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.

Orientador: Joyce Maria Annichino Bizzacchi / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-21T17:55:26Z (GMT). No. of bitstreams: 1 Bassora_FernandaDutraSantiago_D.pdf: 2667928 bytes, checksum: 612538a2c5c4e4e33577d2303de3a9c1 (MD5) Previous issue date: 2012 / Resumo: A trombose venosa é definida como a oclusão de um vaso do sistema venoso. Três fatores básicos para a formação de um trombo no interior dos vasos são: alteração do fluxo sangüíneo, da parede vascular e/ou dos elementos sangüíneos. A trombose venosa e a embolia pulmonar, que ocorre como uma complicação subseqüente representa uma causa importante de morbidade e mortalidade em pacientes hospitalizados. A freqüência da trombose venosa foi estimada em aproximadamente 1/1000 na população em geral. Na maior parte dos casos existe uma tendência para trombose determinada pela presença de um fator causal herdado ou adquirida, ou pela interação desses fatores, bem como por variações genéticas que determinam alterações nos níveis das proteínas pró-coagulantes e anticoagulantes. Dados da literatura têm sugerido a associação de mecanismos inflamatórios com a fisiopatologia da trombose venosa profunda (TVP). Os monócitos, estimulados por citocinas ou endotoxinas, expressam fator tecidual, o maior indutor da coagulação sangüínea, e que também tem a função de sinalização para a mobilidade celular e vascular. Os leucócitos apresentam receptores capazes de ligar e ativar o fator X da coagulação, servindo como via alternativa para a formação de trombina. As plaquetas podem aderir ao endotélio intacto e através da liberação de mediadores e citocinas como interleucina (IL)-1 e Fator de necrose tumoral-a (TNF-a), induzindo a expressão de moléculas de adesão e fator tecidual pela célula endotelial. Com base nestes dados e considerando que a migração leucocitária é um dos principais eventos que caracterizam o processo inflamatório, justificamos a escolha de células mononucleares (monócitos e linfócitos) como células centrais do nosso estudo. Utilizando técnicas de separação de células mononucleares por centrifugação em gradiente de "Ficoll-Hypaque", extração do Ácido ribonucléico (RNA) total, hibridação em Ácido desoxiribonucléico complementar (cDNA) -Microarray, e validação usando a reação em cadeia da polimerase em tempo real quantitativo (qRT-PCR) avaliamos do perfil de expressão gênica de alguns mediadores inflamatórios nessas células e a possível relação com a trombose venosa. Neste trabalho, usando a tecnologia de Microarray encontramos 60 induzidos e 56 genes reprimidos diferencialmente expressos nos pacientes com TVP, estes genes que estavam relacionados à resposta imune, inflamação, proteólise e transcrição. Destes genes diferencialmente expressos, selecionamos nove relacionados com inflamação para validação usando a técnica de qRT-PCR. Destes, somente houve aumento de expressão do gene da caspase 4 (CASP4) nos pacientes com TVP, sendo que, esta diferença se manteve no subgrupo com TVP espontâneo. Neste mesmo subgrupo, também foi verificado o aumento da expressão no gene Elong factor 1, alpha 2(EEF1A2) / Abstract: Venous thrombosis is defined as a vessel occlusion of the venous system. Three basic factors for the formation of a thrombus inside the vessel are: changes in blood flow, vascular wall and / or blood elements. Venous thrombosis and pulmonary embolism, which occurs as a subsequent complication is a major cause of morbidity and mortality in hospitalized patients. The frequency of venous thrombosis was estimated to be approximately 1 / 1000 in the general population. In most cases there is a tendency for thrombosis determined by the presence of a causal factor inherited or acquired, or by the interaction of these factors, as well as genetic variations that determine changes in protein levels of procoagulants and anticoagulants. Literature data have suggested the association of inflammatory mechanisms in the pathophysiology of deep venous thrombosis (DVT). Monocytes, stimulated by cytokines or endotoxin, express tissue factor, the greater inducer of blood coagulation, and also have the function of signaling for cell motility and vascular. Leukocytes have receptors that can bind and activate coagulation factor X, serving as an alternative route for the formation of thrombin. Platelets can adhere to intact endothelium and through the release of mediators and cytokines such as interleukin (IL)-1 and Tumor necrosis factor-a (TNF-a), inducing expression of adhesion molecules and tissue factor by endothelial cells. Based on these data and considering that leukocyte migration is one of the main events that characterize the inflammatory process, we justify the choice of mononuclear cells (monocytes and lymphocytes) as the central cells of our study. Using techniques of separation of mononuclear cells by gradient centrifugation "Ficoll-Hypaque," extraction of total RNA, cDNA-Microarray hybridization, and validation using real time qPCR assessed the gene expression profile of some inflammatory mediators in these cells and the possible relationship with venous thrombosis. In this work using Microarray technology we found 60 genes upregulated and 56 downrelated differentially expressed in patients with DVT. Genes that were related to immune response, inflammation, proteolysis and transcription. Of these differentially expressed genes, we selected nine genes that were related with inflammation to validation using qRT- PCR technique. Just one of then, the caspase 4 (CASP4) genes was differentially increased in DVT patients, this increase kept in patients with spontaneous DVT and an increased of Elong factor 1, alpha 2 (EEF1A2) gene too / Doutorado / Ciencias Biomedicas / Doutora em Ciências Médicas

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.

Immunological and ultrastructural changes in mononuclear cells exposed to Mycobacterium avium serovar-specific glycopeptidolipid (GPL) and the chemically derived R-lipid (lipopeptide fragment) were examined.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24515号 / 医博第4957号 / 新制||医||1064(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 森信 暁雄, 教授 竹内 理, 教授 濵﨑 洋子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Cellular stress is central to the understanding of pathological mechanisms and the development of new therapeutic strategies and serves as a biomarker for disease progression in neurodegeneration, diabetes, cancer, cardiovascular and other chronic diseases. The common cellular stress assay (CSA) based on Seahorse technology in peripheral blood mononuclear cells (PBMCs) shows inconsistent results, which prevents its use as a biomarker for the progression of chronic diseases. Therefore, the aim of this study was to investigate potential factors that affect the CSA in PBMCs. We measured the CSA parameters in PBMCs from study participants and compared the results according to the potential factors, namely, the PBMC isolation method, age, seasonal variation and the gender of the study participants. PBMCs were isolated by OptiPrep® and RobosepTM-S methods. PBMCs isolated with the OptiPrep method showed much higher extracellular acidification and higher respiration compared to Robosep-isolated cells. Moreover, OptiPrep-isolated cells showed a higher number of outliers for the proton production rate (PPR) and a high respiratory quotient, indicating impurities with other cells, such as platelets, and technical inconsistencies. PBMCs from older individuals showed higher maximal respiration, spare capacity and extracellular acidification than younger participants. Additionally, in winter, maximal respiration and spare capacity decreased. From spring until early autumn, spare capacity and maximal respiration continuously increased. Elderly males also showed higher basal respiration, spare capacity and extracellular acidification than females. In conclusion, the findings of this study clearly demonstrate that the results of CSA parameters measured in PBMCs are influenced by the PBMC isolation method, age, seasonal variation and gender. Therefore, we recommend that researchers and physicians properly interpret the results of CSA parameters in PBMCs by considering these factors. It is important to use separate CSA evaluation standards based on the isolation method, age, gender and season-dependent factors. To assess the cellular stress situation in PBMCs, both extracellular acidification and mitochondrial respiration should be taken into account. Further study of additional factors, such as mitochondrial mass, should be conducted to improve the measurement of CSA parameters for the assessment of the real mitochondrial fitness.

No / Recent studies have gained positive results using nanoparticles (NPs) in treating atherosclerosis on animals. But their toxicity and application in treating other heart diseases such as heart failure and endocarditis still need proper investigation. Gold nanoparticles (Au-NPs) were chosen as model substances as they have been successfully used in treating cancer. In this study, we use both H9C2 myoblasts and rat peripheral blood mononuclear cells to determine the influence of Au-NP size on their cytotoxicity and cell apoptosis. H9C2 cells were treated with Au-NPs of a diameter of 5, 20, 40 and 100nmfor 24 hrs before their cell viabilities tested by MTT assay, cell apoptosis measured by flow cytometry, and the generation of reactive oxygen species (ROS) detected by Fluorometric Intracellular ROS Kit. Distribution of the Au-NPs and their effects on the structure of mitochondria and lysosome were detected by electron microscopy. In addition, we obtained rat peripheral blood mononuclear cells and treated them with Au-NPs same with H9C2 cell line. Our results showed NPs of 5, 40, and 100 nm reduced cell viabilities on H9C2 cells while20nm showed no change on cell viability (Ctrl: 100±8.2 vs 20nm: 95.39±9.13, P>0.05, n=6) and some protect effect on ISO induced H9C2 cells apoptosis (ISO: 100±13.5 vs 20nm: 80.19±17.36, P>0.05, n=6). All size of Au-NPs reduced cell viabilities on rat peripheral blood mononuclear cells while 40nm showed the least reduction on cell viability (Ctrl: 100.0±3.0 vs 40nm: 76.31±3.68, P<0.001, n=6) and significant protect effect on ISO-induced rat peripheral monocytes apoptosis (ISO: 100±1.86 vs 40nm: 45.34±10.32, P<0.05, n=6). In addition, 20nm Au-NP showed some protect effect on ROS generation on ISO-induced H9C2 cells (ISO: 100±3.79 vs 20nm: 94.84±4.98, P>0.05, n=6), while 40nm produced more ROS (ISO: 100±3.79 vs 40nm: 141.63±42.81, P>0.05, n=6). Electron microscopy detection showed correlated results in structure. These results on H9C2 cell line are basically in agreeable to our animal study. The protective effect of 20nm may due to its ability to protect ISO-induced ROS generation. The results on rat peripheral monocytes are slightly different to those on H9C2 cells. Further investigation need to focus on the role of NPs size on cell apoptosis by detecting autophagy specific protein through western blotting. / Abstract of conference paper.

Made available in DSpace on 2014-07-29T15:13:47Z (GMT). No. of bitstreams: 1 TESE Julia de Miranda Moraes.pdf: 2895250 bytes, checksum: e061f736f6922f781b2c671bf4f90f32 (MD5) Previous issue date: 2012-08-17 / The aim of this work was to evaluate the regeneration of equine ulnar nerves submitted to neurotomy, silicone tubing and cell therapy with bone marrow mononuclear cells fraction (MCF) or adipose derived mesenchymal stem cells (ADSC). Fifteen adult horses were divided into three groups with five animals each: control group (CG) with the use of saline solution, group with FCM deposition and group with ADSC deposition. The same surgical procedure was performed in all groups, using both nerves of each animal (right and left), establishing two moments of biopsy: on the 13th week on the right limb (CG1, MCF1 and ADSC1) and on the 26th week on the left limb (CG2, MCF2 and ADSC2). The MCF and ADSC were obtained respectively, from bone marrow and adipose tissue from each animal, both used as an authologous implant. After 13 and 26 weeks, biopsies were performed in all groups and immediately it was made some fragments slide imprints for viewing the nanocrystal fluorescent label. The fragments were fixed in 10% buffered formalin for histological analysis, with HE, luxol fast blue, Masson's trichrome, immunohistochemistry against the antibodies neurofilament (NF), S-100, FGF-2 and GDNF. Microscopically it was observed the presence of axonal growth, connective tissue, inflammatory infiltrate, Schwann cells, neural growth factors, myelin sheath and wallerian degeneration. For histologic analysis, it was established qualitative scores and for immunohistochemistry it was performed quantitative analysis with Image J program. It was observed wallerian degeneration reduction, better fascicular reorganization, new collagen increased, myelin sheath early formation, and NF antibody stronger staining in the experimental groups compared to CG. The ADSC group presented the best results than the other groups, showing ADSCs efficiency compared to MCF and CG for peripheral nerve regeneration. However, it was proved a longer time necessity to occur complete regeneration of peripheral nerve after injury. / Este trabalho teve por objetivo avaliar a regeneração do nervo ulnar de equinos, submetidos à neurotomia, tubulização com tubo de silicone e terapia celular com fração de células mononucleares da medula óssea (FCM) ou células-tronco mesenquimais do tecido adiposo (ADSC). Foram utilizados 15 equinos adultos alocados em três grupos, com cinco animais em cada: grupo controle (GC) com utilização de soro fisiológico; grupo com deposição de FCM; e grupo com deposição de ADSC. Foi realizado o mesmo procedimento cirúrgico em todos os grupos, utilizando-se os dois nervos de cada animal (direito e esquerdo), e estabelecido dois momentos de biopsia: na 13ª semana com biopsia no membro direiro (GC1, FCM1 e ADSC1) e na 26ª semana com biopsia no membro esquerdo (GC2, FCM2 e ADSC2). A FCM e a ADSC foram obtidas respectivamente, da medula óssea e tecido adiposo de cada aninal, ambos utilizados como implantes autólogos. Após 13 e 26 semanas, realizaram-se as biopsias de todos os grupos e imediatamente, faziam-se imprints dos fragmentos para visualização do marcador fluorescente nanocristal. Os fragmentos foram fixados em formol tamponado a 10%, para análise histológica, com as coloraçãoes de HE, luxol fast blue, tricrômio de Masson e imuno-histoquímica com os anticorpos neurofilamento (NF), S-100, FGF-2 e GDNF. Microscopicamente avaliou-se a presença de proliferação axonal, tecido conjuntivo, infiltrado inflamatório, células de Schwann, fatores de crescimento neurais, bainha de mielina, e degeneração walleriana. Para a análise histológica estabeleceram-se escores qualitativos e para a imuno-histoquímica realizou-se análise quantitativa com o programa Image J. Foi observada diminuição da degeneração walleriana, melhor reorganização fascicular, aumento do colágeno novo, início de formação de bainha de mielina, além de maior marcação do anticorpo NF nos grupos experimentais em relação ao GC. O grupo ADSC apresentou melhores resultados em relação aos demais, enfatizando a eficiência das ADSCs em relação à FCM e ao GC para a regeneração nervosa periférica. Porém, há a necessidade de um tempo maior para ocorrer completa regeneração do tecido nervoso periférico após lesão.

BACKGROUND: The course of disease for patients with idiopathic pulmonary fibrosis (IPF) is highly heterogeneous. Prognostic models rely on demographic and clinical characteristics and are not reproducible. Integrating data from genomic analyses may identify novel prognostic models and provide mechanistic insights into IPF. METHODS: Total RNA of peripheral blood mononuclear cells was subjected to microarray profiling in a training (45 IPF individuals) and two independent validation cohorts (21 IPF/10 controls, and 75 IPF individuals, respectively). To identify a gene set predictive of IPF prognosis, we incorporated genomic, clinical, and outcome data from the training cohort. Predictor genes were selected if all the following criteria were met: 1) Present in a gene co-expression module from Weighted Gene Co-expression Network Analysis (WGCNA) that correlated with pulmonary function (p < 0.05); 2) Differentially expressed between observed "good" vs. "poor" prognosis with fold change (FC) >1.5 and false discovery rate (FDR) < 2 %; and 3) Predictive of mortality (p < 0.05) in univariate Cox regression analysis. "Survival risk group prediction" was adopted to construct a functional genomic model that used the IPF prognostic predictor gene set to derive a prognostic index (PI) for each patient into either high or low risk for survival outcomes. Prediction accuracy was assessed with a repeated 10-fold cross-validation algorithm and independently assessed in two validation cohorts through multivariate Cox regression survival analysis. RESULTS: A set of 118 IPF prognostic predictor genes was used to derive the functional genomic model and PI. In the training cohort, high-risk IPF patients predicted by PI had significantly shorter survival compared to those labeled as low-risk patients (log rank p < 0.001). The prediction accuracy was further validated in two independent cohorts (log rank p < 0.001 and 0.002). Functional pathway analysis revealed that the canonical pathways enriched with the IPF prognostic predictor gene set were involved in T-cell biology, including iCOS, T-cell receptor, and CD28 signaling. CONCLUSIONS: Using supervised and unsupervised analyses, we identified a set of IPF prognostic predictor genes and derived a functional genomic model that predicted high and low-risk IPF patients with high accuracy. This genomic model may complement current prognostic tools to deliver more personalized care for IPF patients.

As doenças cardiovasculares continuam sendo a primeira causa de morte no Brasil (32%) representando a terceira maior causa de internação hospitalar. Apesar dos avanços terapêuticos das últimas décadas, estudos epidemiológicos consideram o infarto agudo do miocárdio (AMI) uma das maiores causas de morbidade e mortalidade, sendo a maioria, ligados a realização de terapias não adequadas, dos quais 50% das mortes ocorrem nas primeiras 2 horas do ocorrido e 14% morrem antes de receber atendimento médico. O objetivo deste estudo foi de avaliar a técnica de infusão transpericárdica de células mononucleares de medula óssea (CMMO) em suínos. Três suínos fêmeas, pesando 25Kg foram induzidas ao AMI, com auxilio de cateter balão colocado no 1° ramo diagonal da artéria coronária interventricular por 45 minutos, seguido por infusão de 1x108 CMMO marcadas com Hoechst® pela via transpericárdica. O grupo controle foi composto por 3 animais, os quais receberam infusão de 1x108 CMMO marcadas com Hoechst® através da mesma técnica. Os resultados revelaram distribuição homogênea das CMMO no miocárdio, concentrando-se especialmente na área infartada, enquanto que o grupo controle apresentou distribuição homogênea ao longo do miocárdio. Nós concluímos que a técnica transpericárdica é viável para infusão de CMMO em processos de isquemia cardíaca. / Cardiovascular illnesses continue to be the first cause of death in Brazil (32%), representing the third major reason of hospital internment. Although the therapeutics advances in the last decades, epidemiologic studies considered the acute myocardium infarct (AMI) to be one of the most causes of morbidity and mortality (30%), most of, related to the institution of non-adequate therapy, thereby 50% of deaths in the early two hours of the event and 14% dying before any medical assistance. Currently therapies include stent angioplasty, Thrombolytic medication and aortic-coronary venous grafting; while in experimental area the cellular therapy has been largely investigated being the cells infusion technique investigation the most enthusiastic issue. This study aimed to evaluate the transepicardic infusion technique of bone marrow mononuclear cells (BMMC) in swine. Three female swine, averaging 25 kg, were induced to AMI, with the aid of a balloon catheter displaced on the first interventricular diagonal branch of the coronary artery for 45 minutes, following to the infusion of 1x108 BMMC stained with Hoescht® by the transepicardic technique. Sham operation was carried out in three animals (Control group), which received infusion of 1x108 BMMC stained with Hoescht® by the same technique. The results revealed an inhomogeneous distribution of the BMMC in the myocardium, being more concentrated in the infarcted area, while the control group presented a homogeneous distribution along the myocardium. We concluded the transpericardic technique would be acceptable to the infusion of BMMC in cardiac ischemic processes.