Multiple Intravenous Administrations of Human Umbilical Cord Blood Cells Benefit in a Mouse Model of ALS

Background: A promising therapeutic strategy for amyotrophic lateral sclerosis (ALS) is the use of cell-based therapies that can protect motor neurons and thereby retard disease progression. We recently showed that a single large dose (25x10(6) cells) of mononuclear cells from human umbilical cord blood (MNC hUCB) administered intravenously to pre-symptomatic G93A SOD1 mice is optimal in delaying disease progression and increasing lifespan. However, this single high cell dose is impractical for clinical use. The aim of the present pre-clinical translation study was therefore to evaluate the effects of multiple low dose systemic injections of MNC hUCB cell into G93A SOD1 mice at different disease stages. Methodology/Principal Findings: Mice received weekly intravenous injections of MNC hUCB or media. Symptomatic mice received 10(6) or 2.5x10(6) cells from 13 weeks of age. A third, pre-symptomatic, group received 10(6) cells from 9 weeks of age. Control groups were media-injected G93A and mice carrying the normal hSOD1 gene. Motor function tests and various assays determined cell effects. Administered cell distribution, motor neuron counts, and glial cell densities were analyzed in mouse spinal cords. Results showed that mice receiving 10(6) cells pre-symptomatically or 2.5x10(6) cells symptomatically significantly delayed functional deterioration, increased lifespan and had higher motor neuron counts than media mice. Astrocytes and microglia were significantly reduced in all cell-treated groups. Conclusions/Significance: These results demonstrate that multiple injections of MNC hUCB cells, even beginning at the symptomatic disease stage, could benefit disease outcomes by protecting motor neurons from inflammatory effectors. This multiple cell infusion approach may promote future clinical studies.

B Lymphocyte-Induced Maturation Protein-1 Contributes to Intestinal Mucosa Homeostasis by Limiting the Number of IL-17-Producing CD4(+) T Cells

The transcription factor B lymphocyte induced maturation protein-1 (Blimp-1) plays important roles in embryonic development and immunity. Blimp-1 is required for the differentiation of plasma cells, and mice with T cell specific deletion of Blimp-1 (Blimp-1CKO mice) develop a fatal inflammatory response in the colon. Previous work demonstrated that lack of Blimp-1 in CD4(+) and CD8(+) T cells leads to intrinsic functional defects, but little is known about the functional role of Blimp-1 in regulating differentiation of Th cells in vivo and their contribution to the chronic intestinal inflammation observed in the Blimp1CKO mice. In this study, we show that Blimp-1 is required to restrain the production of the inflammatory cytokine IL-17 by Th cells in vivo. Blimp-1CKO mice have greater numbers of IL-17 producing TCR beta(+)CD4(+)cells in lymphoid organs and in the intestinal mucosa. The increase in IL-17 producing cells was not restored to normal levels in wild-type and Blimp-1CKO mixed bone marrow chimeric mice, suggesting an intrinsic role for Blimp-1 in constraining the production of IL-17 in vivo. The observation that Blimp-1 deficient CD4(+) T cells are more prone to differentiate into IL-17(+)/IFN-gamma(+) cells and cause severe colitis when transferred to Rag1-deficient mice provides further evidence that Blimp-1 represses IL-17 production. Analysis of Blimp-1 expression at the single cell level during Th differentiation reveals that Blimp-1 expression is induced in Th1 and Th2 but repressed by TGF-beta in Th17 cells. Collectively, the results described here establish a new role for Blimp-1 in regulating IL-17 production in vivo. The Journal of Immunology, 2012,189: 5682-5693.

Electroluminescence and electric current response spectroscopy applied to the characterization of polymer light-emitting electrochemical cells

Frequency-dependent electroluminescence and electric current response spectroscopy were applied to polymeric light-emitting electrochemical cells in order to obtain information about the operation mechanism regimes of such devices. Three clearly distinct frequency regimes could be identified: a dielectric regime at high frequencies; an ionic transport regime, characterized by ionic drift and electronic diffusion; and an electrolytic regime, characterized by electronic injection from the electrodes and electrochemical doping of the conjugated polymer. From the analysis of the results, it was possible to evaluate parameters like the diffusion speed of electronic charge carriers in the active layer and the voltage drop necessary for operation. (C) 2012 American Institute of Physics. [http://dx.doi.org/10.1063/1.4752438]

Morphological Homogeneity of Neurons: Searching for Outlier Neuronal Cells

We report a morphology-based approach for the automatic identification of outlier neurons, as well as its application to the NeuroMorpho.org database, with more than 5,000 neurons. Each neuron in a given analysis is represented by a feature vector composed of 20 measurements, which are then projected into a two-dimensional space by applying principal component analysis. Bivariate kernel density estimation is then used to obtain the probability distribution for the group of cells, so that the cells with highest probabilities are understood as archetypes while those with the smallest probabilities are classified as outliers. The potential of the methodology is illustrated in several cases involving uniform cell types as well as cell types for specific animal species. The results provide insights regarding the distribution of cells, yielding single and multi-variate clusters, and they suggest that outlier cells tend to be more planar and tortuous. The proposed methodology can be used in several situations involving one or more categories of cells, as well as for detection of new categories and possible artifacts.

Quantum state tomography and quantum logical operations in a three qubits NMR quadrupolar system

In this work, we present an implementation of quantum logic gates and algorithms in a three effective qubits system, represented by a (I = 7/2) NMR quadrupolar nuclei. To implement these protocols we have used the strong modulating pulses (SMP) and the various stages of each implementation were verified by quantum state tomography (QST). The results for the computational base states, Toffolli logic gates, and Deutsch-Jozsa and Grover algorithms are presented here. Also, we discuss the difficulties and advantages of implementing such protocols using the SMP technique in quadrupolar systems.

WBC count and functional changes induced by co-administration of clofazimine and clarithromycin, in single and multiple doses, in Wistar rats

Clofazimine and clarithromycin are used to treat leprosy and infections caused by Mycobacterium avium complex. Little data on the toxicity of co-administration of these two drugs are available. Here we evaluated the potential adverse effects of polytherapy with these two drugs in male Wistar rats by determining WBCs counts and other blood cell counts, neutrophilic phagocytosis, and burst oxidative, by flow cytometry. We observed an increase in WBCs, in multiple-dose regimens, and in polymorphonuclear cells, in both single- clarithromycin only and multiple dose regimens. We also observed a reduction in mononuclear cell counts in single and multiple doses. The drugs seem to reverse the mononuclear and polymorphonuclear cell ratio. An increase in oxidative burst was observed in animals treated with the drugs administered either individually or combined. In conclusion, clofazimine and clarithromycin change WBCs counts. Our results may contribute for a better understanding of the mechanisms related to the effects of co-administrating the two drugs.

Dynamic Range of Vertebrate Retina Ganglion Cells: Importance of Active Dendrites and Coupling by Electrical Synapses

The vertebrate retina has a very high dynamic range. This is due to the concerted action of its diverse cell types. Ganglion cells, which are the output cells of the retina, have to preserve this high dynamic range to convey it to higher brain areas. Experimental evidence shows that the firing response of ganglion cells is strongly correlated with their total dendritic area and only weakly correlated with their dendritic branching complexity. On the other hand, theoretical studies with simple neuron models claim that active and large dendritic trees enhance the dynamic range of single neurons. Theoretical models also claim that electrical coupling between ganglion cells via gap junctions enhances their collective dynamic range. In this work we use morphologically reconstructed multi-compartmental ganglion cell models to perform two studies. In the first study we investigate the relationship between single ganglion cell dynamic range and number of dendritic branches/total dendritic area for both active and passive dendrites. Our results support the claim that large and active dendrites enhance the dynamic range of a single ganglion cell and show that total dendritic area has stronger correlation with dynamic range than with number of dendritic branches. In the second study we investigate the dynamic range of a square array of ganglion cells with passive or active dendritic trees coupled with each other via dendrodendritic gap junctions. Our results suggest that electrical coupling between active dendritic trees enhances the dynamic range of the ganglion cell array in comparison with both the uncoupled case and the coupled case with cells with passive dendrites. The results from our detailed computational modeling studies suggest that the key properties of the ganglion cells that endow them with a large dynamic range are large and active dendritic trees and electrical coupling via gap junctions.

Genotoxic and mutagenic effects of erythrosine B, a xanthene food dye, on HepG2 cells

Erythrosine (ErB) is a xanthene and an US Food and Drug Administration approved dye used in foods, drugs and cosmetics. Although its utilization is permitted, ErB is described as inhibitor of enzymes and protein-protein interactions and is toxic to pituitary and spermatogenesis processes. However, the genotoxicity and mutagenicity of ErB is inconclusive in the literature. This study aimed to analyze the genotoxicity of this dye using the alkaline comet assay and is the first investigation to evaluate ErB mutagenicity using the cytokinesis block micronucleus cytome (CBMN-Cyt) assay in HepG2 cells. These cells were chosen because they produce phase I and phase II enzymes that can mimic in vivo metabolism. The cells were treated with seven concentrations (0.1-70.0 mu g mL(-1)) of ErB, and the results showed genotoxicity at the two highest concentrations and mutagenicity at six concentrations. Furthermore, as micronuclei result from clastogenic and aneugenic processes, while comet assay is often considered more sensitive and detects DNA single strain breaks, we suggest that an aneugenic is responsible for the observed damage. Although ErB is approved for use in the food, cosmetic and pharmaceutical industries, it must be used carefully because it damages the DNA structure. (C) 2012 Elsevier Ltd. All rights reserved.

Ethanol induces vascular relaxation via redox-sensitive and nitric oxide-dependent pathways

We investigated the role of reactive oxygen species (ROS) and nitric oxide (NO) in ethanol-induced relaxation. Vascular reactivity experiments showed that ethanol (0.03-200 mmol/L) induced relaxation in endothelium-intact and denuded rat aortic rings isolated from male Wistar rats. Pre-incubation of intact or denuded rings with L-NAME (non selective NOS inhibitor, 100 mu mol/L), 7-nitroindazole (selective nNOS inhibitor, 100 mu mol/L), ODQ (selective inhibitor of guanylyl cyclase enzyme, I mu mol/L), glibenclamide (selective blocker of ATP-sensitive K+ channels, 3 mu mol/L) and 4-aminopyridine (selective blocker of voltage-dependent K+ channels, 4-AP, 1 mmol/L) reduced ethanol-induced relaxation. Similarly, tiron (superoxide anion (O-2(-)) scavenger, 1 mmol/L) and catalase (hydrogen peroxide (H2O2) scavenger, 300 U/mL) reduced ethanol-induced relaxation to a similar extent in both endothelium-intact and denuded rings. Finally, prodifen (non-selective cytochrome P450 enzymes inhibitor, 10 mu mol/L) and 4-methylpyrazole (selective alcohol dehydrogenase inhibitor, 10 mu mol/L) reduced ethanol-induced relaxation. In cultured aortic vascular smooth muscle cells (VSMCs), ethanol stimulated generation of NO, which was significantly inhibited by L-NAME. In endothelial cells, flow cytometry studies showed that ethanol increased cytosolic Ca2+ concentration ([Ca2+]c), O-2(-) and cytosolic NO concentration ([NO]c). Tiron inhibited ethanol-induced increase in [Ca-2]c and [NO]c. The major new finding of this work is that ethanol induces relaxation via redox-sensitive and NO-cGMP-dependent pathways through direct effects on ROS production and NO signaling. These findings identify putative molecular mechanisms whereby ethanol, at pharmacological concentrations, influences vascular reactivity. (C) 2011 Elsevier Inc. All rights reserved.

A new NO donor failed to release NO and to induce relaxation in the rat basilar artery

Nitric oxide (NO)-donors are pharmacologically active substances that in vivo or in vitro release NO. Their most common side effect is headache caused by cerebral vasodilatation. We previously demonstrated that the new NO-donor Ru(terpy)(bdq)NO](3+) (Terpy), synthesized in our laboratory, induces relaxation of rat aorta. This study aimed to verify the effect of Terpy and sodium nitroprusside (SNP) in basilar artery. We conducted vascular reactivity experiments on endothelium-denuded basilar rings. The concentrations of iron (Fe) and ruthenium (Ru) complex were analyzed in basilar artery lysates after incubation with NO donors by mass spectrometry. We also evaluated the NO released from SNP and Terpy by using confocal microscopy. Interestingly, Terpy did not induce relaxation of the basilar artery. SNP induced relaxation in a concentration-dependent way. NO donors cross the membrane of vascular smooth muscle and entered the cell. In spite of its permeability, Terpy did not release NO in the basilar artery. Otherwise, SNP released NO in the basilar artery cells cytoplasm. Taken together, our results demonstrate that the new NO donor (Terpy) failed to release NO and to induce relaxation in the basilar artery. The NO donor SNP induces vascular relaxation due to NO release in the vascular smooth muscle cells. (C) 2011 Elsevier B.V. All rights reserved.

Impaired beta-adrenoceptor-induced relaxation in small mesenteric arteries from DOCA-salt hypertensive rats is due to reduced K-Ca channel activity

beta-Adrenoceptor (beta-AR)-mediated relaxation plays an important role in the regulation of vascular tone. beta-AR-mediated vascular relaxation is reduced in various disease states and aging. We hypothesized that beta-AR-mediated vasodilatation is impaired in DOCA-salt hypertension due to alterations in the cAMP pathway. beta-AR-mediated relaxation was determined in small mesenteric arteries from DOCA-salt hypertensive and control uninephrectomized (Uni) rats. To exclude nitric oxide (NO) and cyclooxygenase (COX) pathways, relaxation responses were determined in the presence of L-NNA and indomethacin, NO synthase inhibitor and COX inhibitors, respectively. Isoprenaline (ISO)-induced relaxation was reduced in arteries from DOCA-salt compared to Uni rats. Protein kinase A (PKA) inhibitors (H89 or Rp-cAMPS) or adenylyl cyclase inhibitor (SQ22536) did not abolish the difference in ISO-induced relaxation between the groups. Forskolin (adenylyl cyclase activator)-induced relaxation was similar between the groups. The inhibition of IKCa/SKCa channels (TRAM-34 plus UCL1684) or BKCa channels (iberiotoxin) reduced ISO-induced relaxation only in Uni rats and abolished the relaxation differences between the groups. The expression of SKCa channel was decreased in DOCA-salt arteries. The expression of BKCa channel a subunit was increased whereas the expression of BKCa channel p subunit was decreased in DOCA-salt arteries. The expression of receptor for activated C kinase 1 (RACK1), which is a binding protein for BKG, channel and negatively modulates its activity, was increased in DOCA-salt arteries. These results suggest that the impairment of beta-AR-mediated relaxation in DOCA-salt mesenteric arteries may be attributable to altered IKCa/SKCa and/or BKCa channels activities rather than cAMP/PKA pathway. Impaired beta-AR-stimulated BKCa channel activity may be due to the imbalance between its subunit expressions and RACK1 upregulation. (C) 2012 Elsevier Ltd. All rights reserved.

Dynamic symmetry loss of high-frequency hysteresis loops in single-domain particles with uniaxial anisotropy

Understanding how magnetic materials respond to rapidly varying magnetic fields, as in dynamic hysteresis loops, constitutes a complex and physically interesting problem. But in order to accomplish a thorough investigation, one must necessarily consider the effects of thermal fluctuations. Albeit being present in all real systems, these are seldom included in numerical studies. The notable exceptions are the Ising systems, which have been extensively studied in the past, but describe only one of the many mechanisms of magnetization reversal known to occur. In this paper we employ the Stochastic Landau-Lifshitz formalism to study high-frequency hysteresis loops of single-domain particles with uniaxial anisotropy at an arbitrary temperature. We show that in certain conditions the magnetic response may become predominantly out-of-phase and the loops may undergo a dynamic symmetry loss. This is found to be a direct consequence of the competing responses due to the thermal fluctuations and the gyroscopic motion of the magnetization. We have also found the magnetic behavior to be exceedingly sensitive to temperature variations, not only within the superparamagnetic-ferromagnetic transition range usually considered, but specially at even lower temperatures, where the bulk of interesting phenomena is seen to take place. (C) 2011 Elsevier B.V. All rights reserved.

Early transplantation of human immature dental pulp stem cells from baby teeth to golden retriever muscular dystrophy (GRMD) dogs: Local or systemic?

Background:The golden retriever muscular dystrophy (GRMD) dogs represent the best available animal model for therapeutic trials aiming at the future treatment of human Duchenne muscular dystrophy (DMD). We have obtained a rare litter of six GRMD dogs (3 males and 3 females) born from an affected male and a carrier female which were submitted to a therapeutic trial with adult human stem cells to investigate their capacity to engraft into dogs muscles by local as compared to systemic injection without any immunosuppression. Methods Human Immature Dental Pulp Stem Cells (hIDPSC) were transplanted into 4 littermate dogs aged 28 to 40 days by either arterial or muscular injections. Two non-injected dogs were kept as controls. Clinical translation effects were analyzed since immune reactions by blood exams and physical scores capacity of each dog. Samples from biopsies were checked by immunohistochemistry (dystrophin markers) and FISH for human probes. Results and Discussion We analyzed the cells' ability in respect to migrate, engraftment, and myogenic potential, and the expression of human dystrophin in affected muscles. Additionally, the efficiency of single and consecutive early transplantation was compared. Chimeric muscle fibers were detected by immunofluorescence and fluorescent in situ hybridisation (FISH) using human antibodies and X and Y DNA probes. No signs of immune rejection were observed and these results suggested that hIDPSC cell transplantation may be done without immunosuppression. We showed that hIDPSC presented significant engraftment in GRMD dog muscles, although human dystrophin expression was modest and limited to several muscle fibers. Better clinical condition was also observed in the dog, which received monthly arterial injections and is still clinically stable at 25 months of age. Conclusion Our data suggested that systemic multiple deliveries seemed more effective than local injections. These findings open important avenues for further researches.

Hyaluronidase recruits mesenchymal-like cells to the lung and ameliorates fibrosis

Hyaluronidases (HYALs) comprise a group of enzymes that degrade hyaluronic acid (HA). In this report, we reveal that a single intranasal inoculation of HYAL induces an increase in mononuclear cells within the bronchoalveolar space demonstrating a mesenchymal-like phenotype, expressing stem cell antigen-1 (SCA-1), CD44 and CD73 but not CD34, CD45, CD3, CD4, CD8 or CD19. This influx of mesenchymal stem cell (MSC)-like cells was dependent on leukotriene production within the lung parenchyma. These findings prompted experiments demonstrating that HYAL treatment potently blocked bleomycin-induced lung injury and fibrosis while decreasing transforming growth factor (TGF)-β production and collagen deposition. These data suggest that HYAL is a novel and promising tool to use autologous MSC-like cells in the treatment of pulmonary fibrosis.

Toll-like receptor 1 N248S single-nucleotide polymorphism is associated with leprosy risk and regulates immune activation during mycobacterial infection

Conflicting findings about the association between leprosy and TLR1 variants N248S and I602S have been reported. Here, we performed case-control and family based studies, followed by replication in 2 case-control populations from Brazil, involving 3162 individuals. Results indicated an association between TLR1 248S and leprosy in the case-control study (SS genotype odds ratio [OR], 1.81; P = .004) and the family based study (z = 2.02; P = .05). This association was consistently replicated in other populations (combined OR, 1.51; P < .001), corroborating the finding that 248S is a susceptibility factor for leprosy. Additionally, we demonstrated that peripheral blood mononuclear cells (PBMCs) carrying 248S produce a lower tumor necrosis factor/interleukin-10 ratio when stimulated with Mycobacterium leprae but not with lipopolysaccharide or PAM3cysK4. The same effect was observed after infection of PBMCs with the Moreau strain of bacillus Calmette-Guerin but not after infection with other strains. Finally, molecular dynamics simulations indicated that the Toll-like receptor 1 structure containing 248S amino acid is different from the structure containing 248N. Our results suggest that TLR1 248S is associated with an increased risk for leprosy, consistent with its hypoimmune regulatory function.