Preclinical Studies with Umbilical Cord Mesenchymal Stromal Cells in Different Animal Models for Muscular Dystrophy

Umbilical cord mesenchymal stromal cells (MSC) have been widely investigated for cell-based therapy studies as an alternative source to bone marrow transplantation. Umbilical cord tissue is a rich source of MSCs with potential to derivate at least muscle, cartilage, fat, and bone cells in vitro. The possibility to replace the defective muscle cells using cell therapy is a promising approach for the treatment of progressive muscular dystrophies (PMDs), independently of the specific gene mutation. Therefore, preclinical studies in different models of muscular dystrophies are of utmost importance. The main objective of the present study is to evaluate if umbilical cord MSCs have the potential to reach and differentiate into muscle cells in vivo in two animal models of PMDs. In order to address this question we injected (1) human umbilical cord tissue (hUCT) MSCs into the caudal vein of SJL mice; (2) hUCT and canine umbilical cord vein (cUCV) MSCs intra-arterially in GRMD dogs. Our results here reported support the safety of the procedure and indicate that the injected cells could engraft in the host muscle in both animal models but could not differentiate into muscle cells. These observations may provide important information aiming future therapy for muscular dystrophies.

In situ delivery of bone marrow cells and mesenchymal stem cells improves cardiovascular function in hypertensive rats submitted to myocardial infarction

This work aimed to evaluate cardiac morphology/function and histological changes induced by bone marrow cells (BMCs) and cultured mesenchymal stem cells (MSCs) injected at the myocardium of spontaneously hypertensive rats (SHR) submitted to surgical coronary occlusion. Female syngeneic adult SHR, submitted (MI) or not (C) to coronary occlusion, were treated 24 h later with in situ injections of normal medium (NM), or with MSCs (MSC) or BMCs (BM) from male rats. The animals were evaluated after 1 and 30 days by echocardiography, histology of heart sections and PCR for the Y chromosome. Improved ejection fraction and reduced left ventricle infarcted area were observed in MSC rats as compared to the other experimental groups. Treated groups had significantly reduced lesion tissue score, increased capillary density and normal (not-atrophied) myocytes, as compared to NM and C groups. The survival rate was higher in C, NM and MSC groups as compared to MI and BM groups. In situ injection of both MSCs and BMCs resulted in improved cardiac morphology, in a more physiological model of myocardial infarction represented by surgical coronary occlusion of spontaneously hypertensive rats. Only treatment with MSCs, however, ameliorated left ventricle dysfunction, suggesting a positive role of these cells in heart remodeling in infarcted hypertensive subjects.

Leptin's effect on puberty in mice is relayed by the ventral premammillary nucleus and does not require signaling in Kiss1 neurons

Studies m hum ins and rodents indicate that a minimum amount of stored energy is required for normal pubertal development The adipocyte-derived hormone leptin is a key metabolic signal to the neuroendocrine reproductive axis Humans and mice lacking leptin or the leptin receptor (LepR) (ob/ob and db/db mice, respectively) are infertile and fail to enter puberty Leptin administration to leptin-deficient subjects and ob/ob mice induces puberty and restores fertility, but the exact site or sites of leptin action are unclear Here, we found that genetic deletion of LepR selectively from hypothalamic Kiss1 neurons m mice had no effect on puberty or fertility, indicating that direct leptin signaling m Kiss1 neurons is not required for these processes However, bilateral lesions of the ventral premammillary nucleus (PMV) of ob/ob mice blunted the ability of exogenous leptin to induce sexual maturation Moreover, unilateral reexpression of endogenous LepR m PMV neurons was sufficient to induce puberty and improve fertility m female LepR-null mice This LepR reexpression also normalized the increased hypothalamic GnRH content characteristic of leptin-signaling deficiency These data suggest that the PMV is a key site for leptin's permissive action at the onset of puberty and support the hypothesis that the multiple actions of leptin to control metabolism and reproduction at e anatomically dissociated

Expression of an activating mutation in the gene encoding the K(ATP) channel subunit Kir6.2 in mouse pancreatic beta cells recapitulates neonatal diabetes

Neonatal diabetes is a rare monogenic form of diabetes that usually presents within the first six months of life. It is commonly caused by gain-of-function mutations in the genes encoding the Kir6.2 and SUR1 subunits of the plasmalemmal ATP-sensitive K(+) (K(ATP)) channel. To better understand this disease, we generated a mouse expressing a Kir6.2 mutation (V59M) that causes neonatal diabetes in humans and we used Cre-lox technology to express the mutation specifically in pancreatic beta cells. These beta-V59M mice developed severe diabetes soon after birth, and by 5 weeks of age, blood glucose levels were markedly increased and insulin was undetectable. Islets isolated from beta-V59M mice secreted substantially less insulin and showed a smaller increase in intracellular calcium in response to glucose. This was due to a reduced sensitivity of K(ATP) channels in pancreatic beta cells to inhibition by ATP or glucose. In contrast, the sulfonylurea tolbutamide, a specific blocker of K(ATP) channels, closed K(ATP) channels, elevated intracellular calcium levels, and stimulated insulin release in beta-V59M beta cells, indicating that events downstream of K(ATP) channel closure remained intact. Expression of the V59M Kir6.2 mutation in pancreatic beta cells alone is thus sufficient to recapitulate the neonatal diabetes observed in humans. beta-V59M islets also displayed a reduced percentage of beta cells, abnormal morphology, lower insulin content, and decreased expression of Kir6.2, SUR1, and insulin mRNA. All these changes are expected to contribute to the diabetes of beta-V59M mice. Their cause requires further investigation.

Leukotriene B(4) amplifies NF-kappa B activation in mouse macrophages by reducing SOCS1 inhibition of MyD88 expression

Activation of NF-kappa B and 5-lipoxygenase-mediated (5-LO-mediated) biosynthesis of the lipid mediator leukotriene B(4) (LTB(4)) are pivotal components of host defense and inflammatory responses. However, the role of LTB(4) in mediating innate immune responses elicited by specific TLR ligands and cytokines is unknown. Here we have shown that responses dependent on MyD88 (an adaptor protein that mediates signaling through all of the known TLRs, except TLR3, as well as IL-1 beta and IL-18) are reduced in mice lacking either 5-LO or the LTB(4) receptor BTL1, and that macrophages from these mice are impaired in MyD88-dependent activation of NF-kappa B. This macrophage defect was associated with lower basal and inducible expression of MyD88 and reflected impaired activation of STAT1 and overexpression of the STAT1 inhibitor SOCS1. Expression of MyD88 and responsiveness to the TLR4 ligand LPS were decreased by Stat1 siRNA silencing in WT macrophages and restored by Socs1 siRNA in 5-LO-deficient macrophages. These results uncover a pivotal role in macrophages for the GPCR BLT1 in regulating activation of NF-kappa B through Stat1-dependent expression of MyD88.

Intracellular Ca(2+) Regulation During Neuronal Differentiation of Murine Embryonal Carcinoma and Mesenchymal Stem Cells

Changes in intracellular Ca(2+) concentration ([Ca(2+)](i)) play a central role in neuronal differentiation. However, Ca(2+) signaling in this process remains poorly understood and it is unknown whether embryonic and adult stem cells share the same signaling pathways. To clarify this issue, neuronal differentiation was analyzed in two cell lines: embryonic P19 carcinoma stem cells (CSCs) and adult murine bone-marrow mesenchymal stem cells (MSC). We studied Ca(2+) release from the endoplasmic reticulum via intracellular ryanodine-sensitive (RyR) and IP(3)-sensitive (IP(3)R) receptors. We observed that caffeine, a RyR agonist, induced a [Ca(2+)](i) response that increased throughout neuronal differentiation. We also demonstrated a functional coupling between RyRs and L-but not with N-, P-, or Q-type Ca(v)1 Ca(2+) channels, both in embryonal CSC and adult MSC. We also found that agonists of L-type channels and of RyRs increase neurogenesis and neuronal differentiation, while antagonists of these channels have the opposite effect. Thus, our data demonstrate that in both cell lines RyRs control internal Ca(2+) release following voltage-dependent Ca(2+) entry via L-type Ca(2+) channels. This study shows that both in embryonal CSC and adult MSC [Ca(2+)](i) is controlled by a common pathway, indicating that coupling of L-type Ca(2+) channels and RyRs may be a conserved mechanism necessary for neuronal differentiation.

Aerobic exercise training improves Ca(2+) handling and redox status of skeletal muscle in mice

Exercise training is known to promote relevant changes in the properties of skeletal muscle contractility toward powerful fibers. However, there are few studies showing the effect of a well-established exercise training protocol on Ca(2+) handling and redox status in skeletal muscles with different fiber-type compositions. We have previously standardized a valid and reliable protocol to improve endurance exercise capacity in mice based on maximal lactate steady-state workload (MLSSw). The aim of this study was to investigate the effect of exercise training, performed at MLSSw, on the skeletal muscle Ca(2+) handling-related protein levels and cellular redox status in soleus and plantaris. Male C57BL/6J mice performed treadmill training at MLSSw over a period of eight weeks. Muscle fiber-typing was determined by myosin ATPase histochemistry, citrate synthase activity by spectrophotometric assay, Ca(2+) handling-related protein levels by Western blot and reduced to oxidized glutathione ratio (GSH:GSSG) by high-performance liquid chromatography. Trained mice displayed higher running performance and citrate synthase activity compared with untrained mice. Improved running performance in trained mice was paralleled by fast-to-slow fiber-type shift and increased capillary density in both plantaris and soleus. Exercise training increased dihydropyridine receptor (DHPR) alpha 2 subunit, ryanodine receptor and Na(+)/Ca(2+) exchanger levels in plantaris and soleus. Moreover, exercise training elevated DHPR beta 1 subunit and sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) 1 levels in plantaris and SERCA2 levels in soleus of trained mice. Skeletal muscle GSH content and GSH:GSSG ratio was increased in plantaris and soleus of trained mice. Taken together, our findings indicate that MLSSw exercise-induced better running performance is, in part, due to increased levels of proteins involved in skeletal muscle Ca(2+) handling, whereas this response is partially dependent on specificity of skeletal muscle fiber-type composition. Finally, we demonstrated an augmented cellular redox status and GSH antioxidant capacity in trained mice.

PKC beta II inhibition attenuates myocardial infarction induced heart failure and is associated with a reduction of fibrosis and pro-inflammatory responses

Protein kinase C beta II (PKC beta II) levels increase in the myocardium of patients with end-stage heart failure (HF). Also targeted overexpression of PKC beta II in the myocardium of mice leads to dilated cardiomyopathy associated with inflammation, fibrosis and myocardial dysfunction. These reports suggest a deleterious role of PKC beta II in HF development. Using a post-myocardial infarction (MI) model of HF in rats, we determined the benefit of chronic inhibition of PKC beta II on the progression of HF over a period of 6 weeks after the onset of symptoms and the cellular basis for these effects. Four weeks after MI, rats with HF signs that were treated for 6 weeks with the PKC beta II selective inhibitor (beta IIV5-3 conjugated to TAT(47-57) carrier peptide) (3 mg/kg/day) showed improved fractional shortening (from 21% to 35%) compared to control (TAT(47-57) carrier peptide alone). Formalin-fixed mid-ventricle tissue sections stained with picrosirius red, haematoxylin and eosin and toluidine blue dyes exhibited a 150% decrease in collagen deposition, a two-fold decrease in inflammation and a 30% reduction in mast cell degranulation, respectively, in rat hearts treated with the selective PKC beta II inhibitor. Further, a 90% decrease in active TGF beta 1 and a significant reduction in SMAD2/3 phosphorylation indicated that the selective inhibition of PKC beta II attenuates cardiac remodelling mediated by the TGF-SMAD signalling pathway. Therefore, sustained selective inhibition of PKC beta II in a post-MI HF rat model improves cardiac function and is associated with inhibition of pathological myocardial remodelling.

LEACHATE TREATMENT PROCESS AT A MUNICIPAL STABILIZED LANDFILL BY CATALYTIC OZONATION: AN EXPLORATORY STUDY FROM TAGUCHI ORTHOGONAL ARRAY

Catalytic ozonation has been recognized in the scientific community as an efficient technique, reaching elevated rates of recalcitrant organic material mineralization, even at the presence of scavenger species of hydroxyl free radicals. This study presents the most significant factors involving the leachate treatment stabilized by the municipal landfill of the city of Guaratingueta, State of Sao Paulo, Brazil, by using a catalytic ozonation activated by metallic ions Fe(3+), Zn(2+), Mn(2+), Ni(2+) and Cr(3+). The Taguchi L(16) orthogonal array and its associated statistical methods were also used in this study. Among the researched ions, the most notable catalysis was obtained with ferric ion, statistically significant in the reduction of COD with a confidence level of 99.5%.

Ethanol-induced vasoconstriction is mediated via redox-sensitive cyclo-oxygenase-dependent mechanisms

The present study investigated the role of ROS (reactive oxygen species) and COX (cyclooxygenase) in ethanol-induced contraction and elevation of [Ca(2+)](i) (intracellular [Ca(2+)]). Vascular reactivity experiments, using standard muscle bath procedures, showed that ethanol (1-800 mmol/l) induced contraction in endothelium-intact (EC(50): 306 +/- 34 mmol/l) and endothelium-denuded (EC(50): 180 +/- 40 mmol/l) rat aortic rings. Endothelial removal enhanced ethanol-induced contraction. Preincubation of intact rings with L-NAME [N(G)-nitro-L-arginine methyl ester; non-selective NOS (NO synthase) inhibitor, 100 mu mol/l], 7-nitroindazole [selective nNOS (neuronal NOS) inhibitor, 100 mu mol/l], oxyhaemoglobin (NO scavenger, 10 mu mol/l) and ODQ (selective inhibitor of guanylate cyclase enzyme, 1 mu mol/l) increased ethanol-induced contraction. Tiron [O(2)(-) (superoxide anion) scavenger, 1 mmol/l] and catalase (H(2)O(2) scavenger, 300 units/ml) reduced ethanol-induced contraction to a similar extent in both endothelium-intact and denuded rings. Similarly, indomethacin (non-selective COX inhibitor, 10 mu mol/l), SC560 (selective COX- I inhibitor, 1 mu mol/l), AH6809 [PGF(2 alpha) (prostaglandin F(2 alpha))] receptor antagonist, 10 mu mol/l] or SQ29584 [PGH(2)(prostaglandin H(2))/TXA(2) (thromboxane A(2)) receptor antagonist, 3 mu mol/l] inhibited ethanol-induced contraction in aortic rings with and without intact endothelium. In cultured aortic VSMCs (vascular smooth muscle cells), ethanol stimulated generation of O(2)(-) and H(2)O(2). Ethanol induced a transient increase in [Ca(2+)](i), which was significantly inhibited in VSMCs pre-exposed to tiron or indomethacin. Our data suggest that ethanol induces vasoconstriction via redox-sensitive and COX-dependent pathways, probably through direct effects on ROS production and Ca(2+) signalling. These findings identify putative molecular mechanisms whereby ethanol, at high concentrations, influences vascular reactivity. Whether similar phenomena occur in vivo at lower concentrations of ethanol remains unclear.

On Building a Memory Evolutive System for Application to Learning and Cognition Modeling

We address here aspects of the implementation of a memory evolutive system (MES), based on the model proposed by A. Ehresmann and J. Vanbremeersch (2007), by means of a simulated network of spiking neurons with time dependent plasticity. We point out the advantages and challenges of applying category theory for the representation of cognition, by using the MES architecture. Then we discuss the issues concerning the minimum requirements that an artificial neural network (ANN) should fulfill in order that it would be capable of expressing the categories and mappings between them, underlying the MES. We conclude that a pulsed ANN based on Izhikevich`s formal neuron with STDP (spike time-dependent plasticity) has sufficient dynamical properties to achieve these requirements, provided it can cope with the topological requirements. Finally, we present some perspectives of future research concerning the proposed ANN topology.

Vibration-enhanced posture stabilization achieved by tactile supplementation: May blind individuals get extra benefits?

Diminished balance ability poses a serious health risk due to the increased likelihood of falling, and impaired postural stability is significantly associated with blindness and poor vision. Noise stimulation (by improving the detection of sub-threshold somatosensory information) and tactile supplementation (i.e. additional haptic information provided by an external contact surface) have been shown to improve the performance of the postural control system. Moreover, vibratory noise added to the source of tactile supplementation (e.g. applied to a surface that the fingertip touches) has been shown to enhance balance stability more effectively than tactile supplementation alone. In view of the above findings, in addition to the well established consensus that blind subjects show superior abilities in the use of tactile information, we hypothesized that blind subjects may take extra benefits from the vibratory noise added to the tactile supplementation and hence show greater improvements in postural stability than those observed for sighted subjects. If confirmed, this hypothesis may lay the foundation for the development of noise-based assistive devices (e.g. canes, walking sticks) for improving somatosensation and hence prevent falls in blind individuals. (C) 2011 Elsevier Ltd. All rights reserved.

The generalized log-gamma mixture model with covariates: local influence and residual analysis

In a sample of censored survival times, the presence of an immune proportion of individuals who are not subject to death, failure or relapse, may be indicated by a relatively high number of individuals with large censored survival times. In this paper the generalized log-gamma model is modified for the possibility that long-term survivors may be present in the data. The model attempts to separately estimate the effects of covariates on the surviving fraction, that is, the proportion of the population for which the event never occurs. The logistic function is used for the regression model of the surviving fraction. Inference for the model parameters is considered via maximum likelihood. Some influence methods, such as the local influence and total local influence of an individual are derived, analyzed and discussed. Finally, a data set from the medical area is analyzed under the log-gamma generalized mixture model. A residual analysis is performed in order to select an appropriate model.

Influence diagnostics for polyhazard models in the presence of covariates

In this paper, we present various diagnostic methods for polyhazard models. Polyhazard models are a flexible family for fitting lifetime data. Their main advantage over the single hazard models, such as the Weibull and the log-logistic models, is to include a large amount of nonmonotone hazard shapes, as bathtub and multimodal curves. Some influence methods, such as the local influence and total local influence of an individual are derived, analyzed and discussed. A discussion of the computation of the likelihood displacement as well as the normal curvature in the local influence method are presented. Finally, an example with real data is given for illustration.

Direct effect of Plasmodium vivax recombinant vaccine candidates AMA-1 and MSP-1(19) on the innate immune response

The recombinant apical membrane antigen 1 (AMA-1) and 19-kDa fragment of merozoite surface protein (MSP-1(19)) are the lead candidates for inclusion in a vaccine against blood stages of malaria due to encouraging protective studies in humans and animals. Despite the importance of an efficacious malaria vaccine, vaccine-related research has focused on identifying antigens that result in protective immunity rather than determining the nature of anti-malarial immune effector mechanisms. Moreover, emphasis has been placed on adaptive rather than innate immune responses. In this study, we investigated the effect of Plasmodium vivax vaccine candidates Pv-AMA-1 and Pv-MSP-1(19) on the immune response of malaria-naive donors. Maturation of dendritic cells is altered by Pv-AMA-1 but not Pv-MSP-1(19), as observed by differential expression of cell surface markers. In addition, Pv-AMA-1 induced an increased production of MIP-1 alpha/CCL3 and decreased production of TARC/CCL17 levels in both dendritic cells (DCs) and peripheral blood mononuclear cells (PBMCs). Finally, a significant pro-inflammatory response was elicited by Pv-AMA-1-stimulated PBMCs. These results suggest that the recombinant vaccine candidate Pv-AMA-1 may play a direct role on innate immune response and might be involved in parasite destruction. (C) 2007 Elsevier Ltd. All rights reserved.