Adjunctive β2-agonists reverse neuromuscular involvement in murine Pompe disease.

Pompe disease has resisted enzyme replacement therapy with acid α-glucosidase (GAA), which has been attributed to inefficient cation-independent mannose-6-phosphate receptor (CI-MPR) mediated uptake. We evaluated β2-agonist drugs, which increased CI-MPR expression in GAA knockout (KO) mice. Clenbuterol along with a low-dose adeno-associated virus vector increased Rotarod latency by 75% at 4 wk, in comparison with vector alone (P<2×10(-5)). Glycogen content was lower in skeletal muscles, including soleus (P<0.01), extensor digitorum longus (EDL; P<0.001), and tibialis anterior (P<0.05) following combination therapy, in comparison with vector alone. Glycogen remained elevated in the muscles following clenbuterol alone, indicating an adjunctive effect with gene therapy. Elderly GAA-KO mice treated with combination therapy demonstrated 2-fold increased wirehang latency, in comparison with vector or clenbuterol alone (P<0.001). The glycogen content of skeletal muscle decreased following combination therapy in elderly mice (P<0.05). Finally, CI-MPR-KO/GAA-KO mice did not respond to combination therapy, indicating that clenbuterol's effect depended on CI-MPR expression. In summary, adjunctive β2-agonist treatment increased CI-MPR expression and enhanced efficacy from gene therapy in Pompe disease, which has implications for other lysosomal storage disorders that involve primarily the brain.

Assessment of toxicity and biodistribution of recombinant AAV8 vector-mediated immunomodulatory gene therapy in mice with Pompe disease.

A preclinical safety study was conducted to evaluate the short- and long-term toxicity of a recombinant adeno-associated virus serotype 8 (AAV2/8) vector that has been developed as an immune-modulatory adjunctive therapy to recombinant human acid α-glucosidase (rhGAA, Myozyme) enzyme replacement treatment (ERT) for patients with Pompe disease (AAV2/8-LSPhGAApA). The AAV2/8-LSPhGAApA vector at 1.6 × 10(13) vector particles/kg, after intravenous injection, did not cause significant short- or long-term toxicity. Recruitment of CD4(+) (but not CD8(+)) lymphocytes to the liver was elevated in the vector-dosed male animals at study day (SD) 15, and in group 8 animals at SD 113, in comparison to their respective control animals. Administration of the vector, either prior to or after the one ERT injection, uniformly prevented the hypersensitivity induced by subsequent ERT in males, but not always in female animals. The vector genome was sustained in all tissues through 16-week postdosing, except for in blood with a similar tissue tropism between males and females. Administration of the vector alone, or combined with the ERT, was effective in producing significantly increased GAA activity and consequently decreased glycogen accumulation in multiple tissues, and the urine biomarker, Glc4, was significantly reduced. The efficacy of the vector (or with ERT) was better in males than in females, as demonstrated both by the number of tissues showing significantly effective responses and the extent of response in a given tissue. Given the lack of toxicity for AAV2/8LSPhGAApA, further consideration of clinical translation is warranted in Pompe disease.

Physiological And Biochemical Aspects Of The Valve Snap And Valve Closure Responses In The Giant Scallop Placopecten-Magellanicus .1. Physiology

1. Catabolic processes of the phasic and catch parts of the adductor muscle ofPlacopecten magellanicus have been studied in relation to valve snap and valve closure responses. It is concluded that the snap response is powered by both parts of the adductor muscle and the valve closure response is powered exclusively by the catch part. 2. Both parts of the adductor muscle show a high glycolytic potential, reflected by high levels of glycolytic enzymes (Table 1) and high glycogen levels (Table 2). Lactate dehydrogenase could not be detected. In contrast, octopine dehydrogenase shows high activities in both parts of the adductor muscle. It is therefore concluded that a main anaerobic pathway in both tissues is the breakdown of glycogen to octopine. In the catch part, however, a considerable amount of the pyruvate formed from glycogen may also be converted into alanine (see below). The glycolytic flux in the catch part is much higher during the snap response than during valve closure. 3. The absence of phosphoenolpyruvate carboxykinase in the adductor muscle ofP. magellanicus and the observed changes in aspartate, alanine and succinate demonstrate that the energy metabolism in the catch part during valve closure shows great similarities to that which occurs only in the initial stage of anaerobiosis in the catch adductor muscle of the sea musselMytilus edulis L. 4. Arginine kinase activity and arginine phosphate content of the phasic part are much higher than those of the catch part (Tables 1 and 3). This may explain why in the phasic part during the snap response most ATP equivalents are derived from arginine phosphate, and in the catch part during both valve responses most are derived from glycolysis (Table 6). Despite the limited contribution of glycolysis in the phasic part during the snap response, the glycolytic flux increases by a factor of at least 75. 5. Evidence is obtained that octopine is neither transported from one part of the adductor muscle to the other, nor from the adductor muscle to other tissues.

Physiological And Biochemical Aspects Of The Valve Snap And Valve Closure Responses In The Giant Scallop Placopecten-Magellanicus .2. Biochemistry

1. Catabolic processes of the phasic and catch parts of the adductor muscle ofPlacopecten magellanicus have been studied in relation to valve snap and valve closure responses. It is concluded that the snap response is powered by both parts of the adductor muscle and the valve closure response is powered exclusively by the catch part. 2. Both parts of the adductor muscle show a high glycolytic potential, reflected by high levels of glycolytic enzymes (Table 1) and high glycogen levels (Table 2). Lactate dehydrogenase could not be detected. In contrast, octopine dehydrogenase shows high activities in both parts of the adductor muscle. It is therefore concluded that a main anaerobic pathway in both tissues is the breakdown of glycogen to octopine. In the catch part, however, a considerable amount of the pyruvate formed from glycogen may also be converted into alanine (see below). The glycolytic flux in the catch part is much higher during the snap response than during valve closure. 3. The absence of phosphoenolpyruvate carboxykinase in the adductor muscle ofP. magellanicus and the observed changes in aspartate, alanine and succinate demonstrate that the energy metabolism in the catch part during valve closure shows great similarities to that which occurs only in the initial stage of anaerobiosis in the catch adductor muscle of the sea musselMytilus edulis L. 4. Arginine kinase activity and arginine phosphate content of the phasic part are much higher than those of the catch part (Tables 1 and 3). This may explain why in the phasic part during the snap response most ATP equivalents are derived from arginine phosphate, and in the catch part during both valve responses most are derived from glycolysis (Table 6). Despite the limited contribution of glycolysis in the phasic part during the snap response, the glycolytic flux increases by a factor of at least 75. 5. Evidence is obtained that octopine is neither transported from one part of the adductor muscle to the other, nor from the adductor muscle to other tissues.

Physiological stress responses in the edible crab, Cancer pagurus, to the fishery practice of de-clawing

We examined physiological stress responses in the edible crab, Cancer pagurus, subjected to the commercial fishery practice of manual de-clawing. We measured haemolymph glucose and lactate, plus muscular glycogen and glycogen mobilisation, in three experiments where the crabs had one claw removed. In the first, crabs showed physiological stress responses when 'de-clawed' as compared to 'handled only over the short term of 1-10 min. In the second, de-clawing and the presence of a conspecific both increased the physiological stress responses over the longer term of 24 h. In the third, de-clawing was shown to be more stressful than 'induced autotomy' of claws. Further, the former practice caused larger wounds to the body and significantly higher mortality than the latter. Since the fishery practice is to remove both claws, the stress response observed and mortality data reported are conservative.

Rapid change in energy status in fighting animals: causes and effects of strategic decisions

Energetic costs of fighting, such as high lactate or low glucose, have been shown in a range of species to correlate with the decisions made by each opponent, particularly the decision by one opponent, the 'loser', to end the fight by 'giving up'. Studies based on complete fights of differing duration, however, do not provide information on the changes in the physiological correlates of fighting that may take place during the course of the encounter, or how these changes may influence the capability and decisions of the contestants. We interrupted fights between hermit crabs, Pagurus bernhardus, at specific points, and related energy status to the preceding activities. Costs rose quickly with a rapid accumulation of lactic acid in attackers and declining muscular glycogen in defenders. Changes in physiological status appeared much earlier than the changes in behaviour that they may have caused. Furthermore, some physiological changes might have been an effect, rather than the cause, of fight decisions. (c) 2005 The Association for the Study of Animal Behaviour Published by Elsevier Ltd. All rights reserved.

Probabilistic classification of acute myocardial infarction from multiple cardiac markers

Logistic regression and Gaussian mixture model (GMM) classifiers have been trained to estimate the probability of acute myocardial infarction (AMI) in patients based upon the concentrations of a panel of cardiac markers. The panel consists of two new markers, fatty acid binding protein (FABP) and glycogen phosphorylase BB (GPBB), in addition to the traditional cardiac troponin I (cTnI), creatine kinase MB (CKMB) and myoglobin. The effect of using principal component analysis (PCA) and Fisher discriminant analysis (FDA) to preprocess the marker concentrations was also investigated. The need for classifiers to give an accurate estimate of the probability of AMI is argued and three categories of performance measure are described, namely discriminatory ability, sharpness, and reliability. Numerical performance measures for each category are given and applied. The optimum classifier, based solely upon the samples take on admission, was the logistic regression classifier using FDA preprocessing. This gave an accuracy of 0.85 (95% confidence interval: 0.78-0.91) and a normalised Brier score of 0.89. When samples at both admission and a further time, 1-6 h later, were included, the performance increased significantly, showing that logistic regression classifiers can indeed use the information from the five cardiac markers to accurately and reliably estimate the probability AMI. © Springer-Verlag London Limited 2008.

Novel biomarkers in early diagnosis of acute myocardial infarction compared with cardiac troponin T

Aims: To evaluate the role of novel biomarkers in early detection of acute myocardial infarction (MI) in patients admitted with acute chest pain.
Methods and results: A prospective study of 664 patients presenting to two coronary care units with chest pain was conducted over 3 years from 2003. Patients were assessed on admission: clinical characteristics, ECG (electrocardiogram), renal function, cardiac troponin T (cTnT), heart fatty acid binding protein (H-FABP), glycogen phosphorylase-BB, NT-pro-brain natriuretic peptide, D-dimer, hsCRP (high sensitivity C-reactive protein), myeloperoxidase, matrix metalloproteinase-9, pregnancy associated plasma protein-A, soluble CD40 ligand. A =12 h cTnT sample was also obtained. MI was defined as cTnT = 0.03 µg/L. In patients presenting <4 h of symptom onset, sensitivity of H-FABP for MI was significantly higher than admission cTnT (73 vs. 55%; P = 0.043). Specificity of H-FABP was 71%. None of the other biomarkers challenged cTnT. Combined use of H-FABP and cTnT (either one elevated initially) significantly improved the sensitivities of H-FABP or cTnT (85%; P = 0.004). This combined approach also improved the negative predictive value, negative likelihood ratio, and the risk ratio.
Conclusion: Assessment of H-FABP within the first 4 h of symptoms is superior to cTnT for detection of MI, and is a useful additional biomarker for patients with acute chest pain.

PTEN regulates colorectal epithelial apoptosis through Cdc42 signalling.

BACKGROUND:
Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) regulation of the Rho-like GTPase Cdc42 has a central role in epithelial polarised growth, but effects of this molecular network on apoptosis remain unclear.

METHODS:
To investigate the role of Cdc42 in PTEN-dependent cell death, we used flow cytometry, in vitro pull-down assays, poly(ADP ribose) polymerase (PARP) cleavage and other immunoblots in isogenic PTEN-expressing and -deficient colorectal cells (HCT116PTEN(+/+), HCT116PTEN(-/-), Caco2 and Caco2 ShPTEN cells) after transfection or treatment strategies.

RESULTS:
The PTEN knockout or suppression by short hairpin RNA or small interfering RNA (siRNA) inhibited Cdc42 activity, PARP cleavage and/or apoptosis in flow cytometry assays. Transfection of cells with wild-type or constitutively active Cdc42 enhanced PARP cleavage, whereas siRNA silencing of Cdc42 inhibited PARP cleavage and/or apoptosis. Pharmacological upregulation of PTEN by sodium butyrate (NaBt) treatment enhanced Cdc42 activity, PARP cleavage and apoptosis, whereas Cdc42 siRNA suppressed NaBt-induced PARP cleavage. Cdc42-dependent signals can suppress glycogen synthase kinase-ß (GSK3ß) activity. Pharmacological inhibition of GSK3ß by lithium chloride treatment mimicked effects of Cdc42 in promotion of PARP cleavage and/or apoptosis.

CONCLUSION:
Phosphatase and tensin homologue deleted on chromosome 10 may influence apoptosis in colorectal epithelium through Cdc42 signalling, thus providing a regulatory framework for both polarised growth and programmed cell death.

PATHOLOGY AND RESIDUES IN VEAL CALVES TREATED EXPERIMENTALLY WITH CLENBUTEROL

Six veal calves were medicated with clenbuterol at 20 mu g kg bodyweightl day(-1) for 42 days before they were slaughtered, to evaluate the lesions and residues in target organs. Compared with six unmedicated calves the most noticeable changes were tracheal dilatation, decreased uterine weight, slight mucous hypersecretion in the uterus and vagina and depletion of liver glycogen. The highest concentrations of clenbuterol (62 to 128 ng/g(-1)) were recorded in the choroid/retina, and the aqueous humour had the lowest concentration (0.5 to 2.4 ng ml(-1)). The residue concentrations were higher than the maximum residue level set for clenbuterol (0.5 ng g(-1))

FASCIOLA-HEPATICA - THE EFFECT OF THE MICROTUBULE INHIBITORS COLCHICINE AND TUBULOZOLE-C ON THE ULTRASTRUCTURE OF THE ADULT FLUKE

The effect of the microtubule inhibitors colchicine (1 x 10(-3) M) and tubulozole-C(1 x 10(-6) M) on the ultrastructure of adult Fasciola hepatica has been determined in vitro by transmission electron microscopy (TEM), using both intact flukes and tissue-slice material. With colchicine treatment, the apical membrane of the tegument became increasingly convoluted and blebbed, while accumulations of T1 secretory bodies occurred in the basal region of the syncytium, leading to progressively fewer secretory bodies in the syncytium. In the tegumental cells there were distinct accumulations of Tl secretory bodies around the Golgi complexes, which remained active for up to 12 h incubation. Tubulozole-treated flukes showed more severe effects, with initial accumulations of secretory bodies, both at the tegumental apex and base. This was followed in the later time-periods by the sloughing of the tegumental syncytium. In the underlying tegumental cells, the granular endoplasmic reticulum (GER) cisternae were swollen and disrupted, becoming concentrated around the nucleus. The Golgi complexes were dispersed to the periphery of the cells and gradually disappeared from the cytoplasm. After treatment with both drugs, the cell population in the vitelline follicles was altered, with an abnormally large proportion of stem cells and relatively few intermediate type 1 cells. The nurse cell cytoplasm became fragmented and was no longer in contact with the vitelline cells, while the shell globule clusters within the intermediate type 2 and mature cells were loosely packed. In the mature vitelline cells, 'yolk' globules and glycogen deposits became fewer than normal and lipid droplets were observed. The results are discussed in relation to the different modes of action of the two drugs and potential significance of this to anthelmintic (benzimidazole) therapy.

FASCIOLA-HEPATICA - THE EFFECT OF THE MICROFILAMENT INHIBITOR CYTOCHALASIN-B ON THE ULTRASTRUCTURE OF THE ADULT FLUKE

The effect of the microfilament inhibitor cytochalasin B (10 and 100-mu-g/ml) on the ultrastructure of adult Fasciola hepatica was determined in vitro by scanning and transmission electron microscopy (SEM, TEM) using both intact flukes and tissue-slice material. SEM revealed that initial swelling of the tegument led to surface blebbing and limited areas of sloughing after 24 h treatment at 100-mu-g/ml. In the tegumental syncytium, basal accumulations of secretory bodies (especially T2s) were evident in the earlier time periods but declined with longer incubations, until few secretory bodies remained in the syncytium overall. Blebbing of the apical plasma membrane and occasional areas of breakdown and sloughing of the tegument were observed over longer periods of treatment at 100-mu-g/ml. In the tegumental cell bodies, the Golgi complexes gradually decreased in size and activity, and few secretory bodies were produced. In the later time periods, the cells assumed abnormal shapes, the cytoplasm shrinking in towards the nucleus. In the vitelline follicles, a random dispersion of shell protein globules was evident within the intermediate-type cells, rather than their being organized into distinct shell globule clusters. Disruption of this process was more severe at the higher concentration of 100-mu-g/ml and again was more evident in tissue-slice material. In the latter, after prolonged (12 h) exposure to cytochalasin B, the intermediate and mature vitelline cells were filled with loosely packed and expanded shell globule clusters, containing few shell protein globules. The mature vitelline cells continued to lay down "yolk" globules and glycogen deposits. Disruption of the network of processes from the nurse cells was evident at the higher concentration of cytochalasin. Spaces began to appear between the vitelline cells and grew larger with progressively longer incubation periods, and the cells themselves assumed abnormal shapes. A number of binucleate stem cells were observed in tissue-slice material at the longest incubation period (12 h).

Elevated Circulation Levels of an Antiangiogenic SERPIN in Patients with Diabetic Microvascular Complications Impair Wound Healing through Suppression of Wnt Signaling

Wound healing, angiogenesis and hair follicle maintenance are often impaired in the skin of diabetic patients, but the pathogenesis has not been well understood. Here, we report that circulation levels of kallistatin, a member of the serine proteinase inhibitor (SERPIN) superfamily with anti-angiogenic activities, were elevated in Type 2 diabetic patients with diabetic vascular complications. To test the hypothesis that elevated kallistatin levels could contribute to a wound healing deficiency via inhibition of Wnt/β-catenin signaling, we generated kallistatin-transgenic (KS-TG) mice. KS-TG mice had reduced cutaneous hair follicle density, microvascular density, and panniculus adiposus layer thickness as well as altered skin microvascular hemodynamics and delayed cutaneous wound healing. Using Wnt reporter mice, our results showed that Wnt/β-catenin signaling is suppressed in dermal endothelium and hair follicles in KS-TG mice. Lithium, a known activator of β-catenin via inhibition of glycogen synthase kinase-3β, reversed the inhibition of Wnt/β-catenin signaling by kallistatin and rescued the wound healing deficiency in KS-TG mice. These observations suggest that elevated circulating anti-angiogenic serpins in diabetic patients may contribute to impaired wound healing through inhibition of Wnt/β-catenin signaling. Activation of Wnt/β-catenin signaling, at a level downstream of Wnt receptors, may ameliorate the wound healing deficiency in diabetic patients.Journal of Investigative Dermatology accepted article preview online, 24 January 2014. doi:10.1038/jid.2014.40.

Determining optimal duration of seed translocation periods for benthic mussel (Mytilus edulis) cultivation using physiological and behavioural measures of stress

During the benthic cultivation process of Mytilus edulis (blue mussels), wild mussel seed is often transplanted from naturally occurring subtidal beds to sheltered in-shore waters to be grown to a commercial size. The survival of these relaid mussels is ultimately a function of their quality and physiological condition upon relaying and it has been recognised that mussels can suffer from a loss in condition following transportation. We investigated whether the process of being transported to ongrowing plots had a negative effect on the physiological health and resultant behaviour of mussels by simulating transportation conditions in a controlled experiment. Mussels were kept, out of water, in plastic piping to recreate translocation conditions and further, we tested if depth held in a ship hold (0, 1.5 and 3 m) and length of time emersed (12, 24 and 48 h) affected mussel condition and behaviour. Physiological condition was assessed by quantifying mussel tissue pH and whole tissue glucose, glycogen, succinate and propionate concentrations. The rate of byssogenesis was also quantified to estimate recovery following a period of re-immersion. The depth at which mussels were held did not affect any of the physiological indicators of mussel stress but short-term byssus production was affected. Mussels held at 3 m produced fewer byssus threads during the first 72 h following re-immersion compared with mussels at 0 m (i.e. not buried) suggesting that depth held can impede recovery following transportation. Duration of emersion affected all stress indicators. Specifically, mussels held out of water for 48 h had a reduced physiological condition compared with those emersed for just 12 h. This work has practical implications for the benthic cultivation industry and based on these results we recommend that mussels are held out of water for less than 24 h prior to relaying to ensure physiological health and resultant condition is preserved.

Fasciola hepatica:the effect of the microfilament inhibitor cytochalasin B on the ultrastructure of the adult fluke

The effect of the microfilament inhibitor cytochalasin B (10 and 100 micrograms/ml) on the ultrastructure of adult Fasciola hepatica was determined in vitro by scanning and transmission electron microscopy (SEM, TEM) using both intact flukes and tissue-slice material. SEM revealed that initial swelling of the tegument led to surface blebbing and limited areas of sloughing after 24 h treatment at 100 micrograms/ml. In the tegumental syncytium, basal accumulations of secretory bodies (especially T2s) were evident in the earlier time periods but declined with longer incubations, until few secretory bodies remained in the syncytium overall. Blebbing of the apical plasma membrane and occasional areas of breakdown and sloughing of the tegument were observed over longer periods of treatment at 100 micrograms/ml. In the tegumental cell bodies, the Golgi complexes gradually decreased in size and activity, and few secretory bodies were produced. In the later time periods, the cells assumed abnormal shapes, the cytoplasm shrinking in towards the nucleus. In the vitelline follicles, a random dispersion of shell protein globules was evident within the intermediate-type cells, rather than their being organized into distinct shell globule clusters. Disruption of this process was more severe at the higher concentration of 100 micrograms/ml and again was more evident in tissue-slice material. In the latter, after prolonged (12 h) exposure to cytochalasin B, the intermediate and mature vitelline cells were filled with loosely packed and expanded shell globule clusters, containing few shell protein globules. The mature vitelline cells continued to lay down "yolk" globules and glycogen deposits. Disruption of the network of processes from the nurse cells was evident at the higher concentration of cytochalasin. Spaces began to appear between the vitelline cells and grew larger with progressively longer incubation periods, and the cells themselves assumed abnormal shapes. A number of binucleate stem cells were observed in tissue-slice material at the longest incubation period (12 h).