No description available.

La maladie de Parkinson (MP) est caractérisée par la mort des neurones dopaminergiques de la substance noire et la présence de corps de Lewy. Son diagnostic reste sujet à des erreurs notamment aux stades précoces. Les cellules mononucléées sanguines périphériques (PBMC) jouent un rôle dans la cascade délétère et sont le reflet d’événements associés à la MP. Même si elles ne représentent qu’un faible pourcentage, les formes génétiquement déterminées permettent d’identifier des sujets à un stade précoce. Nous avons émis l’hypothèse que les PBMC pouvaient constituer un modèle d’étude reflétant certains mécanismes de la dégénérescence du vivant du patient. Nous avons réalisé des études du transcriptome chez différents groupes de sujets malades ou porteurs de mutations pour y déceler les gènes et voies de signalisation cellulaire dérégulés. Nous avons d’abord étudié le profil d’expression génique de sujets porteurs de la mutation G2019S de LRRK2. L’analyse des puces a permis d’identifier des perturbations de voies impliquées dans la MP comme l’oxydation mitochondriale, l’inflammation et la guidance axonale. Des altérations de la voie des MAPK, du cytosquelette d’actine et du transport vésiculaire ont été notées. La liste des gènes dérégulés permet de séparer les individus selon leur statut génétique. La mutation LRRK2 est associée à un profil d’expression génique dès les stades précoces identifiable dans les PBMC. Nous nous sommes ensuite intéressés à une autre forme de MP avec duplication de SNCA. Nous avons caractérisé la relation entre le génotype et le phénotype clinique des sujets de cette famille. La duplication s’étend sur 4,928 Mb, comporte 31 gènes et résulte d’une recombinaison homologue non allélique. L’analyse de l’expression des gènes présents dans la duplication dans les PBMC d’un sujet à un stade pauci-symptomatique a montré une surexpression de SNCA. Nous avons comparé nos analyses chez les porteurs des mutations LRRK2 et SNCA et chez des parkinsoniens sporadiques. Nos analyses montrent que les sujets LRRK2 et les sujets sporadiques présentent des dérégulations communes de voies de signalisation. En revanche, les voies dérégulées chez le sujet dupliqué reflètent la pathogénie de SNCA comme l’autophagie et les voies lysosomales. Nous nous sommes intéressés à l’expression des 4 isoformes de SNCA dans les PBMC de ces 3 groupes d’individus. Les patients sporadiques et LRRK2 montrent une diminution de l’expression des 4 isoformes de SNCA dans leur PBMC. Chez le sujet dupliqué, on observe uniquement une surexpression de l’isoforme 112. Nous avons ensuite identifié les voies moléculaires associée / Parkinson's disease (PD) is prone to misdiagnosis particularly in the early stages. A better understanding of the deleterious mechanisms is essential to identify therapeutic targets and detect the disease earlier. Peripheral blood mononuclear cells (PBMCs) play a role in the deleterious cascade and reflect molecular events associated with PD. Moreover, the study of genetically determined forms of PD enables the identification of subjects at a very early. We hypothesized that PBMCs could be an interesting model to study some mechanisms reflecting the neurodegeneration even at an early stage of the disease. Therefore, we conducted transcriptomic studies in different groups of PD subjects or patients with mutations in order to detect deregulated genes and signaling pathways.We first studied the gene expression profile of PD subjects with the mutation G2019S of the LRRK2 gene. Analysis of microarrays identified disturbances in cell signaling pathways involved in PD. Alterations in the MAPK pathway, the actin cytoskeleton and vesicular transport, associated with the pathogenesis of LRRK2, were noted. The list of deregulated genes separates individuals based on their genetic status including an asymptomatic subject. G2019S LRRK2 mutation is associated to a particular gene expression profile identifiable in PBMCs even at early stage.Then we investigated another form of genetically determined by duplication of SNCA gene. We better characterized the relationship between genotype and clinical phenotype of the subjects. The duplication extends 4.928 Mb, contains 31 genes and results from non-allelic homologous recombination. The analysis of the expression of genes in the PBMCs of a subject carrying the mutation at preclinical stage showed overexpression of SNCA.We compared PBMCs gene expression of G2019S LRRK2 mutation carriers, SNCA duplication carrier and also sporadic PD patients. Our analysis showed that carriers of the LRRK2 mutation and sporadic PD patients have common deregulated signaling pathways that reflect the PD pathogenesis. By contrast, pathways deregulated in the subject with SNCA duplication reflect the pathogenesis of SNCA. In addition, we looked at the expression of SNCA isoforms in PBMCs of these three groups of individuals. Sporadic and LRRK2 patients showed a decreased expression of four isoforms of SNCA in their PBMCs. However, in the duplicated subject, only isoform 112 was overexpressed.Then we used this technology to identify molecular pathways associated with spino-cerebellar ataxia type 2 (SCA2), which provides rarely a parkinsonian phenotype and compared with subjects with a cerebellar phenotype. Again, we identified deregulation of gene expression associated with SCA2 pathogenesis, such as amyotrophic lateral sclerosis and actin cytoskeleton in PBMCs of subjects with parkinsonian and metabolism of RNA and inositol phosphate in cerebellar subjects.Finally, we looked at gene expression in PBMCs according to the evolutionary and clinical stage of PD including individuals at a very early. We compared their gene expression profiles with more advanced PD patients. From the early stages, we observed a deregulation of ERK/MAPK and PI3K/Akt pathways that control cell survival; these findings underscore the importance of these biological pathways in the development of PD.In conclusion, we demonstrated that PBMCs are an interesting model. The transcriptomic studies can get insight into the mechanisms associated with early stages of degeneration and into biological markers, such as SNCA. This technique could be applied in a larger number of subjects including other neurodegenerative diseases to detect specific diagnostic markers of PD.

Pulmonary hypertension (PH), when it complicates sarcoidosis, carries a poor prognosis, in part because it is difficult to detect early in patients with worsening respiratory symptoms. Pathogenesis of sarcoidosis occurs via incompletely characterized mechanisms that are distinct from the mechanisms of pulmonary vascular remodeling well known to occur in conjunction with other chronic lung diseases. To address the need for a biomarker to aid in early detection as well as the gap in knowledge regarding the mechanisms of PH in sarcoidosis, we used genome-wide peripheral blood gene expression analysis and identified an 18-gene signature capable of distinguishing sarcoidosis patients with PH (n = 8), sarcoidosis patients without PH (n = 17), and healthy controls (n = 45). The discriminative accuracy of this 18-gene signature was 100% in separating sarcoidosis patients with PH from those without it. If validated in a large replicate cohort, this signature could potentially be used as a diagnostic molecular biomarker for sarcoidosis-associated PH.

This thesis reports the results of a large number of experiments which were designed to elucidate the mechanisms whereby Gambian children, suffering from acute Plasmodium falciparum malaria may eventually control their infections. These experiments were carried out in vitro and success or failure of the various test systems was judged by their effect on parasite multiplication. Early in the course of these investiqations it was demonstrated that mononuclear cells from these children could cooperate with antibodies present in their serum to bring about a marked reduction in parasite growth. The efficiency of this antibody-dependent cellular cytotoxicity (ADCC) mechanism was related to levels of parasitaemia in the children, being greater in convalescent children than in those with acute malaria. Attempts were now made to identify the effector cells in this ADCC. Purified T and B cells were ineffective and although purified adherent cells (A) had an effect, it was much less than that mediated by the undepleted mononuclear cell population. Adherent cells were, however, fully effective in ADCC if they were exposed to the supernatant from T cells non-specifically activated by PHA. Thus cell cooperation leading to activation appears to play an important role in this system. Finally, experiments were set up to determine whether activated mononuclear cells could exert an inhibitory effect on parasite multiplication which was independent of anti-malarial antibody. It was shown that depression of parasite growth could be achieved by mononuclear cells, either from the children or from Europeans, if these cells were exposed to supernatants of previously stimulated mononuclear cells. These findings can be assembled to provide a tentative model of the development of protective responses in vivo. Perhaps following phagocytosis of parasite antigens and their presentation on the cell surface, T cells become activated: they may cooperate with B cells to produce parasite specific antibodies; they may also activate other mononuclear cells (non T, non B) to become effector cells. These cells, either alone, or perhaps more efficiently in cooperation with antibody, are able to kill parasites by the release of toxic factors, and the infection is brought under control. Finally, large amounts of specific antibodies of appropriate isotypes are synthesized. Acting as opsonins or by activating complement, they may serve to destroy remaining parasites. Their continued presence, by preventing merozoite penetration, may provide at least a temporary defense against reinfection. It is assumed that Gambian adults who have suffered repeated malaria infections and are now immune are defended by their possession of circulating IgG antibodies and B memory cells of all appropriate specificities.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2016-05-11T16:05:45Z No. of bitstreams: 1 Barbara Maria Nascimento de Oliveira Produção... 2016.pdf: 2363764 bytes, checksum: ec48ade14eccf109aecb436eec7cd66a (MD5) / Approved for entry into archive by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2016-05-11T16:05:58Z (GMT) No. of bitstreams: 1 Barbara Maria Nascimento de Oliveira Produção... 2016.pdf: 2363764 bytes, checksum: ec48ade14eccf109aecb436eec7cd66a (MD5) / Made available in DSpace on 2016-05-11T16:05:58Z (GMT). No. of bitstreams: 1 Barbara Maria Nascimento de Oliveira Produção... 2016.pdf: 2363764 bytes, checksum: ec48ade14eccf109aecb436eec7cd66a (MD5) Previous issue date: 2016 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / INTRODUÇÃO. A interleucina 7 (IL-7) é uma citocina pleiotrópica produzida principalmente por células estromais da medula óssea, células epiteliais tímicas e intestinais. IL-7 e que promove o desenvolvimento de linfócitos T e B e diferenciação de células T memória, especialmente CD4+. Por isso, diversos autores têm estudado a capacidade de IL-7 promover a restauração da população de linfócitos em situações de linfopenia e resposta imune de memória. Cães que desenvolvem leishmaniose visceral e recebem tratamento especfico apresentam cura clínica. No entanto, após a interrupção do tratamento, a maioria dos animais exibe recaída da doença, mesmo na ausência de reinfecção. É possível que esse fenômeno esteja associado a uma dificuldade de estabelecimento de linfócitos T de memória específicos para Leishmania em cães. Em nosso laboratório, tentativas prévias foram realizadas para produzir a IL-7 canina em Escherichia coli, no entanto, o rendimento da proteína biologicamente ativa foi pequeno. OBJETIVO. Por isso, resolveu-se avaliar a viabilidade de produzir IL-7 do sistema baculovírus-células de inseto. MATERIAL E MÉTODOS. No presente trabalho, uma construção de DNA foi concebida contendo uma sequência de nucleotídeos que codificam o peptídeo sinal da proteína GP64 do baculovírus da Autographa californica (AcMNPV), a IL-7 canina (proteína madura) com códons otimizados para a tradução em Trichoplusia ni, um espaçador com 23 aminoácidos e uma cauda de seis histidinas. A construção de DNA elaborada foi sintetizada quimicamente, inserida em um plasmídeo de clonagem (pUC57-GP64caIL-7), subclonada em um plasmídeo carreador (pFastBac1- GP64caIL-7), adequado para o sistema baculovirus-célula de inseto, e transferida para o cromossoma artificial do baculovírus recombinante (bacmídeo). RESULTADOS. A produção de IL-7 recombinante canina (rcaIL-7) em células de inseto infectadas com baculovírus recombinante foi otimizada. Em seguida, a rcaIL-7 foi produzida em células High-five cultivadas em suspensão utilizando multiplicidade de infecção (MOI) de 5 e tempo de infecção (TOI) de 48 h. A proteína recombinantes foi purificada por cromatografia de afinidade em Sepharose-níquel, obtendo-se um rendimento médio de 5,5 mg por litro de cultivo. CONCLUSÃO. RcaIL-7 purificada, avaliada a 40 ng/mL, mostrou-se capaz de promover a proliferação de células mononucleares de sangue periférico de cães sadios, sem estímulo prévio ou concomitante, indicando que a proteína foi produzida biologicamente ativa. Futuros estudos serão realizados para avaliar a capacidade imunomoduladora da rcaIL-7 em cães e determinar a utilidade dessa citocinas no tratamento de enfermidades caninas, como por exemplo, na leishmaniose visceral canina. / INTRODUCTION. The interleukin 7 is a pleiotropic cytokine produced mainly by bone marrow stromal cells, thymus and intestinal epithelial cells. IL-7 promotes the development of T and B limphocytes and differentiation T cells to memory cells, especially T CD4+ cells. For these reasons, several authors have studied the ability of IL- 7 to promote restoration of the lymphocyte population in cases of lymphopenia and memory imune responses. Dogs which develop visceral leishmaniasis and are treated with specific drugs develop only transient clinical cure. After treatment withdrawal, most animals show disease relapse, even in the absence of reinfection. One possible explanation for the lack of control of the infection after treatment in dogs is a difficulty to develop/maintain specific memory T cells. Therefore, it is possible that effective treatments for canine visceral leishmaniasis be developed by manipulating the immune system. Previously, in our laboratory, it was attemped to produce recombinant canine IL- 7 (rcaIL-7) in Escherichia coli, however there was a low yield of biologically active protein. OBJECTIVE. In the current project, the baculovírus-insect cell system was evaluated to produce rcaIL-7. MATERIAL AND METHODS. In this study, a construction of DNA was designed to encode nucleotides of Autographa californica GP64 signal peptide, canine IL-7 (mature protein) with optimized codons for Trichoplusia ni, a 23-amino acid spacer and a six histidine tail. The DNA construct was chemically synthesized, inserted into a cloning plasmid (pUC57-GP64caIL-7), subcloned into a carrier plasmid (pFastBac1-GP64caIL-7), suited to the baculovirus-insect cell expression system, and transfered to an artificial chromosome of the recombinant baculovirus (bacmid). RESULTS. Recombinant protein expression was optimizatized, then, the rcaIL-7 was produced in a cell culture in suspension using multiplicity of infection (MOI) 5 and time of infection (TOI) 48 hours. Subsequently rcaIL-7 was purified by Nickel Sepharose-Affinity chromatography, obtaining an average yield of 5.5 mg per liter of culture. CONCLUSION. The purified recombinant rcaIL-7 at 40 ng/mL was able to induce prolifereation of peripheral blood mononuclear cells of heath adult dog, suggesting it was biologically active. Given the results, the rcaIL-7 may be used in further studies to assess its ability to modulate canine immune system, that would be useful for the development immunotherapy

No description available.

Osteoarthritis (OA) can be debilitating and career-ending for horses. Current treatments offer temporary and symptomatic relief, but potentially deleterious side effects. Bone marrow mononuclear cells (BMNC) are a rich source of macrophage progenitors that are anti-inflammatory and promote inflammation resolution. The objective of this study was to evaluate the ability of intra-articular BMNC therapy to improve clinical signs of naturally occurring equine OA. Horses presenting with clinical and radiographic evidence of moderate OA in a single joint were randomly assigned to 1 of 3 treatments: saline (negative control), triamcinolone (positive control), or BMNC (treatment group). Horses were subjectively and objectively evaluated for lameness and synovial fluid collected (cytology and cytokine/growth factor quantification) at 0, 7, and 21 days post-injection. Data were analyzed using General Estimating Equations with significance set at P<0.05. There were no adverse effects noted in any treatment group. No significant differences in synovial fluid cytology parameters, objective/subjective lameness scores, nor joint circumference were found between treatment groups at any time point. Within treatment groups, joint circumference did not change over time for saline- and triamcinolone-treated horses. However, joint circumference and objective lameness decreased significantly within BMNC-treated horses between Days 0 and 21 and Days 7 and 21. Lameness improved in saline-treated horses from 0 to 21 days, but did not improve in triamcinolone-treated horses. The decreased lameness and lack of adverse effects in the BMNC-treated horses in our study support a larger clinical trial using BMNC. / Master of Science / Osteoarthritis (OA) is a common source of joint pain in people and horses. Current treatments provide only partial and/or temporary relief. As a result, there is an urgent need for more effective and long-lasting treatment options. Arthritis is characterized by uncontrolled joint inflammation and progressive cartilage and bone destruction. Macrophages are cells within normal joints that function to resolve mild inflammation, maintaining joint health. However, when physiologic functions are overwhelmed, macrophages perpetuate inflammation through the recruitment of additional cell types to cope with the increased demands for repair. If this process is appropriately accomplished, macrophages resolve the inflammation, thereby enabling recovery and repair within the joint. Bone marrow aspirate is an excellent source of bone marrow-derived macrophage precursors (bone marrow mononuclear cells or BMNC) that have been shown to reduce joint inflammation and lameness in people and horses. The objective of our clinical trial was to evaluate the ability of intra-articular BMNC to improve clinical signs of naturally occurring OA in horses. BMNC treatment was compared to a placebo injection of saline and a standard-of-care in horses, corticosteroids. There were no adverse effects of BMNC treatment and BMNC-treated horses had significantly reduced joint circumference and lameness after 21 days. Synovial fluid cytology parameters did not differ significantly between treatment groups at any time point. In summary, BMNC are exciting because a horse can be treated with its own cells without the need for specialized equipment, and have the potential to naturally benefit thousands of people and horses suffering from arthritis.

Esta pesquisa avaliou o efeito da transferência passiva dos leucócitos do colostro na imunidade específica de bezerras recém-nascidas. Foram acompanhadas 20 bezerras Holandesas durante o período neonatal, distribuídas em dois grupos experimentais: grupo COL+ recebeu colostro fresco (4L) proveniente de suas respectivas mães; e grupo COL- recebeu colostro congelado e acelular (4L), oriundo de vacas doadoras de colostro. As avaliações foram realizadas antes da mamada do colostro (M0), 1-2 (M1), 7 (M2), 14 (M3), 21 (M4) e 28 dias pós-nascimento (M5). As bezerras foram submetidas ao exame clínico, seguido da colheita das amostras sanguíneas para realização de hemograma, imunofenotipagem e cultivo celular. Os dois grupos foram colostrados com colostro de igual qualidade com relação à concentração de imunoglobulinas (70-120 g/L). A concentração de células do colostro fresco fornecido ao grupo COL+ foi de 1.895.849 células/mL. Não foi possível encontrar diferenças para as funções vitais em relação aos grupos experimentais. O exame específico dos sistemas revelou um caso de broncopneumonia, três de inflamação umbilical e maior frequência de escore de fezes 3 no COL-. As alterações clínicas foram refletidas no eritrograma das bezerras, sendo encontrado menor valor médio para a taxa de hemoglobina (HGB) no COL- em M3. Em relação à idade, observou-se redução gradativa dos valores médios para He (hemácias), HGB, HCT (hematócrito) e índices hematimétricos no primeiro mês de vida. A frequência de bezerras anêmicas foi maior no grupo COL- nos momentos M4 e M5. Em relação ao leucograma, observou-se diferença entre os grupos para linfócitos no M0 e M2 com valores superiores no COL-. Em relação aos momentos foi possível detectar leucocitose por neutrofilia no M0 e M1, observando-se inversão da relação neutrófilo:linfócito a partir desses momentos. Os valores de CD45+CD45RO- foram maiores em M0 no COL-, além disso, observou-se aumento da expressão do marcador de memória celular CD45RO+ do M0 ao M1 nos dois grupos experimentais. O CD3+gamma-delta- aumentou no decorrer do estudo, em contrapartida as células CD3+gamma-delta+ foram menores em M5 com relação ao M0-M3. Foi detectado também aumento dos valores de CD14+MHCII+ no primeiro mês de vida indicando maturação das células apresentadoras de antígeno. Em relação à produção de citocinas pelas células mononucleares sanguíneas, foi possível identificar maior concentração de IFN-gamma em M4, quando as células do COL- foram estimuladas com S. aureus (1 mononuclear:10 bactérias inativadas). A concentração de IL-17 detectada a partir das células do COL+ foi maior em M3, quando as células foram estimuladas com ConA. Com base nos resultados obtidos, pode-se concluir que: a) bezerras COL- apresentaram maior frequência e intensidade de doenças que evoluíram para anemia da inflamação; b) bezerras COL- apresentaram maior número absoluto de linfócitos, representadas especialmente pela subpopulação CD3+gamma-delta+ nos episódios de maior frequência de diarreias; c) Linfócitos de memória CD45RO+ aumentaram após a colostragem em ambos os grupos, sugerindo que outros componentes acelulares do colostro podem apresentar papel fundamental no desenvolvimento da resposta imunológica de bezerras recém-nascidas; d) a subpopulação CD3+gamma-delta- e as células CD14+MHCII- e CD14+MHCII+ aumentaram durante o primeiro mês de vida, indicando maturação imunológica; e) as células mononucleares das bezerras não responderam ao Herpesvírus Bovino tipo 1, porém responderam aos estímulos bacterianos, especialmente para a Escherichia coli; a interpretação do leucograma em conjunto com a análise das variações apresentadas para as citocinas inflamatórias IFN-gamma e IL-17 permitem afirmar que as bezerras apresentaram resposta inflamatória retardada e de menor magnitude no COL-. / This study evaluated the effect of leukocytes passive transference from bovine colostrum in specific immunity of newborn calves. During neonatal period, 20 Holstein calves were followed. Animals were distributed in two experimental groups: COL+ which received fresh colostrum (4L) from their mothers, and COL- which received frozen and acellular colostrum (4L) that came from donor cows. The evaluations were performed in the following moments: before colostrum intake (M0), 1-2 (M1), 7 (M2), 14 (M3), 21 (M4) and 28 days after birth (M5). Heifers were submitted to clinical examination. Then, blood samples were harvested for hemogram, immunophenotyping and cell culture. Both groups were fed with the same quality of colostrum (immunoglobulin concentration 70-120 g/L). The cell concentration of fresh colostrum that was provided to COL+ group was 1.895.849 cells/mL. It was not possible to detect differences in vital functions concerning the experimental groups. The system specific examination reveled one case of bronchopneumonia, three cases of umbilical inflammation and major rates of diarrhea score 3 in group COL-. Clinical alterations were reflected in calves erythrogram. It was found lower mean value for hemoglobin (HGB) in M3 for COL-. Regarding age, a gradual reduction in mean values for erythrocytes, HGB, HCT (hematocrit) and hematimetric rates were observed in the first month of life. The frequency of anemic heifers was higher in COL- group at moments M4 and M5. Regarding leukogram, it was observed difference between groups for lymphocytes in M0 and M2 with higher values in COL-. Concerning moments, it was possible to detect leukocytosis by neutrophilia from M0 up to M1 and inversion of neutrophil:lymphocyte relation from this moment. Values of CD45+CD45RO- was higher in M0 for COL-, furthermore, increase of cellular memory marker expression CD45RO+ was observed from M0 to M1 in both groups. The CD3+gamma-delta- increased during the study. On the other hand, CD3+gamma-delta+ were lower in M5 in relation to M0-M3. Increase of CD14+MHCII+ values were also detected in the first month of life, indicating maturation of antigen presenting cells. Regarding cytokine production by mononuclear cells of heifers blood, it was possible to identify higher concentration of IFN-gamma in M4 when cells of COL- were stimulated with S. aureus (1 mononuclear: 10 inactivated bacteria). The concentration of IL-17 detected from COL+ cells was higher in M3, when cells were stimulated with ConA. Based on these results, it can be concluded that: a) COL- heifers presented higher frequency and intensity of diseases that evolved to anemia of inflammation; b) COL- heifers presented higher lymphocyte absolute number, represented specially by CD3+gamma-delta+ subsets in episodes of higher frequency of diarrhea; c) memory lymphocytes CD45RO+ increased after colostrum intake in both groups, suggesting that other acellular colostrum components can present fundamental role in development of immunological response in newborn heifers; d) the subset of CD3+gamma-delta- and the cells CD14+MHCII- and CD14+MHCII+ increased during the first month of life, indicating immunological maturation; e) heifers mononuclear cells did not respond for herpes virus bovine type 1, however, responded for bacterial stimulus, specially Escherichia coli. The interpretation of leukogram with the variation of presented analyses for inflammatory cytokines IFN-gamma and IL-17, allow to state that heifers presented delayed inflammatory response and of lesser magnitude in COL-.

A necessidade de tratamentos mais eficazes e menos invasivos para os pacientes, em adição à capacidade de diferenciação celular da medula óssea sugere que o transplante de células mononucleares totais poderia ser uma das melhores formas de tratamento para as diversas patologias existentes. Entretanto, vários fatores implicam sobre a viabilidade das células da medula óssea dos quais destacamos a ausência de padronização de protocolo de criopreservação que permita a manutenção da viabilidade celular, sendo altamente necessário o desenvolvimento de estudos nesta área. Deste modo, neste estudo, após a anestesia de um grupo de animais foi realizada punção da medula óssea, separação das células mononucleares e avaliação da viabilidade. Foram testados oito meios diferentes para criopreservação das células. O meio de congelamento A é composto por 20% dimetilsulfóxido (DMSO), 40% Dulbecco´s Modified Eagle´s Médium (DMEM) e 40% de Plasma Autólogo, o meio de congelamento B contém 20% dimetilsulfóxido (DMSO), 40% Roswell Park Memorial Institute (RPMI) e 40% de Plasma Autólogo, o meio de congelamento C tem em sua composição 20% dimetilsulfóxido (DMSO), 40% soro fetal bovino (SFB) e 40% plasma autólogo, o meio de congelamento D é composto de 20% dimetilsulfóxido (DMSO) e 80% de plasma autólogo, o meio E constitui-se de 5% dimetilsulfóxido (DMSO), 47,5% de Dulbecco\'s Modified Eagle\'s Médium (DMEM) e 47,5% de plasma autólogo, o meio F contém 5% dimetilsulfóxido (DMSO), 47,5% de Roswell Park Memorial Institute (RPMI) e 47,5% de plasma autólogo, o meio G contém 5% dimetilsulfóxido (DMSO), 47,5% de soro fetal bovino (SFB) e 47,5% de plasma autólogo e o meio H constitui-se de 5% dimetilsulfóxido (DMSO) e 95% de plasma autólogo. Após as análises realizadas pela técnica de citometria de fluxo, o meio mais eficiente na criopreservação das células mononucleares de suínos foi o protocolo D por possuir maior concentração de plasma autólogo e crioprotetor. / The necessity of the most efficient and less invasive treatments for the patients, in addition to the capacity of cellular differentiation of the bone marrow suggests that the transplant of mononuclear total cells might be one of the best treatment for several pathologies. Meantime, several factors influence on the viability of the cells of the bone marrow as the absence of standardization of criopreservação protocol which could allow the maintenance of the cellular viability, being an important issue of investigation. In this study, after the anaesthesia of a group of animals, samples of bone marrow were collected, following the separation of the mononuclear cells and evaluation of the viability. Eight different protocols were tested for cells criopreservation. The protocol A by 20% dimetilsulfóxido (DMSO), 40% Dulbecco\'s Modified Eagle\'s Médium (DMEM) and 40% autologus plasma, B protocol conatained 20% dimetilsulfóxido (DMSO), 40% Roswell Park Memorial Institute (RPMI) and 40% autologus plasma, the C protocol was composed 20 % dimetilsulfóxido (DMSO), 40% bovine fetal serum (SFB) e 40% autologus plasma, D protocol was made using 20% dimetilsulfóxido (DMSO) and 80% autologus plasma, E protocol was constituted by 5% dimetilsulfóxido (DMSO), 47,5% de Dulbecco\'s Modified Eagle\'s Médium (DMEM) and 47,5% autólogo plasma, the F contains 5% dimetilsulfóxido (DMSO), 47,5% de Roswell Park Memorial Institute (RPMI) and 47,5% autologo plasma, the protocol G was compoed by 5% dimetilsulfóxido (DMSO), 47,5% de bovine feta serum (SFB) and 47,5% autologus plasma and the H protocol was 5% dimetilsulfóxido (DMSO) and 95% autologus plasma. After flux citometer analyses, the most efficient protocol in criopreservation of the mononuclear cells of pigs was the protocol D which was composed the by the most amount of autologus plasma and crioprotectant.

In cardiovascular disease the repair response is insufficient to restore blood flow, leading to the death of muscle and loss of tissue function. Therefore, strategies to augment the endogenous cell response and its effects may help improve tissue recovery and function. In this study we explored the use of tissue-engineered collagen matrices for augmenting endogenous regenerative processes after myocardial infarction. Treatment with the sLeX-collagen matrix reduced inflammation and apoptosis and had a positive regenerative effect on the infarcted mouse heart, through improved vascular density and possibly enhanced cardiomyogenesis. Additionally, we investigated the effects of cryopreservation on generating circulating angiogenic cells (CACs) from peripheral blood mononuclear cells (PBMCs), as a potential source of stem cells that could be used in combination with our collagen scaffold. Our findings show that despite PBMCs experiencing phenotypic changes after cryopreservation, they may still be used to generate the same therapeutic CACs as freshly procured PBMCs.