Both Interleukin-3 and Interleukin-6 Are Necessary for Better Ex Vivo Expansion of CD133+Cells From Umbilical Cord Blood

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Umbilical cord blood (UCB), an ideal source for transplantable hematopoietic stem cells (HSC), is readily available and is rich in progenitor cells. Identification of conditions favoring UCB-HSC ex vivo expansion and of repopulating potential remains a major challenge in hematology. CD133+ cells constitute an earlier, less-differentiated HSC group with a potentially higher engraftment capacity. The presence of SCF, Flt3-L, and TPO are essential for CD133+ and/or CD34+ cells ex vivo expansion; however, IL-3 and IL-6 influence has not yet been clearly established. We investigated this influence on CD133+ cells from UCB ex vivo expansion and the effect of these cytokines upon cell phenotype. Immediately after isolation an 85% of CD133+ cell purity was obtained, diminishing after 4 and 8 days of ex vivo expansion. CD133+ fold-increase was higher using IMDM with SCF, Flt3-L, and TPO (BM)+IL-3 or BM+IL-3+IL-6 on day 8 (13.83- and 17.47-fold increase, respectively). BM+IL-6 presented no significant difference from BM alone. We demonstrated that 5.1% of the CD133+ cells expressed IL-6 receptor (IL-6R) after isolation. After 4 and 8 days in culture, the percentage of CD133+ cells that expressed IL-6R was as follows: BM alone (9.8% and 22.02%, respectively); BM+IL-3 (8.33% and 16.74%); BM+IL-6 (9.2% and 17.67%); and BM+IL-3+IL-6 (12.5% and 61.20%). Cell cycle analysis revealed quiescent cells after isolation, 95.5% CD133+ cells in the G0/G1 phase. Regardless of culture period or cytokine incubation, CD133+ cell cycle altered to 70% of CD133+ in the G0/G1 phase. Colony-forming unit (CFU) doubled in BM+IL-3+IL-6 after 8 days of incubation compared with BM group. SOX-2 and NANOG-relative gene expression was detected on day 0 after isolation. BM+IL-6 prevented the decrease in NANOG and SOX-2 gene expression level compared to BM+IL-3 or BM+IL-3+IL-6 incubated cells. Our results indicated that UCB-isolated CD133+ cells were better ex vivo expanded in the presence of SCF, Flt3-L, TPO, IL-3+IL-6. IL-3 probably promotes higher CD133+ cell expansion and IL-6 maintains immature phenotype.o TEXTO COMPLETO DESTE ARTIGO, ESTARÁ DISPONÍVEL À PARTIR DE AGOSTO DE 2015.193413421Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Second-harmonic Generation Microscopy Used To Evaluate The Effect Of The Dimethyl Sulfoxide In The Cryopreservation Process In Collagen Fibers Of Differentiated Chondrocytes

Cartilaginous lesions are a significant public health problem and the use of adult stem cells represents a promising therapy for this condition. Cryopreservation confers many advantages for practitioners engaged in cell-based therapies. However, conventional slow freezing has always been associated with damage and mortality due to intracellular ice formation, cryoprotectant toxicity, and dehydration. The aim of this work is to observe the effect of the usual Dimethyl Sulfoxide (DMSO) cryopreservation process on the architecture of the collagen fiber network of chondrogenic cells from mesenchymal stem cells by Second Harmonic Generation (SHG) microscopy. To perform this study we used Mesenchymal Stem Cells (MSC) derived from adipose tissue which presents the capacity to differentiate into other lineages such as osteogenic, adipogenic and chondrogenic lineages. Mesenchymal stem cells obtained after liposuction were isolated digested by collagenase type I and characterization was carried out by differentiation of mesodermic lineages, and flow cytometry using specific markers. The isolated MSCs were cryopreserved by the DMSO technique and the chondrogenic differentiation was carried out using the micromass technique. We then compared the cryopreserved vs non-cryopreserved collagen fibers which are naturally formed during the differentiation process. We observed that noncryopreserved MSCs presented a directional trend in the collagen fibers formed which was absent in the cryopreserved MSCs. We confirmed this trend quantitatively by the aspect ratio obtained by Fast Fourier Transform which was 0.76 for cryopreserved and 0.52 for non-cryopreserved MSCs, a statistical significant difference. © 2012 Copyright Society of Photo-Optical Instrumentation Engineers (SPIE).8226The Society of Photo-Optical Instrumentation Engineers (SPIE),Becker and Hickel GmbH,Boston Electronics Corp.,Carl Zeiss MicroImaging GmbH,Chroma Technologies Corp.Hunziker, E.B., Articular cartilage repair: Basic science and clinical progress. A review of the current status and prospects (2002) Osteoarthritis Cartilage, 10, pp. 432-463Liu, Y., Shu, X.Z., Prestwich, G.D., Osteochondral defect repair with autologous bone marrow-derived mesenchymal stem cells in an injectable, in situ, cross-linked synthetic extracellular matrix (2006) Tissue Eng, 12, pp. 3405-3416Buckwalter, J.A., Mankin, H.J., Articular cartilage: Degeneration and osteoarthritis, repair, regeneration, and transplantation (1998) Instr Course Lect, 47, pp. 487-504Knutsen, G., Engebretsen, L., Ludvigsen, T.C., Drogset, J.O., Grontvedt, T., Solheim, E., Strand, T., Johansen, O., Autologous chondrocyte implantation compared with microfracture in the knee. A randomized trial (2004) J Bone Joint Surg Am, 86 A, pp. 455-464Bouwmeester, S.J., Beckers, J.M., Kuijer, R., Van Der Linden, A.J., Bulstra, S.K., Long-term results of rib perichondrial grafts for repair of cartilage defects in the human knee (1997) Int Orthop, 21, pp. 313-317Brittberg, M., Lindahl, A., Nilsson, A., Ohlsson, C., Isaksson, O., Peterson, L., Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation (1994) N Engl J Med, 331, pp. 889-895Ferretti, M., Marra, K.G., Kobayashi, K., Defail, A.J., Chu, C.R., Controlled in vivo degradation of genipin crosslinked polyethylene glycol hydrogels within osteochondral defects (2006) Tissue Eng, 12, pp. 2657-2663Pittenger, M.F., Mackay, A.M., Beck, S.C., Jaiswal, R.K., Douglas, R., Mosca, J.D., Moorman, M.A., Marshak, D.R., Multilineage potential of adult human mesenchymal stem cells (1999) Science, 284, pp. 143-147Nasef, A., Ashammakhi, N., Fouillard, L., Immunomodulatory effect of mesenchymal stromal cells: Possible mechanisms (2008) Regen Med, 3, pp. 531-546Ishige, I., Nagamura-Inoue, T., Honda, M.J., Harnprasopwat, R., Kido, M., Sugimoto, M., Nakauchi, H., Tojo, A., Comparison of mesenchymal stem cells derived from arterial, venous, and Wharton's jelly explants of human umbilical cord (2009) Int J Hematol, 90, pp. 261-269Chamberlain, J., Yamagami, T., Colletti, E., Theise, N.D., Desai, J., Frias, A., Pixley, J., Almeida-Porada, G., Efficient generation of human hepatocytes by the intrahepatic delivery of clonal human mesenchymal stem cells in fetal sheep (2007) Hepatology, 46, pp. 1935-1945Campagnoli, C., Roberts, I.A., Kumar, S., Bennett, P.R., Bellantuono, I., Fisk, N.M., Identification of mesenchymal stem/progenitor cells in human first-trimester fetal blood, liver, and bone marrow (2001) Blood, 98, pp. 2396-2402Erices, A., Conget, P., Minguell, J.J., Mesenchymal progenitor cells in human umbilical cord blood (2000) Br J Haematol, 109, pp. 235-242Mello, M.A., Tuan, R.S., Effects of TGF-beta1 and triiodothyronine on cartilage maturation: In vitro analysis using long-term high-density micromass cultures of chick embryonic limb mesenchymal cells (2006) J Orthop Res, 24, pp. 2095-2105Honorati, M.C., Cattini, L., Facchini, A., VEGF production by osteoarthritic chondrocytes cultured in micromass and stimulated by IL-17 and TNF-alpha (2007) Connect Tissue Res, 48, pp. 239-245Gruber, H.E., Chow, Y., Hoelscher, G.L., Ingram, J.A., Zinchenko, N., Norton, H.J., Sun, Y., Hanley Jr., E.N., Micromass culture of human anulus cells: Morphology and extracellular matrix production (2010) Spine (Phila Pa 1976), 35, pp. 1033-1038Wescoe, K.E., Schugar, R.C., Chu, C.R., Deasy, B.M., The role of the biochemical and biophysical environment in chondrogenic stem cell differentiation assays and cartilage tissue engineering (2008) Cell Biochem Biophys, 52, pp. 85-102Xiang, Y., Zheng, Q., Jia, B., Huang, G., Xie, C., Pan, J., Wang, J., Ex vivo expansion, adipogenesis and neurogenesis of cryopreserved human bone marrow mesenchymal stem cells (2007) Cell Biology International, 31, pp. 444-450Ock, S.A., Rho, G.J., Effect of dimethyl sulfoxide (DMSO) on cryopreservation of porcine mesenchymal stem cells (pMSCs) (2011) Cell Transplant, 20, pp. 1231-1239Fleming, K.K., Hubel, A., Cryopreservation of hematopoietic and non-hematopoietic stem cells (2006) Transfus Apher Sci, 34, pp. 309-315Buchanan, S.S., Gross, S.A., Acker, J.P., Toner, M., Carpenter, J.F., Pyatt, D.W., Cryopreservation of stem cells using trehalose: Evaluation of the method using a human hematopoietic cell line (2004) Stem Cells Dev, 13, pp. 295-305Schenke-Layland, K., Riemann, I., Damour, O., Stock, U.A., Konig, K., Two-photon microscopes and in vivo multiphoton tomographs - Powerful diagnostic tools for tissue engineering and drug delivery (2006) Advanced Drug Delivery Reviews, 58, pp. 878-896Kiviranta, P., Rieppo, J., Korhonen, R.K., Julkunen, P., Toyras, J., Jurvelin, J.S., Collagen network primarily controls Poisson's ratio of bovine articular cartilage in compression (2006) J Orthop Res, 24, pp. 690-699Zhang, Z.X., Guan, L.X., Zhang, K., Wang, S., Cao, P.C., Wang, Y.H., Wang, Z., Dai, L.J., Cytogenetic analysis of human bone marrow-derived mesenchymal stem cells passaged in vitro (2007) Cell Biology International, 31, pp. 645-648Cicchi, R., Kapsokalyvas, D., De Giorgi, V., Maio, V., Van Wiechen, A., Massi, D., Lotti, T., Pavone, F.S., Scoring of collagen organization in healthy and diseased human dermis by multiphoton microscopy (2010) Journal of Biophotonics, 3, pp. 34-4

Natural type II collagen hydrogel, fibrin sealant, and adipose-derived stem cells as a promising combination for articular cartilage repair

CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESObjective. Articular cartilage is an avascular tissue with limited ability of self-regeneration and the current clinical treatments have restricted capacity to restore damages induced by trauma or diseases. Therefore, new techniques are being tested for cartilage repair, using scaffolds and/or stem cells. Although type II collagen hydrogel, fibrin sealant, and adipose-derived stem cells (ASCs) represent suitable alternatives for cartilage formation, their combination has not yet been investigated in vivo for focal articular cartilage defects. We performed a simple experimental procedure using the combination of these 3 compounds on cartilage lesions of rabbit knees. Design. The hydrogel was developed in house and was first tested in vitro for chondrogenic differentiation. Next, implants were performed in chondral defects with or without ASCs and the degree of regeneration was macroscopically and microscopically evaluated. Results. Production of proteoglycans and the increased expression of collagen type II (COL2 alpha 1), aggrecan (ACAN), and sex-determining region Y-box 9 (SOX9) confirmed the chondrogenic character of ASCs in the hydrogel in vitro. Importantly, the addition of ASC induced a higher overall repair of the chondral lesions and a better cellular organization and collagen fiber alignment compared with the same treatment without ASCs. This regenerating tissue also presented the expression of cartilage glycosaminoglycan and type II collagen. Conclusions. Our results indicate that the combination of the 3 compounds is effective for articular cartilage repair and may be of future clinical interest.84439443CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESCONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESSem informaçãoSem informaçãoSem informaçãoThe authors would like to thank Prof. Benedicto de Campos Vidal from University of Campinas for providing collagen type II, and Tereza Salles and Paulo Latuf Filho for their valuable technical assistance. We also thank Raquel Foglio for English review and Michel Moraes for figure formatting and design. This work was supported by Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq/MCT), Fundavao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) and Fundacao de Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES)

Vitamin D3 Induces Ido+ Tolerogenic Dcs And Enhances Treg, Reducing The Severity Of Eae

Background: A growing body of evidence supports the hypothesis that vitamin D is an important environmental factor in the etiology of T-cell-mediated autoimmune diseases such as multiple sclerosis (MS). Aim: The purpose of this study was exploring the mechanisms underlying the beneficial effect of vitamin D3 in encephalomyelitis (EAE). Methods: We treated monophasic experimental autoimmune EAE, induced in Lewis rat, with vitamin D3 and adoptively transfer tolerogenic bone marrow-derived DCs generated in the presence of vitamin D3. Results: This study provides evidence that the in vivo administration of vitamin D3, as well as the adoptive transfer of vitamin D3-induced IDO+ immature/tolerogenic dendritic cells, leads to a significant increase in the percentage of CD4+CD25+Foxp3+ regulatory T cells in the lymph nodes in a rat model of MS, experimental autoimmune EAE. Concomitant with the increase in this cell population, there is a significant decrease in the number of autoreactive T cells in the central nervous system. Bone marrow-derived DCs cultivated in the presence of vitamin D3 present a tolerogenic profile with high IL-10, TNFα, and IDO expression and decreased MHC-II and CD80 expression. The adoptive transfer of IDO + DCs induces a significant increase in the percentage of CD4+CD25+Foxp3+ T cells in the lymph nodes, comparable with vitamin D3 treatment. Conclusion: These mechanisms contribute actively to the generation of a microenvironment in the lymph nodes that suppresses the activation of encephalitogenic T cells, resulting in the downregulation of the inflammatory response in the central nervous system. © 2013 Blackwell Publishing Ltd.194269277Hafler, D.A., Slavik, J.M., Anderson, D.E., O'Connor, K.C., De Jager, P., Baecher-Allan, C., Multiple sclerosis (2005) Immunol Rev, 204, pp. 208-231van der Mei, I.A., Ponsonby, A.L., Blizzard, L., Dwyer, T., Regional variation in multiple sclerosis prevalence in Australia and its association with ambient ultraviolet radiation (2001) Neuroepidemiology, 20, pp. 168-174Wallin, M.T., Page, W.F., Kurtzke, J.F., Multiple sclerosis in US veterans of the Vietnam era and later military service: Race, sex, and geography (2004) Ann Neurol, 55, pp. 65-71Cantorna, M.T., Hayes, C.E., DeLuca, H.F., 1,25-Dihydroxyvitamin D3 reversibly blocks the progression of relapsing encephalomyelitis, a model of multiple sclerosis (1996) Proc Natl Acad Sci U S A, 93, pp. 7861-7864Spach, K.M., Hayes, C.E., Vitamin D3 confers protection from autoimmune encephalomyelitis only in female mice (2005) J Immunol, 175, pp. 4119-4126Becklund, B.R., Hansen Jr, D.W., Deluca, H.F., Enhancement of 1,25-dihydroxyvitamin D3-mediated suppression of experimental autoimmune encephalomyelitis by calcitonin (2009) Proc Natl Acad Sci U S A, 106, pp. 5276-5281Santos, L.M., al-Sabbagh, A., Londono, A., Weiner, H.L., Oral tolerance to myelin basic protein induces regulatory TGF-beta-secreting T cells in Peyer's patches of SJL mice (1994) Cell Immunol, 157, pp. 439-447Hou, S.W., Liu, C.Y., Li, Y.H., Fasudil ameliorates disease progression in experimental autoimmune encephalomyelitis, acting possibly through antiinflammatory effect (2012) CNS Neurosci Ther, 18, pp. 909-917Spach, K.M., Nashold, F.E., Dittel, B.N., Hayes, C.E., IL-10 signaling is essential for 1,25-dihydroxyvitamin D3-mediated inhibition of experimental autoimmune encephalomyelitis (2006) J Immunol, 177, pp. 6030-6037Bettelli, E., Carrier, Y., Gao, W., Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells (2006) Nature, 441, pp. 235-238Langrish, C.L., Chen, Y., Blumenschein, W.M., IL-23 drives a pathogenic T cell population that induces autoimmune inflammation (2005) J Exp Med, 201, pp. 233-240Park, H., Li, Z., Yang, X.O., A distinct lineage of CD4 T cells regulates tissue inflammation by producing interleukin 17 (2005) Nat Immunol, 6, pp. 1133-1141O'Connor, R.A., Prendergast, C.T., Sabatos, C.A., Cutting edge: Th1 cells facilitate the entry of Th17 cells to the central nervous system during experimental autoimmune encephalomyelitis (2008) J Immunol, 181, pp. 3750-3754Muthian, G., Raikwar, H.P., Rajasingh, J., Bright, J.J., 1,25 Dihydroxyvitamin-D3 modulates JAK-STAT pathway in IL-12/IFNgamma axis leading to Th1 response in experimental allergic encephalomyelitis (2006) J Neurosci Res, 83, pp. 1299-1309Tang, J., Zhou, R., Luger, D., Calcitriol suppresses antiretinal autoimmunity through inhibitory effects on the Th17 effector response (2009) J Immunol, 182, pp. 4624-4632Adorini, L., Penna, G., Dendritic cell tolerogenicity: A key mechanism in immunomodulation by vitamin D receptor agonists (2009) Hum Immunol, 70, pp. 345-352Mora, J.R., Iwata, M., von Andrian, U.H., Vitamin effects on the immune system: Vitamins A and D take centre stage (2008) Nat Rev Immunol, 8, pp. 685-698Hewison, M., Freeman, L., Hughes, S.V., Differential regulation of vitamin D receptor and its ligand in human monocyte-derived dendritic cells (2003) J Immunol, 170, pp. 5382-5390Piemonti, L., Monti, P., Sironi, M., Vitamin D3 affects differentiation, maturation, and function of human monocyte-derived dendritic cells (2000) J Immunol, 164, pp. 4443-4451Szeles, L., Keresztes, G., Torocsik, D., 1,25-dihydroxyvitamin D3 is an autonomous regulator of the transcriptional changes leading to a tolerogenic dendritic cell phenotype (2009) J Immunol, 182, pp. 2074-2083Correale, J., Ysrraelit, M.C., Gaitan, M.I., Vitamin D-mediated immune regulation in multiple sclerosis (2011) J Neurol Sci, 311, pp. 23-31Tang, Q., Bluestone, J.A., The Foxp3 +  regulatory T cell: A jack of all trades, master of regulation (2008) Nat Immunol, 9, pp. 239-244Awasthi, A., Carrier, Y., Peron, J.P., A dominant function for interleukin 27 in generating interleukin 10-producing anti-inflammatory T cells (2007) Nat Immunol, 8, pp. 1380-1389Carrier, Y., Yuan, J., Kuchroo, V.K., Weiner, H.L., Th3 cells in peripheral tolerance. II. TGF-beta-transgenic Th3 cells rescue IL-2-deficient mice from autoimmunity (2007) J Immunol, 178, pp. 172-178Sakaguchi, S., Ono, M., Setoguchi, R., Foxp3 +  CD25 +  CD4 +  natural regulatory T cells in dominant self-tolerance and autoimmune disease (2006) Immunol Rev, 212, pp. 8-27Anderton, S.M., Liblau, R.S., Regulatory T cells in the control of inflammatory demyelinating diseases of the central nervous system (2008) Curr Opin Neurol, 21, pp. 248-254O'Connor, R.A., Anderton, S.M., Foxp3 +  regulatory T cells in the control of experimental CNS autoimmune disease (2008) J Neuroimmunol, 193, pp. 1-11Awasthi, A., Murugaiyan, G., Kuchroo, V.K., Interplay between effector Th17 and regulatory T cells (2008) J Clin Immunol, 28, pp. 660-670Barrat, F.J., Cua, D.J., Boonstra, A., In vitro generation of interleukin 10-producing regulatory CD4(+) T cells is induced by immunosuppressive drugs and inhibited by T helper type 1 (Th1)- and Th2-inducing cytokines (2002) J Exp Med, 195, pp. 603-616Ureta, G., Osorio, F., Morales, J., Rosemblatt, M., Bono, M.R., Fierro, J.A., Generation of dendritic cells with regulatory properties (2007) Transplant Proc, 39, pp. 633-637Penna, G., Roncari, A., Amuchastegui, S., Expression of the inhibitory receptor ILT3 on dendritic cells is dispensable for induction of CD4(+)Foxp3(+) regulatory T cells by 1,25-dihydroxyvitamin D-3 (2005) Blood, 106, pp. 3490-3497Farias, A.S., Martins-de-Souza, D., Guimaraes, L., Proteome analysis of spinal cord during the clinical course of monophasic experimental autoimmune encephalomyelitis (2012) Proteomics, 12, pp. 2656-2662Farias, A.S., Talaisys, R.L., Blanco, Y.C., Regulatory T cell induction during Plasmodium chabaudi infection modifies the clinical course of experimental autoimmune encephalomyelitis (2011) PLoS ONE, 6, pp. e17849Ramsdell, F., Foxp3 and natural regulatory T cells: Key to a cell lineage? (2003) Immunity, 19, pp. 165-168Sucher, R., Fischler, K., Oberhuber, R., IDO and regulatory T cell support are critical for cytotoxic T lymphocyte-associated Ag-4 Ig-mediated long-term solid organ allograft survival (2012) J Immunol, 188, pp. 37-46O'Sullivan, B.J., Pai, S., Street, S., Immunotherapy with costimulatory dendritic cells to control autoimmune inflammation (2011) J Immunol, 187, pp. 4018-4030Sun, Y., Chin, Y.E., Weisiger, E., Cutting edge: Negative regulation of dendritic cells through acetylation of the nonhistone protein STAT-3 (2009) J Immunol, 182, pp. 5899-5903Enioutina, E.Y., Bareyan, D., Daynes, R.A., Vitamin D3-mediated alterations to myeloid dendritic cell trafficking in vivo expand the scope of their antigen presenting properties (2007) Vaccine, 25, pp. 1236-1249Pallotta, M.T., Orabona, C., Volpi, C., Indoleamine 2,3-dioxygenase is a signaling protein in long-term tolerance by dendritic cells (2011) Nat Immunol, 12, pp. 870-878Steinbrink, K., Wolfl, M., Jonuleit, H., Knop, J., Enk, A.H., Induction of tolerance by IL-10-treated dendritic cells (1997) J Immunol, 159, pp. 4772-4780Chen, W., IDO: More than an enzyme (2011) Nat Immunol, 12, pp. 809-811Smolders, J., Peelen, E., Thewissen, M., The relevance of vitamin D receptor gene polymorphisms for vitamin D research in multiple sclerosis (2009) Autoimmun Rev, 8, pp. 621-62

Use of The second harmonic generation microscopy to evaluate chondrogenic differentiation of mesenchymal stem cells for cartilage repair

Articular cartilage injury remains one of the major concerns in orthopedic surgery. Mesenchymal stem cell (MSC) transplantation has been introduced to avoid some of the side effects and complications of current techniques.. With the aim to evaluate chondrogenic differentiation of mesenchymal stem cells, we used Second Harmonic Generation (SHG) microscopy to analyze the aggregation and orientation of collagen fibrils in the hyaline cartilage of rabbit knees. The experiment was performed using implants with type II collagen hydrogel (a biomaterial that mimics the microenvironment of the cartilage), one implant containing MSC and one other without MSC (control). After 10 weeks, the rabbit knees were dissected and fibril collagen distribution and spatial organization in the extracellular matrix of the lesions were verified by SHG. The result showed significant differences, whereas in histological sections of the cartilaginous lesions with MSC the collagen fibers are organized and regular; in the control sections the collagen fibers are more irregular, with absence of cells. A macroscopic analysis of the lesions confirmed this difference, showing a greater percentage of lesions filling in knees treated with MSC than in the knees used as controls. This study demonstrates that SHG microscopy will be an excellent tool to help in the evaluation of the effectiveness of MSC-based cell therapy for cartilage repair.Conference on Multiphoton Microscopy in the Biomedical Sciences XII822

Vitamin D3 Induces Ido+ Tolerogenic Dcs And Enhances Treg, Reducing The Severity Of Eae.

A growing body of evidence supports the hypothesis that vitamin D is an important environmental factor in the etiology of T-cell-mediated autoimmune diseases such as multiple sclerosis (MS). The purpose of this study was exploring the mechanisms underlying the beneficial effect of vitamin D3 in encephalomyelitis (EAE). We treated monophasic experimental autoimmune EAE, induced in Lewis rat, with vitamin D3 and adoptively transfer tolerogenic bone marrow-derived DCs generated in the presence of vitamin D3. This study provides evidence that the in vivo administration of vitamin D3, as well as the adoptive transfer of vitamin D3 -induced IDO(+) immature/tolerogenic dendritic cells, leads to a significant increase in the percentage of CD4(+) CD25(+) Foxp3(+) regulatory T cells in the lymph nodes in a rat model of MS, experimental autoimmune EAE. Concomitant with the increase in this cell population, there is a significant decrease in the number of autoreactive T cells in the central nervous system. Bone marrow-derived DCs cultivated in the presence of vitamin D3 present a tolerogenic profile with high IL-10, TNFα, and IDO expression and decreased MHC-II and CD80 expression. The adoptive transfer of IDO (+) DCs induces a significant increase in the percentage of CD4(+) CD25(+) Foxp3(+) T cells in the lymph nodes, comparable with vitamin D3 treatment. These mechanisms contribute actively to the generation of a microenvironment in the lymph nodes that suppresses the activation of encephalitogenic T cells, resulting in the downregulation of the inflammatory response in the central nervous system.19269-7

"Why caipirinha?"- the online via chat laddering technique CAN answer

As customers are becoming increasingly connected to the internet, this means that they are available for online interviews, thus opening up a space for investigating research methods, especially qualitative research, in an attempt to identify how to adapt data collecting instruments to the so-called “connected customer era”. In this context, the focus of this article is on the application viability analysis of the laddering technique used online and in real-time chat by asking the following question: “Why caipirinha?”. Conducting online in-depth interviews through the MSN Messenger and Skype (the most commonly used chat tools in Brazil), 23 attributes, 22 consequences and 13 values were identified, resulting in 133 ladders, 71 of which reached the value level. Along with friends/mates, Integration, Entertainment and Fun, in addition to Alcohol, Insouciance/ relaxation and Pleasure constitute the most frequent ladders. Concerning the application itself, the participants gave positive feedback, even though some of them did not feel satisfied because they became tired. Convenience, objectivity, disinhibition, easy scheduling and flexibility were identified. The viability of online in-depth interviewing via real-time chats was confirmed, raising the question of the possibility of it achieving other qualitative research techniques