Avaliação da resposta inflamatória local após aplicação de células tronco mesenquimais alogênicas em tendão flexor digital superficial de equinos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Application of the general linear model to assess the effect of missing data on bone marrow mononuclear cell therapy with infusion timing and follow-up MRI

With most clinical trials, missing data presents a statistical problem in evaluating a treatment's efficacy. There are many methods commonly used to assess missing data; however, these methods leave room for bias to enter the study. This thesis was a secondary analysis on data taken from TIME, a phase 2 randomized clinical trial conducted to evaluate the safety and effect of the administration timing of bone marrow mononuclear cells (BMMNC) for subjects with acute myocardial infarction (AMI).^ We evaluated the effect of missing data by comparing the variance inflation factor (VIF) of the effect of therapy between all subjects and only subjects with complete data. Through the general linear model, an unbiased solution was made for the VIF of the treatment's efficacy using the weighted least squares method to incorporate missing data. Two groups were identified from the TIME data: 1) all subjects and 2) subjects with complete data (baseline and follow-up measurements). After the general solution was found for the VIF, it was migrated Excel 2010 to evaluate data from TIME. The resulting numerical value from the two groups was compared to assess the effect of missing data.^ The VIF values from the TIME study were considerably less in the group with missing data. By design, we varied the correlation factor in order to evaluate the VIFs of both groups. As the correlation factor increased, the VIF values increased at a faster rate in the group with only complete data. Furthermore, while varying the correlation factor, the number of subjects with missing data was also varied to see how missing data affects the VIF. When subjects with only baseline data was increased, we saw a significant rate increase in VIF values in the group with only complete data while the group with missing data saw a steady and consistent increase in the VIF. The same was seen when we varied the group with follow-up only data. This essentially showed that the VIFs steadily increased when missing data is not ignored. When missing data is ignored as with our comparison group, the VIF values sharply increase as correlation increases.^

CD14+ blood monocytes can differentiate into functionally mature CD83+ dendritic cells.

Dendritic cells are potent antigen-presenting cells that initiate primary immune responses. Although dendritic cells derive from bone marrow stem cells, the intermediate stages in their development remain unknown. In this study, plastic-adherent blood monocytes (CD14+, CD1a-) cultured for 7 days with granulocyte-monocyte colony-stimulating factor, interleukin 4, and tumor necrosis factor alpha were shown to differentiate into CD1a+ CD83+ dendritic cells. These cells displayed all phenotypic and morphologic characteristics of mature dendritic cells and were the most potent stimulatory cells in allogeneic mixed leukocyte reactions. The identification of specific culture conditions that generate large numbers of dendritic cells from purified monocytes uncovers an important step in dendritic cell maturation that will allow the further characterization of their role in autoimmune diseases, graft rejection, and human immunodeficiency virus infection.

Combined radioimmunotherapy and chemotherapy of human colon carcinoma grafted in nude mice, advantages and limitations.

In order to determine if 5-fluorouracil (5FU) could potentiate the effect of radioimmunotherapy (RIT), nude mice bearing subcutaneous human colon carcinoma xenografts were treated by 1 or 2 intravenous injection(s) of subtherapeutic doses of 131I labeled F(ab')2 from anti-carcinoembryonic antigen monoclonal antibodies combined with 5 daily intraperitoneal injections of 5FU. Control mice received either 131I F(ab')2 alone, 5FU alone or no treatment. RIT alone induced significant tumor regression, while 5FU alone gave only minimal tumor growth inhibition. The combined treatment group also resulted in long-term tumor regression with tumors remaining significantly smaller than in the RIT alone group. There was however, no significant difference in tumor recurrence time between the groups treated with RIT alone or with RIT + 5FU. Myelotoxicity, the major side effect of RIT, detected by the decrease of peripheral white blood cells (WBC), was shown to be almost identical between the groups receiving only RIT or only 5FU. Surprisingly, there was no cumulative bone marrow toxicity in animals which received 5FU before RIT. Furthermore, in the latter group, the WBC levels after RIT were significantly higher than in the control group receiving only RIT. Taken together, the results demonstrate the higher therapeutic efficiency of RIT as compared to 5FU in this model. They do not show, however, that the combination of the two forms of treatment can induce longer tumor remission. Interestingly, the WBC results suggest that 5FU given before RIT can have a radioprotective effect on bone marrow, possibly by selecting radioresistant bone marrow stem cells.

Secondary prevention of type 1 diabetes mellitus: stopping immune destruction and promoting ß-cell regeneration

Type 1 diabetes mellitus results from a cell-mediated autoimmune attack against pancreatic ß-cells. Traditional treatments involve numerous daily insulin dosages/injections and rigorous glucose control. Many efforts toward the identification of ß-cell precursors have been made not only with the aim of understanding the physiology of islet regeneration, but also as an alternative way to produce ß-cells to be used in protocols of islet transplantation. In this review, we summarize the most recent studies related to precursor cells implicated in the regeneration process. These include embryonic stem cells, pancreas-derived multipotent precursors, pancreatic ductal cells, hematopoietic stem cells, mesenchymal stem cells, hepatic oval cells, and mature ß-cells. There is controversial evidence of the potential of these cell sources to regenerate ß-cell mass in diabetic patients. However, clinical trials using embryonic stem cells, umbilical cord blood or adult bone marrow stem cells are under way. The results of various immunosuppressive regimens aiming at blocking autoimmunity against pancreatic ß-cells and promoting ß-cell preservation are also analyzed. Most of these regimens provide transient and partial effect on insulin requirements, but new regimens are beginning to be tested. Our own clinical trial combines a high dose immunosuppression with mobilized peripheral blood hematopoietic stem cell transplantation in early-onset type 1 diabetes mellitus.

L’implication du Stromal Cell-derived Factor-1 alpha dans le remodelage cardiaque une semaine après un infarctus du myocarde

L’injection de cellules souches provenant de la moelle osseuse est reconnue pour améliorer la fonction ventriculaire ainsi que le remodelage cicatriciel après un infarctus du myocarde (IM). Le Stromal Cell-derived factor-1 alpha (SDF-1 alpha), une chimiokine induite par l’ischémie cardiaque, représente une grande importance en raison de son rôle dans le recrutement de cellules inflammatoires et de cellules souches de la moelle osseuse vers les sites endommagés. Quoique les recherches sur le rôle de la chimiokine SDF-1 alpha dans le remodelage ventriculaire se multiplient, son implication dans la phase aiguë du remodelage reste inexplorée. Le but de la présente étude est de déterminer l’effet du SDF-1 alpha sur la taille de la cicatrice, l’hypertrophie cardiaque ainsi que la fonction ventriculaire chez des rats et des souris une semaine après un IM. La stratégie utilisée implique l’administration de l’AMD3100 (1 mg/kg, 24 heures après l’IM, pendant 6 jours), l’antagoniste sélectif du récepteur du SDF-1 alpha, le CXCR4. Ce récepteur est couplé à une protéine G alpha i et induit la migration et la prolifération cellulaire. Chez les rats du groupe IM, l’expression de la chimiokine a été détectée surtout dans les cellules musculaires lisses et les cellules endothéliales des vaisseaux cicatriciels. Le profil d’expression de la chimiokine dans le cœur infarci indique un gradient de concentration vers la cicatrice. Une semaine après l’IM, le traitement avec l’AMD3100 a diminué la taille de la cicatrice, résultant en une amélioration de la fonction ventriculaire et une diminution de l’élévation de l’expression de l’ARNm de l’ANP dans le ventricule gauche non infarci (VGNI). Chez les souris, le traitement avec l’AMD3100 a engendré les mêmes effets, soit une diminution de la taille de la cicatrice ainsi qu’une amélioration de la fonction ventriculaire. La réduction de la taille de la région infarcie chez les souris traitées avec l’AMD3100 est associée avec une atténuation de l’infiltration des neutrophiles dans la région ischémique. Ces résultats suggèrent que le blocage pharmacologique de l’axe SDF-1 alpha/CXCR4 lors de la phase aiguë du remodelage ventriculaire après un IM diminue la taille de la cicatrice et améliore la fonction ventriculaire, en partie, par la diminution de la réaction inflammatoire.

Evaluation of oxytocin pharmacokinetic : pharmacodynamic profile and establishment of its cardiomyogenic potential in swine

La thérapie cellulaire est une avenue pleine de promesses pour la régénération myocardique, par le remplacement du tissu nécrosé, ou en prévenant l'apoptose du myocarde survivant, ou encore par l'amélioration de la néovascularisation. Les cellules souches de la moelle osseuse (CSMO) expriment des marqueurs cardiaques in vitro quand elles sont exposées à des inducteurs. Pour cette raison, elles ont été utilisées dans la thérapie cellulaire de l'infarctus au myocarde dans des études pre-cliniques et cliniques. Récemment, il a été soulevé de possibles effets bénéfiques de l'ocytocine (OT) lors d’infarctus. Ainsi, l’OT est un inducteur de différenciation cardiaque des cellules souches embryonnaires, et cette différenciation est véhiculée par la voie de signalisation du monoxyde d’azote (NO)-guanylyl cyclase soluble. Toutefois, des données pharmacocinétiques de l’OT lui attribue un profil non linéaire et celui-ci pourrait expliquer les effets pharmacodynamiques controversés, rapportés dans la lttérature. Les objectifs de ce programme doctoral étaient les suivants : 1) Caractériser le profil pharmacocinétique de différents schémas posologiques d'OT chez le porc, en développant une modélisation pharmacocinétique / pharmacodynamique plus adaptée à intégrer les effets biologiques (rénaux, cardiovasculaires) observés. 2) Isoler, différencier et trouver le temps optimal d’induction de la différenciation pour les CSMO porcines (CSMOp), sur la base de l'expression des facteurs de transcription et des protéines structurales cardiaques retrouvées aux différents passages. 3) Induire et quantifier la différenciation cardiaque par l’OT sur les CSMOp. 4) Vérifier le rôle du NO dans cette différenciation cardiaque sur les CSMOp. Nous avons constaté que le profil pharmacocinétique de l’OT est mieux expliqué par le modèle connu comme target-mediated drug disposition (TMDD), parce que la durée du séjour de l’OT dans l’organisme dépend de sa capacité de liaison à son récepteur, ainsi que de son élimination (métabolisme). D'ailleurs, nous avons constaté que la différenciation cardiomyogénique des CSMOp médiée par l’OT devrait être induite pendant les premiers passages, parce que le nombre de passages modifie le profile phénotypique des CSMOp, ainsi que leur potentiel de différenciation. Nous avons observé que l’OT est un inducteur de la différenciation cardiomyogénique des CSMOp, parce que les cellules induites par l’OT expriment des marqueurs cardiaques, et l'expression de protéines cardiaques spécifiques a été plus abondante dans les cellules traitées à l’OT en comparaison aux cellules traitées avec la 5-azacytidine, qui a été largement utilisée comme inducteur de différenciation cardiaque des cellules souches adultes. Aussi, l’OT a causé la prolifération des CMSOp. Finalement, nous avons observé que l'inhibition de la voie de signalisation du NO affecte de manière significative l'expression des protéines cardiaques spécifiques. En conclusion, ces études précisent un potentiel certain de l’OT dans le cadre de la thérapie cellulaire cardiomyogénique à base de cellules souches adultes, mais soulignent que son utilisation requerra de la prudence et un approfondissement des connaissances.

L'impact des cellules souches issues de la moelle sur la néovascularisation dans un modèle de souris de rétinopathie induite par l'oxygène

La rétinopathie induite par l’oxygène (RIO) est un modèle animal semblable aux rétinopathies vue chez l’homme. Dans ce modèle, une destruction des microvaisseaux rétiniens est suivie d’une néovascularisation pathologique qui chez l’homme peut mener à un détachement de la rétine et subséquemment une perte de vision. Afin de remédier à cette revascularisation anarchique, un traitement de cellules souches (hématopoïétiques et mésenchymateuses) a été effectué chez des souris soumises à ce modèle. Les cellules injectées ont pu migrer à la rétine et induire une revascularisation saine (surtout les cellules souches mésenchymateuses). L’injection du milieu de culture de ces cellules induit aussi une revascularisation semblable à celle vue chez les souris traitées avec les cellules indiquant que l’effet thérapeutique des cellules semble être accompli par l’entremise de facteurs paracrines. Ces résultats suggèrent que ces cellules peuvent jouer un rôle au niveau de l’angiogénèse et indiquent un potentiel thérapeutique pour les rétinopathies.

Influência do tipo de parto na hematologia do cordão umbilical de cães

O sangue do cordão umbilical humano tem sido crescentemente utilizado como fonte de células-tronco. Os modelos experimentais de células-tronco da medula óssea, em cães, têm propiciado informações importantes para transplantes medulares em humanos. Vários trabalhos citam a influência do tipo de parto nas características do sangue do cordão umbilical (SCU) humano. Entretanto, não existem relatos sobre a colheita do sangue do cordão umbilical de cães. O objetivo deste ensaio é avaliar a influência do tipo de parto na hematologia do cordão umbilical de cães. No presente protocolo experimental, foram estudados 54 fetos de cães, ao final da vida intra-uterina, provenientes de parto normal (n=24) e cesariana (n=30). A colheita de sangue do cordão umbilical foi realizada com seringa de cinco mL contendo solução anticoagulante EDTA (1mg/1mL sangue). em seguida, a contagem global de hemácias, leucócitos, plaquetas, a determinação da concentração de hemoglobina, taxa de hematócrito, os índices eritrocitários foram realizados no contador automático de células. A contagem diferencial de leucócitos foi determinada em esfregaços de SCU corados com May-Grunwald-Giemsa (MGG). Com relação ao eritrograma e ao plaquetograma, não houve diferença significativa entre as amostras obtidas em cesarianas e partos normais. Os valores do leucograma do SCU colhido em partos normais foram superiores àqueles obtidos em cesarianas (P<0,05). Portanto, o tipo de parto influencia os valores hematológicos do cordão umbilical de cães.

Implante autólogo de células-tronco mesenquimais do tecido adiposo no tratamento de tendinites experimentais em equinos: avaliação clínica, ultrassonográfica, histopatológia e imunoistoquímica

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Implante de células-tronco mesenquimais autólogas, associadas ao plasma rico em plaquetas em tendinites experimentais de equinos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Co-cultivo de embriões bovinos com células-tronco adultas derivadas de tecido adiposo

A produção in vitro de embriões (PIV) é uma biotecnologia utilizada para aumentar o potencial reprodutivo de animais geneticamente superiores, os embriões produzidos in vitro são de qualidade inferior aos produzidos in vivo, por isso técnicas tentam melhorar os índices de embriões produzidos in vitro. Uma técnica é o sistema de co-cultivo com células somáticas que removem metabólitos tóxicos e protegem contra o stress oxidativo. As células-tronco mesenquimais derivadas de tecido adiposo (CTA) são células multipotentes que segregam fatores de crescimento e citocinas. As células-tronco foram utilizadas em co-cultivo in vitro de embriões bovinos em diferentes concentrações com o objetivo de melhorar o protocolo de PIVE. CTAs foram submetidas à diferenciação em três linhagens mesenquimais, e foi realizada a imunofenotipagem de marcadores específicos de membrana das CTMs. A taxa de clivagem foi avaliada no segundo dia após a fertilização e taxa de blastocistos no sétimo dia, quando foram armazenados para contagem do número total de células e expressão gênica. Os resultados foram analisados por ANOVA, Teste-t e pós-teste de Fisher, adotando um nível de significância de 5%. O tratamento do co-cultivo com CTAs influenciou significativamente a formação de blastocisto, o número total de células de embriões e a expressão gênica correlacionada a pluripotência e metabolismo de carboidratos. Estes resultados mostraram aumento da taxa de produção e qualidade dos embriões produzidos in vitro em co-cultivo com CTAs em relação ao co-cultivo com células da granulosa. Os resultados deste trabalho indicam também que a presença constante de CTAs em co-cultivo é superior ao condicionamento com CTAs. Os efeitos verificados das CTAs podem ocorrer através de fatores solúveis ou via exossomos secretados pelas CTAs. Estudos futuros são necessários para esclarecer a possível via causadora dos efeitos positivos verificados neste trabalho pelas CTAs em co-cultivo.

Análise da integridade genômica por meio do ensaio cometa e teste do micronúcleo em células-tronco mesenquimais derivadas do tecido adiposo

Pós-graduação em Biociências - FCLAS

Análise da integridade genômica por meio do ensaio cometa e teste do micronúcleo em células-tronco mesenquimais derivadas do tecido adiposo

Pós-graduação em Biociências - FCLAS

Benzene induces gene-duplicating but not gene-inactivating mutations at the glycophorin A locus in exposed humans.

Occupational exposure to benzene is known to cause leukemia, but the mechanism remains unclear. Unlike most other carcinogens, benzene and its metabolites are weakly or nonmutagenic in most simple gene mutation assays. Benzene and its metabolites do, however, produce chromosomal damage in a variety of systems. Here, we have used the glycophorin A (GPA) gene loss mutation assay to evaluate the nature of DNA damage produced by benzene in 24 workers heavily exposed to benzene and 23 matched control individuals in Shanghai, China. The GPA assay identifies stem cell or precursor erythroid cell mutations expressed in peripheral erythrocytes of MN-heterozygous subjects, distinguishing the NN and N phi mutant variants. A significant increase in the NN GPA variant cell frequency (Vf) was found in benzene-exposed workers as compared with unexposed control individuals (mean +/- SEM, 13.9 +/- 1.7 per million cells vs. 7.4 +/- 1.1 per million cells in control individuals; P = 0.0002). In contrast, no significant difference existed between the two groups for the N phi Vf (9.1 +/- 0.9 vs. 8.8 +/- 1.8 per million cells; P = 0.21). Further, lifetime cumulative occupational exposure to benzene was associated with the NN Vf (P = 0.005) but not with the N phi Vf (P = 0.31), suggesting that NN mutations occur in longer-lived bone marrow stem cells. NN variants result from loss of the GPA M allele and duplication of the N allele, presumably through recombination mechanisms, whereas NO variants arise from gene inactivation, presumably due to point mutations and deletions. Thus, these results suggest that benzene produces gene-duplicating mutations but does not produce gene-inactivating mutations at the GPA locus in bone marrow cells of humans exposed to high benzene levels. This finding is consistent with data on the genetic toxicology of benzene and its metabolites and adds further weight to the hypothesis that chromosome damage and mitotic recombination are important in benzene-induced leukemia.