

OBJECTIVE: To investigate how extracorporal cholesterol lowering therapy affects circulating leptin levels in patients with ravenous hunger after treatment and permanent weight gain. DESIGN: A case report. SUBJECT: 51 y old caucasian male patient with moderate chronic renal failure. MEASUREMENTS: Serum Leptin concentration (RIA, Linco Research Inc, St. Louis, MO, USA), total cholesterol, low density lipoprotein cholesterol, blood glucose levels, calorie intake by food records. RESULTS: During treatment total cholesterol was reduced by 50%. Serum Leptin levels showed a 42% reduction at the end of treatment, that by far exceeds the physiological diurnal variation. Calorie intake was significantly increased on days of treatment. CONCLUSION: We conclude that this artificial reduction in circulating leptin plays an important role in the pathogenesis of ravenous hunger and weight gain under extracorporal cholesterol lowering therapy in this case.

Effect of an individualised training programme during weight reduction on body composition: a randomised trial.

OBJECTIVE: To study the effect of a standardised training programme focusing on maintenance of fat free mass during weight reduction by energy reduction in obese children. DESIGN: Randomised trial of physical training programme and dietary advice (group A) versus dietary advice alone (group B). SUBJECTS: Thirty obese children and adolescents (14 group A, 16 group B) participated in the 12 week long programme; 20 children (10 group A, 10 group B) were also reassessed after one year. MEASUREMENTS: Fat free mass was estimated from the resistance index, obtained by bioelectrical impedance analysis at baseline, after four, eight, and 12 weeks in all subjects, and after one year in 20 subjects. RESULTS: The mean (SD) change in fat free mass was significantly different between the two groups after 12 weeks (group A, 2.68 (3.74) kg; group B, 0.43 (1.65) kg). The change in body weight after one year was inversely correlated with the change in fat free mass after 12 weeks (r = -0. 44), as assessed in the 20 subjects. CONCLUSIONS: A standardised training programme as used in this study can prevent reduction in fat free mass during weight loss in obese children. Reduction in fat free mass during weight reduction might be a risk factor for regain of weight.

Effects of intensified lifestyle modification on the need for further revascularization after coronary angioplasty.

BACKGROUND: In patients with coronary artery disease (CAD), a rate of restenosis as high as 50% is observed after percutaneous transluminal coronary angioplasty (PTCA). Frequently, this results in further revascularization procedures. Lifestyle intervention has been shown to slow the progression of CAD and to reduce cardiovascular events after myocardial infarction. However, no information exists whether such treatment influences the rate of restenosis in patients with CAD. The present study was performed to investigate the effects of an intensified lifestyle intervention on the need for further revascularization procedures in patients with established CAD after successful PTCA. DESIGN: A total of 60 patients were included and randomized to either conventional treatment by cardiologists and general practitioners or additional intensified lifestyle intervention in a diabetes and metabolism outpatient clinic for 12 months. The mean observation time after successful PTCA was 26 months. The primary outcome variable was the need for further revascularization procedures because of clinical restenosis. Secondary outcome variables were lifestyle-related measures. RESULTS: Intervention resulted in a reduction in body weight and blood pressure, and in increased physical activity. Furthermore, nutritional habits were changed towards less fat intake, and body composition changed towards a higher proportion of fat-free mass. The need for further revascularization procedures was reduced from a total of 14 out of 32 in the conventionally treated group to 3 out of 28 in the intervention group. This resulted in an event-free survival probability of 0.89 in the intervention group and 0.57 in the control group (P = 0.0055, log rank) with a resulting relative risk of 0.26 (95% CI 0.09-0.74). CONCLUSION: In conclusion, our data strongly suggest that intensified lifestyle modification is able to reduce the need for further revascularization procedures after PTCA in patients with CAD.

Primary myeloid leukemia presenting concomitantly with primary multiple myeloma: two cases and an update of the literature.

We report one case of primary acute myelogenous leukemia (AML) and one case of refractory anemia with excess blasts in transformation (RAEB-T) each presenting concomitantly with multiple myeloma, an unusual finding. The twin diagnoses in each patient were confirmed by cytochemical and immunohistochemical studies, and in one of our cases, by ultrastructural, flow cytometric, and molecular studies. The last three methods have not been previously used to document this phenomenon.

Forearm composition contributes to differences in reactive hyperaemia between healthy men and women.

BACKGROUND: Post-ischaemic reactive hyperaemia in the forearm has been suggested as a marker of resistance vessel function. The contribution of forearm composition to the kinetics of reactive hyperaemia is largely unknown. The body composition of men and women differs in that women have a higher body fat content and less lean body mass. METHODS: In the present study, we investigated whether the kinetics of reactive hyperaemia in the forearm in 14 healthy subjects (seven men and seven women) show gender-specific differences and whether forearm composition contributes to such differences. RESULTS: Peak reactive hyperaemic flow as well as 1-min-flow debt repayment (measured by venous occlusion plethysmography) were significantly higher in male than in female study participants. This difference was explained to > 60% by gender-specific differences in forearm relative muscle mass (as determined by magnetic resonance imaging). The half-life of the reactive hyperaemic response, on the other hand, was not different between men and women and did not show an association with forearm muscle. CONCLUSION: Our results demonstrate that forearm composition must be considered if peak reactive hyperaemic or flow debt repayment is used as a target, and that dynamic measurements of the reactive hyperaemic process are more suitable to describe the function of resistance arteries than single-point observations.

Effects of low-dose L-arginine on insulin-mediated vasodilatation and insulin sensitivity.

The present study was carried out to evaluate the effect of a low-dose intravenous supplementation of L-arginine on insulin-mediated vasodilatation and insulin sensitivity. The study was performed in healthy subjects (n = 7) and patients with obesity (n = 9) and non-insulin-dependent diabetes mellitus (NIDDM) (n = 9). Insulin-mediated vasodilatation was measured by venous occlusion plethysmography during the insulin suppression test, evaluating insulin sensitivity. Experiments were performed twice in each subject in the presence or absence of a concomitant infusion of L-arginine (0.52 mg kg-1 min-1). L-Arginine restored the imparied insulin-mediated vasodilatation observed in obesity (22.4 +/- 4.1%, P < 0.01 vs. without L-arginine) and NIDDM (20.3 +/- 3.2%, P < 0.01 vs. without L-arginine). In healthy subjects, no effect on insulin mediated-vasodilatation was observed (24.8 +/- 3.1% vs. 21.4 +/- 3.1%). Insulin sensitivity was improved significantly (P < 0.001) in all three groups by infusion of L-arginine. No effect of L-arginine was observed on insulin, insulin-like growth factor I (IGF-I), free fatty acids (FFAs) or C-peptide levels during the insulin suppression test. Our data indicate that defective insulin-mediated vasodilatation in obesity and NIDDM can be normalized by intravenous L-arginine. Furthermore, L-arginine improves insulin sensitivity in obese patients and NIDDM patients as well as in healthy subjects, indicating a possible mechanism that is different from the restoration of insulin-mediated vasodilatation.