Alfredo M. Goes

Polymyxin B as inhibitor of LPS contamination of Schistosoma mansoni recombinant proteins in human cytokine analysis

BackgroundRecombinant proteins expressed in Escherichia coli vectors are generally contaminated with endotoxin. In this study, we evaluated the ability of Polymyxin B to neutralize the effect of LPS present as contaminant on Schistosoma mansoni recombinant proteins produced in E. coli in inducing TNF-α and IL-10. Peripheral blood mononuclear cells from individuals chronically infected with S. mansoni were stimulated in vitro with recombinant Sm22.6, Sm14 and P24 antigens (10 μg/mL) in the presence of Polymyxin B (10 μg/mL).ResultsThe levels of cytokines were measured using ELISA. There was greater than 90 % reduction (p < 0.05) in the levels of TNF-α and IL-10 when Polymyxin B was added to the cultures stimulated with LPS. In cultures stimulated with S. mansoni recombinant proteins in the presence of Polymyxin B, a reduction in the levels of TNF-α and IL-10 was also observed. However, the percentage of reduction was lower when compared to the cultures stimulated with LPS, probably because these proteins are able to induce the production of these cytokines by themselves.ConclusionThis study showed that Polymyxin B was able to neutralize the effect of endotoxin, as contaminant in S. mansoni recombinant antigens produced in E. coli, in inducing TNF-α and IL-10 production.

The type III inositol 1,4,5-trisphosphate receptor preferentially transmits apoptotic Ca2+ signals into mitochondria.

There are three isoforms of the inositol 1,4,5- trisphosphate receptor (InsP3R), each of which has a distinct effect on Ca2+ signaling. However, it is not known whether each isoform similarly plays a distinct role in the activation of Ca2+-mediated events. To investigate this question, we examined the effects of each InsP3R isoform on transmission of Ca2+ signals to mitochondria and induction of apoptosis. Each isoform was selectively silenced using isoform-specific small interfering RNA in Chinese hamster ovary cells, which express all three InsP3R isoforms. ATP-induced cytosolic Ca2+ signaling patterns were altered, regardless of which isoform was silenced, but in a different fashion depending on the isoform. ATP also induced Ca2+ signals in mitochondria, which were inhibited more effectively by silencing the type III InsP3R than by silencing either the type I or type II isoform. The type III isoform also co-localized most strongly with mitochondria. When apoptosis was induced by activation of either the extrinsic or intrinsic apoptotic pathway, induction was reduced most effectively by silencing the type III InsP3R. These findings provide evidence that the type III isoform of the InsP3R plays a special role in induction of apoptosis by preferentially transmitting Ca2+ signals into mitochondria.

Time-dependent migration of systemically delivered bone marrow mesenchymal stem cells to the infarcted heart.

In this study the time course of homing and the body distribution of systemically delivered bone marrow mesenchymal stem cells (BM-MSCs) after myocardial infarction (MI) were evaluated. BM-MSCs were isolated from Wistar rats, expanded in vitro, and their phenotypical characterization was performed by flow cytometer. Rats were randomly divided into three groups: control, sham MI, and MI. BM-MSCs (5 × 106) were labeled with 99mTc-HMPAO and injected through the tail vein 7 days after MI. Gamma camera imaging was performed at 5, 15, 30, and 60 min after cell inoculation. Due to the 99mTc short half-life, cell migration and location were also evaluated in heart sections using DAPI-labeled cells 7 days after transplantation. Phenotypical characterization showed that BM-MSCs were CD90+, CD73+, CD54+, and CD45-. Five minutes after 99mTc-HMPAO-labeled cell injection, they were detected in various tissues. The cells migrated mainly to the lungs (approximately 70%) and, in small amounts, to the heart, kidneys, spleen, and bladder. The number of cells in the heart and lungs decreased after 60 min. MI markedly increased the amount of cells in the heart, but not in the lungs, during the period of observation (4.55 ± 0.32 vs. 6.34 ± 0.67% of uptake in infarcted hearts). No significant differences were observed between control and sham groups. Additionally, 7 days after DAPI-labeled cells injection, they were still detected in the heart but only in infarcted areas. These results suggest that the migration of systemically delivered BM-MSCs to the heart is time dependent and MI specifically increases BM-MSCs homing to injured hearts. However, the systemic delivery is limited by cell entrapment in the lungs.

Nuclear Ca2+ regulates cardiomyocyte function

In the heart, cytosolic Ca(2+) signals are well-characterized events that participate in the activation of cell contraction. In contrast, nuclear Ca(2+) contribution to cardiomyocyte function remains elusive. Here, we examined functional consequences of buffering nuclear Ca(2+) in neonatal cardiomyocytes. We report that cardiomyocytes contain a nucleoplasmic reticulum, which expresses both ryanodine receptor (RyR) and inositol 1,4,5-trisphosphate receptor (InsP(3)R), providing a possible way for active regulation of nuclear Ca(2+). Adenovirus constructs encoding the Ca(2+) buffer protein parvalbumin were targeted to the nucleus with a nuclear localization signal (Ad-PV-NLS) or to the cytoplasm with a nuclear exclusion signal (Ad-PV-NES). A decrease in the amplitude of global Ca(2+) transients and RyR-II expression, as well as an increase in cell beating rate were observed in Ad-PV-NES and Ad-PV-NLS cells. When nuclear Ca(2+) buffering was imposed nuclear enlargement, increased calcineurin expression, NFAT translocation to the nucleus and subcellular redistribution of atrial natriuretic peptide were observed. Furthermore, prolongation of action potential duration occurred in adult ventricular myocytes. These results suggest that nuclear Ca(2+) levels underlie the regulation of specific protein targets and thereby modulate cardiomyocyte function. The local nuclear Ca(2+) signaling and the structures that control it constitute a novel regulatory motif in the heart.

Three-dimensional culture of rat BMMSCs in a porous chitosan-gelatin scaffold: A promising association for bone tissue engineering in oral reconstruction.

OBJECTIVE this study investigated the in vitro effects of a chitosan-gelatin scaffold on growth and osteogenic differentiation of rat bone marrow mesenchymal stem cells (BMMSCs) in three-dimensional (3D) cultures and evaluated the biomaterial biocompatibility and degradability after its grafting into tooth sockets of rats. DESIGN a porous chitosan-gelatin scaffold cross-linked by glutaraldehyde was synthesised and characterised by light (LM), scanning electronic microscopy (SEM), energy dispersion spectroscopy (EDS) and X-ray diffraction (XRD). Rat BMMSCs were isolated, expanded and seeded onto scaffold using Dulbeccos Modified Eagles Medium (DMEM) with or without an osteogenic supplement. Cell viability by MTT assay, alkaline phosphatase (ALP) activity and morphological LM and SEM analysis were performed after 1, 3, 8 and 14 days in culture. Free-cell scaffolds were implanted into tooth sockets of Lewis rats after upper first molars extraction. Fifteen male recipient rats were sacrificed after 5, 21 and 35 days for histological analysis. RESULTS scaffold characterisation revealed the porous structure, organic and amorphous content. This biomaterial promoted the adhesion, spreading and in vitro viability of the BMMSCs. Osteogenic-supplemented media did not improve the cellular response compared to DMEM. The biomaterial presented high biocompatibility and slow biodegradation in vivo. Remains of biomaterial were still observed at 21 and 35 days after implantation. However, on the 21st day, alveolar bone and epithelial healing were completely established. CONCLUSIONS these results indicate that chitosan-gelatin support the adhesion and osteogenic differentiation of rat BMMSCs and offer adequate physico-chemical and biological properties for use as scaffolds in bone tissue engineering-related strategies.

Local injection of BDNF producing mesenchymal stem cells increases neuronal survival and synaptic stability following ventral root avulsion

The present study proposed to graft mesenchymal stem cells (MSCs), which continuously produce BDNF, into the spinal cord ventral horn, after ventral root avulsion. Neurotrophin expression was naturally achieved by culturing MSCs in an undifferentiated state for at least 10 weeks. Lewis rats were subjected to unilateral avulsion of lumbar ventral roots, receiving 3 x 10(5) cells injected through the lateral funiculus. Two weeks after surgery, the animals were sacrificed and neuronal survival, astroglial reaction and synaptic inputs within the motor nucleus analyzed. The results indicated that the MSCs treatment significantly rescued avulsed motoneurons. Such neuronal survival was related to in vivo mRNA up regulation as well as expression of BDNF and GDNF. Such increase was correlated to the preservation of synaptophysin- positive nerve terminals. Thus it was proposed that when maintained undifferentiated for a period of 10 weeks, MSCs may be used as a continuous source of BDNF, positively influencing neuronal survival and synaptic plasticity.

Induction of protective immunity and modulation of granulomatous hypersensitivity in mice using PIII, an anionic fraction of Schistosoma mansoni adult worm

This study was performed in order to define Schistosoma mansoni antigens that are able to function as modulator agents in the granulomatous hypersensitivity to parasite eggs in BALB/c and C57BL/6 mice. A fraction of S. mansoni, designated PIII, derived from adult worm antigen preparation (SWAP) was obtained using anion-exchange chromatography on an FPLC system. Immunization of mice with PIII in the presence of Corynebacterium parvum and Al(OH)3 as adjuvant induced an immune response in this animals as determined by ELISA and spleen cell proliferation assays against S. mansoni antigens SEA, SWAP and PIII. In addition, PIII caused a significant degree of protection against a challenge infection in immunized mice as observed by the decrease on worm burden recovered from the portal system. We also showed that PIII profoundly inhibited the vigorous anamnestic granulomatous response to eggs in the liver and lungs. This suppression correlated with a significant decrease in granuloma size. From these results we conclude that the PIII preparation contains antigens that can mediate protective anti-parasite immunity and downregulate granulomatous hypersensitivity to S. mansoni eggs.

Effect of a Three-Dimensional Chitosan Porous Scaffold on the Differentiation of Mesenchymal Stem Cells into Chondrocytes

Cartilage tissue has a poor capacity for self-repair, especially in the case of severe cartilage damage due to trauma or age-related degeneration. Cell-based tissue engineering using scaffolds has provided an option for the repair of cartilage tissue. The present work demonstrates that a three-dimensional (3D) chitosan scaffold increases the efficiency of the adhesion and differentiation of mesenchymal stem cells (MSCs) after the addition of a chondrogenic medium. These culture conditions promoted MSC differentiation into chondrocytes during the first 9 weeks of monolayer or 3D culture in a scaffold composed of chitosan or chitosan/gelatin. The results demonstrated that a chitosan scaffold caused a reduction in alkaline phosphatase production and an increase in the collagen concentration indicating phenotypic changes in the cells. In support of these results, the production of collagen type II by the MSCs cultured in the chitosan scaffold increased after 3 weeks of culture, indicating the beginning of differentiation. However, the addition of gelatin to the chitosan scaffold did not improve on the results obtained with chitosan alone. These results suggest that this 3D chitosan scaffold is a promising candidate for biomaterial implants designed to promote MSC colonization and has applications in regenerative medicine.

Osteogenic differentiation of mesenchymal stem cells from osteopenic rats subjected to physical activity with and without nitric oxide synthase inhibition

Physical activity has potent and complex effects on bones. We hypothesized that physical activity has a positive effect upon osteopenic rat bones because it stimulates osteogenic differentiation of bone marrow mesenchymal stem cells (MSCs). We also postulated that local nitric oxide concentrations mediate the effects of physical activity on bones. The objective of this study was to investigate the osteogenic differentiation in vitro of MSCs from osteopenic female rats subjected to physical activity with and without nitric oxide synthase inhibition. We used MSCs from the femurs of Wistar female rats divided into six groups: Group 1, sham-operated (control); Group 2, sedentary osteopenic; Group 3, active osteopenic; Group 4, sham-operated with L-NAME; Group 5, sedentary osteopenic with L-NAME; and Group 6, active osteopenic with L-NAME. The cells were cultured at 37 degrees C and 5% CO2. Cells were phenotypically characterized with anti-CD45, anti-CD90, anti-CD73, and anti-CD54 using a FACScan cytometer. MSCs were cultured in osteogenic medium for 7, 14 and 21 days. Alkaline phosphatase activity, the capacity of dimethylthiazol conversion in formazan crystals, collagen synthesis and the number of mineralized nodules were analyzed. The means of all of the variables were compared using the SNK test. MSCs did not express CD45 in 96.94% of the cells, but there was expression of CD73, CD54 and CD90 in 93.99%, 95.10% and 86.77% of the cells, respectively. MSCs from osteopenic rats showed less osteogenic differentiation. Surprisingly, physical activity increased the osteogenic differentiation of MSCs in osteopenic rats. Inhibition of nitric oxide synthase in vivo had a negative effect upon the osteogenic potential of MSCs from normal rats and from osteopenic rats subjected to physical activity. Our results suggest that nitric oxide stimulates MSCs osteogenic differentiation and that nitric oxide mediates the beneficial effects of physical activity upon MSCs osteogenic differentiation.

Retinal incorporation and differentiation of mesenchymal stem cells intravitreally injected in the injured retina of rats

PURPOSE To evaluate the pattern of retinal integration and differentiation of mesenchymal stem cells (MSCs) injected into the vitreous cavity of rat eyes with retinal injury. METHODS Adult rat retinas were submitted to laser damage followed by transplantation of DAPI-labeled BM-MSCs grafts. To assess the integration and differentiation of BM-MSCs in laser-injured retina, host retinas were evaluated 2.4 and 8 weeks after injury/transplantation. RESULTS Our results demonstrated that the grafted cells survived in the retina for at least 8 weeks and almost all BM-MSCs migrated and incorporated into the neural retina, specifically in the outer nuclear layer (ONL), inner nuclear layer (INL) and ganglion cell layer (GCL) while a subset of grafted cells were found in the subretinal space posttransplantation. At 8 weeks immunohistochemical analysis with several retinal specific markers revealed that the majority of the grafted cells expressed rhodopsin, a rod photoreceptor marker, followed by parvalbumin, a marker for bipolar and amacrine cells. A few subsets of cells were able to express a glial marker, glial fibrillary acidic protein. However, grafted cells failed to express pan-cytokeratin, a retinal pigment epithelium marker. CONCLUSIONS These results suggest the potential of BM-MSCs to differentiate into retinal neurons. Taken together, these findings might be clinically relevant for future mesenchymal stem cell therapy studies concerning retinal degeneration repair.