Células-tronco mesenquimais aplicadas nas fases inflamatória e proliferativa da cicatrização de feridas cutâneas / Mesenchymal stem cells applied to the inflammatory and proliferative phases of wound healing

A cicatrização de feridas é um processo que requer a interação de várias células da derme e epiderme. O objetivo deste trabalho foi avaliar qual o momento da aplicação das células das ADSCs em feridas cutâneas agudas que faria diferença na cicatrização nos primeiros sete dias da lesão. As células-tronco foram isoladas do tecido adiposo de camundongos C57Bl/6 GFP+. Para tanto, foram utilizados 49 camundongos C57Bl/6, divididos em quatro grupos: grupo I (GI/controle; n=14); grupo II (GII; n=14): ADSCs injetadas no d0; grupo III (GIII; n=14): ADSCs injetadas no terceiro dia; e Grupo IV (GIV; n=7): ADSCs injetadas no quinto dia. As avaliações clínicas ocorreram nos dias zero, três, cinco e sete, e as histopatológicas nos dias cinco e sete. Na metodologia proposta, foi observado que o uso de ADSCs aumenta a vascularização, a formação de tecido de granulação, a colagenização e incrementa o número de folículos pilosos em apenas sete dias de avaliação. Além disso, o momento da aplicação das células não repercutiu diferenças significativas nas fases inflamatória e proliferativa do processo de cicatrização das feridas cutâneas.(AU)

Wound healing is a process that requires the interaction of various cells in the dermis and epidermis. The aim of this study was to evaluate the action of ADSCs in the treatment of acute wounds in order to understand if application time of the cells results in a difference in healing the first seven days of injury. The stem cells were isolated from adipose tissue of C57BL / 6 mice GFP +. Thus, we used 49 mice C57BL / 6 divided into four groups: Group I (GI / control, n=14); Group II (GII; n=14): ADSCs injected to the d0; Group III (GIII; n=14): ADSCs injected on the 3rd day, and Group IV (GIV; n=7): ADSCs injected day 5(d5). Clinical evaluations were performed on days 0, 3, 5 and 7 and the histopathology on days 5 and 7. In the proposed methodology, the use of ADSCs increased vascularization, formation of granulation tissue, collagen deposition and increases the number of hair follicles in just seven days of evaluation. In addition, the time of application of the cells did not affect significant differences in the inflammatory and the proliferative phase of wound healing skin.(AU)

Transrectal endoscopy with three different techniques of rectal suture associate: NOTES survival study with liver biopsy in a swine model / Endoscopia transretal para biópsia hepática associada com três diferentes técnicas de sutura retal: estudo da NOTES em modelo experimental suíno

A cirurgia endoscópica por orifícios naturais (NOTES) representa um novo conceito de cirurgia, caracterizada por ausência de incisões abdominais. Os acessos mais comumente usados são o transvaginal e o transgástrico. Entretanto, as rotas transcolônica e transretal representam alternativas promissoras. O presente estudo objetiva avaliar três diferentes técnicas de sutura retal em três suínos submetidos a NOTES transretal para biópsia hepática, avaliando-se concomitantemente as repercussões clínicas e hematológicas. Sob anestesia geral, foi realizada uma incisão transversal no reto para a passagem do endoscópio até a cavidade abdominal em todos os animais para a realização da biópsia hepática. Cada animal recebeu um tipo de sutura retal: sutura em dois planos; reforço com tela de polipropileno ou reforço com membrana de pericárdio bovino. A NOTES transretal em modelo experimental suíno não apresentou implicações clínicas e hematológicas importantes, o que demonstra um acesso alternativo para biópsia hepática. Nenhum animal apresentou sinais de peritonite, aderências ou deiscência de pontos. O uso de reforço com pericárdio bovino para a sutura retal apresenta um atraso na cicatrização quando comparado com a sutura convencional ou com o uso de tela de polipropileno.(AU)

Recombinant expression and purification of the bovine acidic Seminal Fluid Protein

The acidic Seminal Fluid Protein (aSFP), a 12.9 kDa protein is a maker for bovine semen freezability possibly due to its antioxidant activity and effect on sperm mitochondrial function. However, its precise function on sperm preservation during freezing thaw is poorly understood. The use of recombinant DNA technology allows new approaches on the study of function and structure of proteins, and its production in procaryote systems offers several advantages. The present work describes the recombinant expression of the bovine aSFP and its binding properties. A cDNA library from the bovine seminal vesicle was used as template for amplification of the aSFP coding region. The amplicon was cloned into a pET23a (+) vector and transformed into E.coli BL21 pLysS strain. The recombinant expression was obtained in E coli. One step ion immobilized affinity chromatography was performed, resulting in high yield of purified protein. To determine the bioactivity of the r aSFP, the protein was incubated in different concentrations with 10 7 spermtozoa at 37°C for 5 h. Western blotting and fluorescence microscopy analyses showed the ability of the recombinant aSFP to attach to the spermatozoa. Based on our results, the described method can be used to obtain mg levels of recombinant aSFP.

Bone regeneration in mandible defect with autograft bone and cell suspension from bone marrow in rabbits / Regeneração óssea em defeitos mandibulares com auto-enxerto ósseo e suspensão celular da medula óssea em coelhos

The objective of this study was to investigate the bone regeneration of a "gold standard" (autograft) from iliac crest associated with cellular therapy in rabbits. A bone defect was created with 10x5x5mm in 28 rabbit mandibles. The control group animals (n=14) were repaired with autograft of iliac crest and the experimental group animals (n=14) received iliac crest autograft in association with mononuclear cells from the bone marrow of the femur. Weekly radiographs were taken of the surgery region and histological analyses was performed in seven animals in each group at 15 days and in seven animals of each group at 30 days after the surgery. A gradual increase of bone density was observed and the experimental animals presented the bone bridge in 85.7 percent (6/7) of the cases, while only 42.8 percent (3/7) of the animals in the control group presented this structure 28 days after the surgery. The histopathological parameters analyzed did not show any statistical difference between the control and experimental group in 15 and 30 days of analysis. The results suggest that the mononuclear cells from the marrow bone can better support the autograft regeneration in mandible defects in rabbits.

Avaliou-se a regeneração óssea de auto-enxerto, considerado "padrão ouro" da crista ilíaca associado à terapia celular da medula óssea em coelhos. Foi criado um defeito ósseo de 10x5x5mm na mandíbula de 28 coelhos, distribuídos em grupo-controle com, 14 animais, reparados com auto-enxerto de crista ilíaca, e grupo experimental com, 14 animais, em que o auto-enxerto foi associado a células mononucleares da medula óssea autógena do fêmur. Foram realizadas radiografias semanais da região operada e análise histológica em sete animais de cada grupo aos 15 e em sete de cada grupo aos 30 dias do pós-operatório. Houve aumento gradativo da densidade óssea, e 85,7 por cento (6/7) dos animais do grupo experimental e 42,8 por cento (3/7) do grupo-controle apresentaram formação de ponte óssea 28 dias após a cirurgia. Na análise histopatológica aos 15 dias, os enxertos foram facilmente visualizados e a atividade das células fagocitárias foi intensa. Já aos 30 dias, a visualização foi mais difícil e, quando possível, apenas um resquício foi visualizado. Os resultados sugerem que a adição de células mononucleares da medula óssea favorece a regeneração do auto-enxerto em defeitos mandibulares de coelhos.

Repeated in vivo hydrocortisone treatment promotes a dual modulation of cytokeratin expression by mouse thymic epithelial cells.

Previous studies showed that a single dose of hydrocortisone in mice was able to transiently upregulate the expression of cytokeratins (CKs) 3 and 10 by medullary epithelial cells of the mouse thymus. Herein we studied these cells (specifically recognized by immunocytochemistry with the anti-CK monoclonal antibody KL1) following a series of repeated injections of the glucocorticoid hormone. A progressive dual (up and down) modulation of KL1+ medullary epithelial cells was observed with a late appearance of KL1 immunoreactivity in the thymic cortex. The data indicate that a single versus repeated exposure to high doses of glucocorticoid hormone may trigger different circuits regulating intrathymic CK expression. Lastly, the model described herein may be regarded as promising in studies concerning the effect of repeated stress conditions upon the thymus.

Automated technique to evaluate thymocyte adhesion to thymic epithelial cells.

Thymocyte adhesion to thymic epithelial cells is a relevant issue during intrathymic T-cell differentiation, and directly intervenes in the generation and expansion of the T-cell repertoire. In view of these data, it was apparent the usefulness of an automated strategy to evaluate the degree of thymic epithelial cell-thymocyte adhesion. This prompted us to develop an ELISA procedure (using an anti-Thy1 reagent) to determine the degree of thymocyte adhesion onto cultured thymic epithelial cells. The procedure described herein is simple, non-radioactive and reproducible. Additionally, it can potentially be applied to quantitate the degree of thymocyte adhesion to any cellular or non-cellular substrate (for example, extracellular matrix). Moreover, it detected fluctuations of thymocyte adhesion secondary to glucocorticoid treatment of epithelial cells. Thus, it can be regarded as a further tool to analyze intrathymic interactions.

Functional gap junctions in thymic epithelial cells are formed by connexin 43.

A multiparametric study was carried out to investigate the presence and possible role of communicating junctions in the thymus, particularly in the thymic epithelium, the major component of the thymic microenvironment. The presence of direct cell-cell communication mediated by gap junctions was demonstrated in human and murine thymic epithelial cells (TEC) by means of in situ and in vitro immunohistochemical labeling as well as in vitro fluorochrome injection and double whole-cell patch clamp experiments. Moreover, both immuno- and Northern blot studies revealed that the gap junction protein connexin 43 and its mRNA were present in TEC. Importantly, we showed that thymic endocrine activity, as ascertained by thymulin production, could be specifically down-modulated in vitro by a gap junction inhibitor, octanol. We also investigated the existence of gap junctions between TEC and thymocytes. In thymic nurse cells we were able to detect cell-cell communication, although only a minor percentage of epithelial/thymocyte pairs were coupled in a given moment. In contrast, intercellular communication was not detected between cultured phagocytic cells of the thymic reticulum and the respective rosetting thymocytes. We suggest that gap junctions formed by connexin 43 may represent a novel (and rather cell type-specific) pathway for intrathymic cellular communication, including TEC/TEC as well as possible TEC/thymocyte interactions.

Hydrocortisone increases the numbers of KL1+ cells, a discrete thymic epithelial cell subset characterized by high molecular weight cytokeratin expression.

The thymic epithelium, a major component of the thymic microenvironment, is a heterogeneous tissue bearing distinct monoclonal antibody-defined subsets. Among these, KL1+ cells represent a mouse medullary subpopulation characterized by high mol wt cytokeratin expression. Given the fact that thymic epithelial cells (TEC) express glucocorticoid receptors and that glucocorticoid hormones are known to modulate the expression of keratins, we decided to study the in vivo effects of hydrocortisone on KL1+ cells in normal and autoimmune mice. Within 24 h after a single injection of this steroid we observed a significant increase in the number of KL1+ cells. Interestingly, this effect was reversible and was no longer detected 7 days after treatment. Parallel studies analyzing the effects of hydrocortisone on the secretion of thymulin, a chemically defined thymic hormone revealed a transient decrease in serum levels of this hormone, but with different kinetics than the effects on KL1+ cells. Ontogenetic studies showed that the responsiveness of TEC to hydrocortisone, in terms of high mol wt cytokeratin expression, appeared late in fetal life and disappeared in aging animals. Importantly, aging, but also young adult, autoimmune mice were not responsive. In vitro experiments using a mouse TEC line confirmed the data observed in vivo demonstrating that the increase in KL1+ cells is a direct effect of hydrocortisone on TEC. The bulk of the data presently reported demonstrates that glucocorticoid hormone can act on TEC modulating the expression of both secretory and cytoskeletal protein families.