Human multipotent adipose-derived stem cells restore dystrophin expression of Duchenne skeletal-muscle cells in vitro

Background information. DMD (Duchenne muscular dystrophy) is a devastating X-linked disorder characterized by progressive muscle degeneration and weakness. The use of cell therapy for the repair of defective muscle is being pursued as a possible treatment for DMD. Mesenchymal stem cells have the potential to differentiate and display a myogenic phenotype in vitro. Since liposuctioned human fat is available in large quantities, it may be an ideal source of stem cells for therapeutic applications. ASCs (adipose-derived stem cells) are able to restore dystrophin expression in the muscles of mdx (X-linked muscular dystrophy) mice. However, the outcome when these cells interact with human dystrophic muscle is still unknown. Results. We show here that ASCs participate in myotube formation when cultured together with differentiating human DMD myoblasts, resulting in the restoration of dystrophin expression. Similarly, dystrophin was induced when ASCs were co-cultivated with DMD myotubes. Experiments with GFP (green fluorescent protein)-positive ASCs and DAPI (4,6-diamidino-2-phenylindole)-stained DMD myoblasts indicated that ASCs participate in human myogenesis through cellular fusion. Conclusions. These results show that ASCs have the potential to interact with dystrophic muscle cells, restoring dystrophin expression of DMD cells in vitro. The possibility of using adipose tissue as a source of stem cell therapies for muscular diseases is extremely exciting.

Exercise prevents the effects of experimental arthritis on the metabolism and function of immune cells

Active lymphocytes (LY) and macrophages (M Phi) are involved in the pathophysiology of rheumatoid arthritis (RA) Due to its anti-inflammatory effect. physical exercise may be beneficial in RA by acting on the immune system (IS) Thus, female Wistar rats with type II collagen-induced arthritis (CIA) were submitted to swimming training (6 weeks. 5 days/week. 60 min/day) and some biochemical and immune parameters, such as the metabolism of glucose and glutamine and function of LY and M. were evaluated In addition, plasma levels of some hormones and of interleukin-2 (IL-2) were also determined Results demonstrate that CIA increased lymphocyte proliferation (1.9- and 1 7-fold, respectively, in response to concanavalin A (ConA) and lipopolysaccharide (LPS)), as well as macrophage H(2)O(2) production (1 6-fold), in comparison to control Exercise training prevented the activation of immune cells, induced by CIA. and established a pattern of substrate utilization similar to that described as normal for these cells. Exercise also promoted an elevation of plasma levels of corticosterone (22 2%), progesterone (1 7-fold) and IL-2 (2 6-fold) Our data suggest that chronic exercise is able to counterbalance the effects of CIA on cells of the IS. reinforcing the proposal that the benefits of exercise may not be restricted to aerobic capacity and/or strength improvement Copyright (C) 2010 John Wiley & Sons, Ltd

Organized Crime and Terrorism: From the Cells Towards Political Communication, A Case Study

During the first half of 2006 the city of Sao Paulo suffered three series of violent attacks against the security forces, civilians, and the government. The violent campaign also included a massive rebellion in prisons and culminated in the kidnapping of a journalist and the broadcast of a manifesto from the criminal organization PCC threatening the police and the government. Right after, the main device used to contain organized crime in the prisons was declared unconstitutional. This episode represents a prototypical example of the use of media-focused terrorism by organized crime for projection into the political communication arena.

SJL dystrophic mice express a significant amount of human muscle proteins following systemic delivery of human adipose-derived stromal cells without immunosuppression

Limb-girdle muscular dystrophies (LGMDs) are a heterogeneous group of disorders characterized by progressive degeneration of skeletal muscle caused by the absence of or defective muscular proteins. The murine model for limb-girdle muscular dystrophy 2B (LGMD2B), the SJL mice, carries a deletion in the dysferlin gene that causes a reduction in the protein levels to 15% of normal. The mice show muscle weakness that begins at 4-6 weeks and is nearly complete by 8 months of age. The possibility of restoring the defective muscle protein and improving muscular performance by cell therapy is a promising approach for the treatment of LGMDs or other forms of progressive muscular dystrophies. Here we have injected human adipose stromal cells (hASCs) into the SJL mice, without immunosuppression, aiming to assess their ability to engraft into recipient dystrophic muscle after systemic delivery; form chimeric human/mouse muscle fibers; express human muscle proteins in the dystrophic host and improve muscular performance. We show for the first time that hASCs are not rejected after systemic injection even without immunosuppression, are able to fuse with the host muscle, express a significant amount of human muscle proteins, and improve motor ability of injected animals. These results may have important applications for future therapy in patients with different forms of muscular dystrophies.

Tick saliva inhibits the chemotactic function of MIP-1 alpha and selectively impairs chemotaxis of immature dendritic cells by down-regulating cell-surface CCR5

Ticks are blood-feeding arthropods that secrete immunomodulatory molecules through their saliva to antagonize host inflammatory and immune responses. As dendritic cells (DCs) play a major role in host immune responses, we studied the effects of Rhipicephalus sanguineus tick saliva on DC migration and function. Bone marrow-derived immature DCs pre-exposed to tick saliva showed reduced migration towards macrophage inflammatory protein (MIP)-1 alpha, MIP-1 beta and regulated upon activation, normal T cell expressed and secreted (RANTES) chemokines in a Boyden microchamber assay. This inhibition was mediated by saliva which significantly reduced the percentage and the average cell-surface expression of CC chemokine receptor CCR5. In contrast, saliva did not alter migration of DCs towards MIP-3 beta, not even if the cells were induced for maturation. Next, we evaluated the effect of tick saliva on the activity of chemokines related to DC migration and showed that tick saliva per se inhibits the chemotactic function of MIP-1 alpha, while it did not affect RANTES, MIP-1 beta and MIP-3 beta. These data suggest that saliva possibly reduces immature DC migration, while mature DC chemotaxis remains unaffected. In support of this, we have analyzed the percentage of DCs on mice 48 h after intradermal inoculation with saliva and found that the DC turnover in the skin was reduced compared with controls. Finally, to test the biological activity of the saliva-exposed DCs, we transferred DCs pre-cultured with saliva and loaded with the keyhole limpet haemocyanin (KLH) antigen to mice and measured their capacity to induce specific T cell cytokines. Data showed that saliva reduced the synthesis of both T helper (Th)1 and Th2 cytokines, suggesting the induction of a non-polarised T cell response. These findings propose that the inhibition of DCs migratory ability and function may be a relevant mechanism used by ticks to subvert the immune response of the host. (c) 2007 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Tick saliva induces regulatory dendritic cells: MAP-kinases and Toll-like receptor-2 expression as potential targets

Ticks (Acari: Ixodidae) are bloodsucking ectoparasitic arthropods of human and veterinary medical importance. Tick saliva has been shown to contain a wide range of bioactive molecules with vasodilatory, antihemostatic, and immunomodulatory activities. We have previously demonstrated that saliva from Rhipicephalus sanguineus ticks inhibits the maturation of dendritic cells (DCs) stimulated with LPS. Here we examined the mechanism of this immune subversion, evaluating the effect of tick saliva on Toll-like receptor (TLR)-4 signalling pathway in bone marrow-derived DCs. We demonstrated that R. sanguineus tick saliva impairs maturation of DCs stimulated with LIPS, a TLR-4 ligand, leading to increased production of interleukin (IL)-10 and reduced synthesis of IL-12p70 and TNF-alpha. The immunomodulatory effect of the tick saliva on the production of pro-inflammatory cytokines by DCs stimulated with LPS was associated with the observation that tick saliva inhibits the activation of the ERK 1/2 and p38 MAP kinases. These effects were independent of the expression of TLR-4 on the surface of DCs. Additionally, saliva-treated DCs also presented a similar pattern of cytokine modulation in response to other TLR ligands. Since the recent literature reports that several parasites evade immune responses through TLR-2-mediated production of IL-10, we evaluated the effect of tick saliva on the percentage of TLR-2(+) DCs stimulated with the TLR-2 ligand lipoteicoic acid (LTA). The data showed that the population of DCs expressing TLR-2 was significantly increased in DCs treated with LTA plus saliva. In addition, tick saliva alone increased the expression of TLR-2 in a dose- and time-dependent manner. Our data suggest that tick saliva induces regulatory DCs, which secrete IL-10 and low levels of IL-12 and TNF-alpha when stimulated by TLR ligands. Such regulatory DCs are associated with expression of TLR-2 and inhibition of ERK and p38, which promotes the production of IL-10 and thus down-modulates the host`s immune response, possibly favouring susceptibility to tick infestations. (C) 2009 Elsevier B.V. All rights reserved.

Characterization and kinetics of sulfide-oxidizing autotrophic denitrification in batch reactors containing suspended and immobilized cells

Sulfide-oxidizing autotrophic denitrification is an advantageous alternative over heterotrophic denitrification, and may have potential for nitrogen removal of low-strength wastewaters, such as anaerobically pre-treated domestic sewage. This study evaluated the fundamentals and kinetics of this process in batch reactors containing suspended and immobilized cells. Batch tests were performed for different NO(x)(-)/S(2-) ratios and using nitrate and nitrite as electron acceptors. Autotrophic denitrification was observed for both electron acceptors, and NO(x)(-)/S(2-) ratios defined whether sulfide oxidation was complete or not. Kinetic parameter values obtained for nitrate were higher than for nitrite as electron acceptor. Zero-order models were better adjusted to profiles obtained for suspended cell reactors, whereas first-order models were more adequate for immobilized cell reactors. However, in the latter, mass transfer physical phenomena had a significant effect on kinetics based on biochemical reactions. Results showed that sulfide-oxidizing autotrophic denitrification can be successfully established for low-strength wastewaters and have potential for nitrogen removal from anaerobically pre-treated domestic sewage.

Topology optimization for designing strain-gauge load cells

Load cells are used extensively in engineering fields. This paper describes a novel structural optimization method for single- and multi-axis load cell structures. First, we briefly explain the topology optimization method that uses the solid isotropic material with penalization (SIMP) method. Next, we clarify the mechanical requirements and design specifications of the single- and multi-axis load cell structures, which are formulated as an objective function. In the case of multi-axis load cell structures, a methodology based on singular value decomposition is used. The sensitivities of the objective function with respect to the design variables are then formulated. On the basis of these formulations, an optimization algorithm is constructed using finite element methods and the method of moving asymptotes (MMA). Finally, we examine the characteristics of the optimization formulations and the resultant optimal configurations. We confirm the usefulness of our proposed methodology for the optimization of single- and multi-axis load cell structures.

On-line fault diagnostic system for proton exchange membrane fuel cells

In this paper, a supervisor system, able to diagnose different types of faults during the operation of a proton exchange membrane fuel cell is introduced. The diagnosis is developed by applying Bayesian networks, which qualify and quantify the cause-effect relationship among the variables of the process. The fault diagnosis is based on the on-line monitoring of variables easy to measure in the machine such as voltage, electric current, and temperature. The equipment is a fuel cell system which can operate even when a fault occurs. The fault effects are based on experiments on the fault tolerant fuel cell, which are reproduced in a fuel cell model. A database of fault records is constructed from the fuel cell model, improving the generation time and avoiding permanent damage to the equipment. (C) 2007 Elsevier B.V. All rights reserved.

Recombinant rabies virus glycoprotein synthesis in bioreactor by transfected Drosophila melanogaster S2 cells carrying a constitutive or an inducible promoter

S2 cell populations (S2AcRVGP2K and S2MtRVGP-Hy) were selected after transfection of gene expression vectors carrying the cDNA encoding the rabies virus glycoprotein (RVGP) gene under the control of the constitutive (actin) or inductive (metallothionein) promoters. These cell populations were cultivated in a 1 L bioreactor mimicking a large scale bioprocess. Cell cultures were carried out at 90 rpm and monitored/controlled for temperature (28 degrees C) and dissolved oxygen (10 or 50% air saturation). Cell growth attained similar to 1.5-3 x 10(7) cells/mL after 3-4 clays of cultivation. The constitutive synthesis of RVGP in S2AcRVGP2K cells led to values of 0.76 mu g/10(7) cells at day 4 of culture. The RVGP synthesis in S2MtRVGP-Hy cell fraction increased upon CuSO(4) induction attaining specific productivities of 1.5-2 mu g/10(7) cells at clays 4-5. RVGP values in supernatant as a result of cell lysis were always very low (<0.2 mu g/mL) indicating good integrity of cells in culture. Overall the RVGP productivity was of 1.5-3 mg/L. Our data showed an important influence of dissolved oxygen on RVGP synthesis allowing a higher and sustained productivity by S2MtRVGP-Hy cells when cultivated with a DO of 10% air saturation. The RVGP productivity in bioreactors shown here mirrors those previously observed for T-flasks and shaker bottles and allow the preparation of the large RVGP quantities required for studies of structure and function. (C) 2010 Elsevier B.V. All rights reserved.

Retamycin production by immobilized cells of Streptomyces olindensis ICB20 in repeated-batch cultures

Repeated-batch cultures of Ca-alginate immobilized cells of Streptomyces olindensis ICB20 for retamycin production were carried out in two different bioreactors: a basket-type stirred tank reactor (BSTR) and a bubble column reactor (BCR). Higher average values of retamycin content (R) and productivity (P-R) were achieved in the BSTR cultures (about 1.7 AU and 0.031 AU h(-1), respectively) compared to those obtained in the BCR cultures (about 0.6 AU and 0.012 AU h(-1), respectively). The BCR, on the other hand, presented significantly better operation stability than the BSTR, which makes the former much more promising regarding future industrial applications. (C) 2008 Elsevier Ltd. All rights reserved.

Open-circuit voltages: Theoretical and experimental optimizations of rear passivated silicon solar cells using Fz and Cz wafers

The theoretical and experimental open-circuit voltage optimizations of a simple fabrication process of silicon solar cells n(+)p with rear passivation are presented. The theoretical results were obtained by using an in-house developed program, including the light trapping effect and metal-grid optimization. On the other hand, the experimental steps were monitored by the photoconductive decay technique. The starting materials presented thickness of about 300 pm and resistivities: FZ (0.5 Omega cm), Cz-type 1 (2.5 Omega cm) and Cz-type 2 (3.3 Omega cm). The Gaussian profile emitters were optimized with sheet resistance between 55 Omega/sq and 100 Omega/sq, and approximately 2.0 mu m thickness in accordance to the theoretical results. Excellent implied open-circuit voltages of 670.8 mV, 652.5 mV and 662.6 mV, for FZ, Cz-type 1 and Cz-type 2 silicon wafers, respectively, could be associated to the measured lifetimes that represents solar cell efficiency up to 20% if a low cost anti-reflection coating system, composed by random pyramids and SiO(2) layer, is considered even for typical Cz silicon. (C) 2009 Elsevier Ltd. All rights reserved.

Antioxidant response of Nicotiana tabacum cv. Bright Yellow 2 cells to cadmium and nickel stress

Plant cell cultures are a suitable model system for investigation of the physiological mechanisms of tolerance to environmental stress. We have determined the effects of Cd (0.1 and 0.2 mM CdCl(2)) and Ni (0.075 and 0.75 mM NiCl(2)) on Nicotiana tabacum L. cv. Bright Yellow (TBY-2) cell suspension cultures over a 72-h period. Inhibition of growth, loss of cell viability and lipid peroxidation occurred, in general, only when the TBY-2 cells were grown at 0.2 mM CdCl(2) and at 0.75 mM NiCl(2). At 0.1 mM CdCl(2), a significant increase in growth was determined at the end of the experiment. Increases in the activities of all of the four enzymatic antioxidant defence systems tested, were induced by the two concentrations of Cd and Ni, but at different times during the period of metal exposure. Overall, the cellular antioxidant responses to Cd and Ni were similar and were apparently sufficient to avoid oxidative stress at the lower concentrations of Cd and Ni. The activities of glutathione reductase and glutathione S-transferase increased early but transiently, whereas the activities of catalase and guaiacol peroxidase increased in the latter half of the experimental period. Therefore it is likely that the metabolism of reduced glutathione was enhanced during the initial onset of the stress, while catalase and guaiacol-type peroxidase appeared to play a more important role in the antioxidant response once the stress became severe.

Plasmodium vivax recombinant vaccine candidate AMA-1 plays an important role in adaptive immune response eliciting differentiation of dendritic cells

The Apical Membrane Antigen-1 (AMA-1) is a well-characterized and functionally important merozoite protein and is currently considered a major candidate antigen for a malaria vaccine. Previously, we showed that AMA-1 has an influence on cellular immune responses of malaria-naive subjects, resulting in an alternative activation of monocyte-derived dendritic cells and induction of a pro-inflammatory response by stimulated PBMCs. Although there is evidence, from human and animal malaria model systems that cell-mediated immunity may contribute to both protection and pathogenesis, the knowledge on cellular immune responses in vivax malaria and the factors that may regulate this immunity are poorly understood. In the current work, we describe the maturation of monocyte-derived dendritic cells of P. vivax naturally infected individuals and the effect of P. vivax vaccine candidate Pv-AMA-1 on the immune responses of the same donors. We show that malaria-infected subjects present modulation of DC maturation, demonstrated by a significant decrease in expression of antigen-presenting molecules (CD1a, HLA-ABC and HLA-DR), accessory molecules (CD40, CD80 and CD86) and Fc gamma RI (CD64) receptor (P <= 0.05). Furthermore, Pv-AMA-1 elicits an upregulation of CD1a and HLA-DR molecules on the surface of monocyte-derived dendritic cells (P=0.0356 and P=0.0196, respectively), and it is presented by AMA-1-stimulated DCs. A significant pro-inflammatory response elicited by Pv-AMA-1-pulsed PBMCs is also demonstrated, as determined by significant production of TNF-alpha, IL-12p40 and IFN-gamma (P <= 0.05). Our results suggest that Pv-AMA-1 may partially revert DC down-modulation observed in infected subjects, and exert an important role in the initiation of pro-inflammatory immunity that might contribute substantially to protection. (c) 2009 Elsevier Ltd. All rights reserved.

Requirement for PLC gamma 2 in IL-3 and GM-CSF-Stimulated MEK/ERK Phosphorylation in Murine and Human Hematopoietic Stem/Progenitor Cells

Even though the involvement of intracellular Ca(2+) (Ca(i)(2+)) in hematopoiesis has been previously demonstrated, the relationship between Ca(i)(2+) signaling and cytokine-induced intracellular pathways remains poorly understood. Herein, the molecular mechanisms integrating Ca(2+) signaling with the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway in primary murine and human hematopoietic stem/progenitor cells stimulated by IL-3 and GM-CSF were studied. Our results demonstrated that IL-3 and GM-CSF stimulation induced increased inositol 1,4,5-trisphosphate (IP(3)) levels and Ca(i)(2+) release in murine and human hematopoietic stem/ progenitor cells. In addition, Ca(i)(2+) signaling inhibitors, such as inositol 1,4,5-trisphosphate receptor antagonist (2-APB), PKC inhibitor (GF109203), and CaMKII inhibitor (KN-62), blocked phosphorylation of MEK activated by IL-3 and GM-CSF, suggesting the participation of Ca(2+)-dependent kinases in MEK activation. In addition, we identify phospholipase C gamma 2 (PLC gamma 2) as a PLC gamma responsible for the induction of Ca(2+) release by IL-3 and GM-CSF in hematopoietic stem/progenitor cells. Furthermore, the PLCg inhibitor U73122 significantly reduced the numbers of granulocyte-macrophage colony-forming units after cytokine stimulation. Similar results were obtained in both murine and human hematopoietic stem/progenitor cells. Taken together, these data indicate a role for PLC gamma 2 and Ca(2+) signaling through the modulation of MEK in both murine and human hematopoietic stem/ progenitor cells. J. Cell. Physiol. 226: 1780-1792, 2011. (C) 2010 Wiley-Liss, Inc.