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ABSTRACT: Epidemiological studies report opposing influences of infection on childhood B-cell acute lymphoblastic leukemia (B-ALL). Although infections in the first year of life appear to exert the largest impact on leukemia risk, the effect of early pathogen exposure on the fetal preleukemia cells (PLC) that lead to B-ALL has yet to be reported. Using cytomegalovirus (CMV) infection as a model early-life infection, we show that virus exposure within 1 week of birth induces profound depletion of transplanted E2A-PBX1 and hyperdiploid B-ALL cells in wild-type recipients and in situ-generated PLC in Eµ-ret mice. The age-dependent depletion of PLC results from an elevated STAT4-mediated cytokine response in neonates, with high levels of interleukin (IL)-12p40-driven interferon (IFN)-γ production inducing PLC death. Similar PLC depletion can be achieved in adult mice by impairing viral clearance. These findings provide mechanistic support for potential inhibitory effects of early-life infection on B-ALL progression and could inform novel therapeutic or preventive strategies.
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Modelos Animais de Doenças , Leucemia-Linfoma Linfoblástico de Células Precursoras B , Animais , Camundongos , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Infecções por Citomegalovirus , Pré-Leucemia/genética , Pré-Leucemia/patologia , Camundongos Endogâmicos C57BL , Animais Recém-Nascidos , DiploideRESUMO
High-risk neuroblastoma remains a profound clinical challenge that requires eradication of neuroblastoma cells from a variety of organ sites, including bone marrow, liver, and CNS, to achieve a cure. While preclinical modeling is a powerful tool for the development of novel cancer therapies, the lack of widely available models of metastatic neuroblastoma represents a significant barrier to the development of effective treatment strategies. To address this need, we report a novel luciferase-expressing derivative of the widely used Th-MYCN mouse. While our model recapitulates the non-metastatic neuroblastoma development seen in the parental transgenic strain, transplantation of primary tumor cells from disease-bearing mice enables longitudinal monitoring of neuroblastoma growth at distinct sites in immune-deficient or immune-competent recipients. The transplanted tumors retain GD2 expression through many rounds of serial transplantation and are sensitive to GD2-targeted immune therapy. With more diverse tissue localization than is seen with human cell line-derived xenografts, this novel model for high-risk neuroblastoma could contribute to the optimization of immune-based treatments for this deadly disease.
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Neuroblastoma , Camundongos , Humanos , Animais , Proteína Proto-Oncogênica N-Myc , Camundongos Transgênicos , Neuroblastoma/terapia , Neuroblastoma/tratamento farmacológico , Adaptação Fisiológica , AclimataçãoRESUMO
Chromosome dimers occur in bacterial cells as a result of the recombinational repair of DNA. In most bacteria, chromosome dimers are resolved by XerCD site-specific recombination at the dif (deletion-induced filamentation) site located in the terminus region of the chromosome. Caulobacter crescentus, a Gram-negative oligotrophic bacterium, also possesses Xer recombinases, called CcXerC and CcXerD, which have been shown to interact with the Escherichia colidif site in vitro Previous studies on Caulobacter have suggested the presence of a dif site (referred to in this paper as dif1CC ), but no in vitro data have shown any association with this site and the CcXer proteins. Using recursive hidden Markov modeling, another group has proposed a second dif site, which we call dif2CC , which shows more similarity to the dif consensus sequence. Here, by using a combination of in vitro experiments, we compare the affinities and the cleavage abilities of CcXerCD recombinases for both dif sites. Our results show that dif2CC displays a higher affinity for CcXerC and CcXerD and is bound cooperatively by these proteins, which is not the case for the original dif1CC site. Furthermore, dif2CC nicked substrates are more efficiently cleaved by CcXerCD, and deletion of the site results in about 5 to 10% of cells showing an altered cellular morphology.IMPORTANCE Bacteria utilize site-specific recombination for a variety of purposes, including the control of gene expression, acquisition of genetic elements, and the resolution of dimeric chromosomes. Failure to resolve dimeric chromosomes can lead to cell division defects in a percentage of the cell population. The work presented here shows the existence of a chromosomal resolution system in C. crescentus Defects in this resolution system result in the formation of chains of cells. Further understanding of how these cells remain linked together will help in the understanding of how chromosome segregation and cell division are linked in C. crescentus.
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Caulobacter crescentus/genética , Cromossomos Bacterianos/genética , Proteínas de Bactérias/fisiologia , Divisão Celular , Segregação de Cromossomos , Recombinação Genética , Resposta SOS em GenéticaRESUMO
Indoleamine 2,3-dioxygenase (IDO) induces immunological tolerance in physiological and pathological conditions. Therefore, we used dermal fibroblasts with stable IDO expression as a cell therapy to: (i) Investigate the factors determining the efficacy of this cell therapy for autoimmune diabetes in non-obese diabetic (NOD) mice; (ii) Scrutinize the potential immunological mechanisms. Newly diabetic NOD mice were randomly injected with either 10 × 10(6) (10M) or 15 × 10(6) (15M) IDO-expressing dermal fibroblasts. Blood glucose levels (BGLs), body weight, plasma kynurenine levels, insulitis severity, islet beta cell function, autoreactive CD8(+) T cells, Th17 cells and regulatory T cells (Tregs) were then investigated in these mice. IL-1ß and cleaved caspase-3 levels were assessed in islets co-cultured with IDO-expressing fibroblasts. BGLs in 83% mice treated with 15M IDO-expressing fibroblasts recovered to normal up to 120 days. However, only 17% mice treated with 10M IDO-expressing cells were reversed to normoglycemia. A 15M IDO-expressing fibroblasts significantly reduced infiltrated immune cells in islets and recovered the functionality of remaining islet beta cells in NOD mice. Additionally, they successfully inhibited autoreactive CD8(+) T cells and Th17 cells as well as increased Tregs in different organs of NOD mice. Islet beta cells co-cultured with IDO-expressing fibroblasts had reduced IL-1ß levels and cell apoptosis. Both cell number and IDO enzymatic activity contributes to the efficiency of IDO cell therapy. Optimized IDO-expressing fibroblasts successfully reverse the progression of diabetes in NOD mice through induction of Tregs as well as inhibition of beta cell specific autoreactive CD8(+) T cells and Th17 cells. J. Cell. Physiol. 231: 1964-1973, 2016. © 2016 Wiley Periodicals, Inc.
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Diabetes Mellitus Experimental/imunologia , Fibroblastos/enzimologia , Hiperglicemia/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Células Secretoras de Insulina/imunologia , Animais , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/metabolismo , Feminino , Hiperglicemia/imunologia , Células Secretoras de Insulina/enzimologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Linfócitos T Reguladores/imunologiaRESUMO
The presence of supernumerary chromosomes is the only abnormality shared by all patients diagnosed with high-hyperdiploid B cell acute lymphoblastic leukemia (HD-ALL). Despite being the most frequently diagnosed pediatric leukemia, the lack of clonal molecular lesions and complete absence of appropriate experimental models have impeded the elucidation of HD-ALL leukemogenesis. Here, we report that for 23 leukemia samples isolated from moribund Eµ-Ret mice, all were characterized by non-random chromosomal gains, involving combinations of trisomy 9, 12, 14, 15, and 17. With a median gain of three chromosomes, leukemia emerged after a prolonged latency from a preleukemic B cell precursor cell population displaying more diverse aneuploidy. Transition from preleukemia to overt disease in Eµ-Ret mice is associated with acquisition of heterogeneous genomic abnormalities affecting the expression of genes implicated in pediatric B-ALL. The development of abnormal centrosomes in parallel with aneuploidy renders both preleukemic and leukemic cells sensitive to inhibitors of centrosome clustering, enabling targeted in vivo depletion of leukemia-propagating cells. This study reveals the Eµ-Ret mouse to be a novel tool for investigating HD-ALL leukemogenesis, including supervision and selection of preleukemic aneuploid clones by the immune system and identification of vulnerabilities that could be targeted to prevent relapse.
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Modelos Animais de Doenças , Leucemia-Linfoma Linfoblástico de Células Precursoras B , Animais , Camundongos , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Aneuploidia , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Centrossomo/patologia , DiploideRESUMO
Understanding the extracellular matrix (ECM) effect on pancreatic ß cells is critical to optimise the derivation of functional ß cells for transplantation and understand mechanisms that control islet neogenesis and glucose homeostasis. We assessed the effect of natural ECMs [collagen I, collagen IV, laminin and fibronectin (FN)] on rat islets of Langerhans' morphology, adhesion, viability, functionality and islet specific genes expression after 7 days in vitro culture. However, we could not detect a significant difference on the other parameters in these ECMs and islets interaction. To examine islets interactions, we used a synthetic three dimensional surface composed of electrospun polyamide nanofibres. Laminin-coated nanofibrillar surfaces, but not laminin or nanosurface alone, induced comparable expression of the Ins1 and Ins2 genes in adult ß cells. Using a glucose challenge test, a marked response of insulin secretion by islets occurred that were cultured on laminin-coated nanofibrillar surfaces.We contend that the reestablishment of cellular interactions by the combination of nanomaterials and natural ECMs can be useful in maintaining in vitro islet functions.
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Ilhotas Pancreáticas/fisiologia , Laminina/química , Nanofibras/química , Animais , Adesão Celular , Sobrevivência Celular , Materiais Revestidos Biocompatíveis/química , Técnicas Eletroquímicas , Expressão Gênica , Ilhotas Pancreáticas/citologia , Lactonas , Masculino , Ratos Wistar , Técnicas de Cultura de TecidosRESUMO
Common infections have long been proposed to play a role in the development of pediatric B-cell acute lymphoblastic leukemia (B-ALL). However, epidemiologic studies report contradictory effects of infection exposure on subsequent B-ALL risk, and no specific pathogen has been definitively linked to the disease. A unifying mechanism to explain the divergent outcomes could inform disease prevention strategies. We previously reported that the pattern recognition receptor (PRR) ligand Poly(I:C) exerted effects on B-ALL cells that were distinct from those observed with other nucleic acid-based PRR ligands. Here, using multiple double-stranded RNA (dsRNA) moieties, we show that the overall outcome of exposure to Poly(I:C) reflects the balance of opposing responses induced by its ligation to endosomal and cytoplasmic receptors. This PRR response biology is shared between mouse and human B-ALL and can increase leukemia-initiating cell burden in vivo during the preleukemia phase of B-ALL, primarily through tumor necrosis factor α signaling. The age of the responding immune system further influences the impact of dsRNA exposure on B-ALL cells in both mouse and human settings. Overall, our study demonstrates that potentially proleukemic and antileukemic effects can each be generated by the stimulation of pathogen recognition pathways and indicates a mechanistic explanation for the contrasting epidemiologic associations reported for infection exposure and B-ALL.
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Leucemia-Linfoma Linfoblástico de Células Precursoras B , Transdução de Sinais , Camundongos , Humanos , Animais , Criança , Ligantes , RNA de Cadeia Dupla/farmacologia , Linfócitos BRESUMO
BACKGROUND: Several studies have demonstrated the derivation of multi- or pluripotent stem cells from testicular cells of both newborn and adult mice by a spontaneous conversion process, when these cells are cultured in vitro for an extended time. To obtain a better and robust derivation, we have attempted to identify small molecules (SMs) that induce reprogramming of testicular cells in culture into germline-derived pluripotent stem cells (gPSCs). METHODS: We tested several SMs based on previous reports that have shown enhancement of establishment of induced pluripotent stem cells or embryonic stem cells (ESCs) on mouse NMRI (outbred strain) and C57BL/6 (inbred strain) testicular cells. After appearance of ESC-like colonies at Day 6, they were passaged on mitotically arrested mouse embryonic fibroblasts in mouse ESC medium in the absence or presence of SMs up to Day 14. The generated cells were characterized using a variety of experimental approaches. RESULTS: The application of several SMs involved in pluripotent reprogramming led to the discovery that CHIR99021 (CHIR), a glycogen synthase kinase-3 (GSK-3) inhibitor, promotes efficient derivation of gPSCs from neonatal mouse NMRI and C57BL/6 testes. The pluripotency of the generated cell lines has been confirmed by in vitro spontaneous and direct differentiation toward cardiac and neural lineages, and formation of chimeras after injection of gPSCs into blastocysts. We have shown that the generated gPSCs could be maintained and expanded under chemically defined serum and feeder-free conditions by inhibition of both the extracellular signal-regulated kinases (Erk1/2) and GSK-3. CONCLUSIONS: To our knowledge, this is the first report of a simple and efficient protocol to reprogram gPSCs from testicular cells solely by inhibition of GSK-3 in two strains of mice with different genetic backgrounds. Additionally, this brings us closer to eliminating the need for genetic modification in pluripotent reprogramming. Future studies will determine whether the inhibition of GSK-3 could affect the generation of naïve gPSCs lines in other mammals.
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Regulação Enzimológica da Expressão Gênica , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Células-Tronco Pluripotentes/citologia , Testículo/metabolismo , Animais , Animais Recém-Nascidos , Técnicas de Cultura de Células , Células-Tronco Embrionárias/citologia , Inibidores Enzimáticos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Quinase 3 da Glicogênio Sintase/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Piridinas/farmacologia , Pirimidinas/farmacologia , Testículo/efeitos dos fármacos , Fatores de TempoRESUMO
Fibroblasts, or their homolog stromal cells, are present in most tissues and play an essential role in tissue homeostasis and regeneration. As a result, fibroblast-based strategies have been widely employed in tissue engineering. However, while considered to have immunosuppressive properties, the survival and functionality of allogeneic fibroblasts after transplantation remain controversial. Here, we evaluated innate and adaptive immune responses against allogeneic fibroblasts following intradermal injection into different immune-deficient mouse strains. While allogeneic fibroblasts were rejected 1 week after transplantation in immunocompetent mice, rejection did not occur in immunodeficient γ chain-deficient NOD-SCID (NSG) mice. T-cell- and B-cell-deficient RAG1 knockout mice showed greater loss of fibroblasts by day 5 after transplantation compared with NSG mice (P ≤ 0.05) but prolonged persistence compared with wild-type recipient (P ≤ 0.005). Loss of fibroblasts correlated with the expression of proinflammatory chemokine genes and infiltration of myeloid cells in the transplantation site. Depletion of macrophages and neutrophils delayed rejection, revealing the role of innate immune cells in an early elimination of fibroblasts that is followed by T-cell-mediated rejection in the second week. These findings indicate that the application of allogeneic fibroblasts in tissue engineering products requires further improvements to overcome cell rejection by innate and adaptive immune cells.
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Rejeição de Enxerto , Transplante de Células-Tronco Hematopoéticas , Animais , Fibroblastos , Imunidade , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante de Pele , Transplante HomólogoRESUMO
Introduction: Measurement of pancreatic beta cell mass in animal models is a common assay in diabetes researches. Novel whole-organ clearance methods in conjunction with transgenic mouse models hold tremendous promise to improve beta cell mass measurement methods. Here, we proposed a refined method to estimate the beta cell mass using a new transgenic Tg(Pdx1-GFP) mouse model and a recently developed free-of-acrylamide clearing tissue (FACT) protocol. Methods: First, we generated and evaluated a Tg(Pdx1-GFP) transgenic mouse model. Using the FACT protocol in our model, we could quantify the beta cell mass and alloxan-induced beta cell destruction in whole pancreas specimens. Results: Compiled fluorescent images of pancreas resulted in enhanced beta cell mass characterization in FACT-cleared sections (2928869±120215 AU) compared to No-FACT cleared sections (1292372±325632 AU). Additionally, the total number of detected islets with this method was significantly higher than the other clearance methods (155.7 and 109, respectively). Using this method, we showed green fluorescent protein (GFP) expression confined to beta cells in Tg(Pdx1-GFP) transgenic. This enhanced GFP expression enabled us to accurately measure beta cell loss in a beta cell destruction model. The results suggest that our proposed method can be used as a simple, and rapid assay for beta cell mass measurement in islet biology and diabetes studies. Conclusion: The Tg(Pdx1-GFP) transgenic mouse in conjunction with the FACT protocol can enhance large-scale screening studies in the field of diabetes.
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BACKGROUND AIMS: The aim was to investigate the therapeutic effect of granulocyte-colony-stimulating factor (G-CSF) administration following implantation of autologous bone marrow mononuclear cells (BM MNC) for patients with lower limb ischemia. METHODS: The design was a randomized controlled trial. Fifteen patients with severe chronic limb ischemia were treated with autologous BM MNC [without G-CSF (MNC-G-CSF) or combined with G-CSF administration for 5 days following transplantation (MNC+G-CSF)]. RESULTS: All clinical parameters, including ankle brachial index, visual analog scale and pain-free walking distance, showed a mean improvement from baseline, which was measured at 4 and 24 weeks after transplantation in both groups. However, in three (20%) patients, the clinical course did not improve and limb salvage was not achieved. No significant difference was observed among the patients treated in the MNC-G-CSF and MNC+G-CSF groups. No severe adverse reactions were reported during the study period. No relationship was observed between both the numbers of viable MNC or CD34+ cells and the clinical outcome. CONCLUSIONS: Autologous transplantation of BM MNC into ischemic lower limbs is safe, feasible and efficient for patients with severe peripheral artery disease. However, the administration of G-CSF following cell transplantation does not improve the effect of BM MNC implantation and therefore would not have any beneficial value in clinical applications of such cases.
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Transplante de Medula Óssea , Fator Estimulador de Colônias de Granulócitos/administração & dosagem , Isquemia/terapia , Leucócitos Mononucleares/metabolismo , Extremidade Inferior/cirurgia , Adulto , Idoso , Índice Tornozelo-Braço , Contagem de Células , Progressão da Doença , Estudos de Viabilidade , Feminino , Seguimentos , Fator Estimulador de Colônias de Granulócitos/efeitos adversos , Humanos , Injeções Intramusculares , Isquemia/patologia , Isquemia/fisiopatologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/patologia , Leucócitos Mononucleares/transplante , Extremidade Inferior/patologia , Extremidade Inferior/fisiopatologia , Masculino , Pessoa de Meia-Idade , Recuperação de Função Fisiológica , Transplante Autólogo , Resultado do TratamentoRESUMO
To determine the optimal vitrification conditions for sheep cumulus-oocyte complexes (COC), good-quality isolated COC were randomly divided into non-vitrified control, conventional straw, cryotop and solid-surface vitrification groups. In the conventional and cryotop methods, the vitrified COC were respectively loaded by conventional straw and cryotop, whereas in the solid-surface group, the vitrified COC were loaded by cryotop and then cooled before plunging in liquid nitrogen. The results indicated that the mean percentage of viability of vitrified-warmed COC was higher in both cryotop groups than that of the conventional group (83.84+/-2.85 and 78.56+/-1.72 versus 63.43+/-1.48%, P<0.05). In the cryotop group, although the mean percentage of oocyte maturation was similar to that in the control group (48.81+/-3.09 versus 51.94+/-3.01%), it was significantly higher than the other vitrification groups (48.81+/-3.09 versus 36.60+/-1.69 and 6.09+/-2.51%, respectively, P<0.05). However, the expression of maturation genes (GDF9, BMP15) was retarded after vitrification. Among the vitrification groups, the cryotop group had better expression. BMPRII was also expressed highly in the control, whereas ALK5 was similar in all groups. In conclusion, direct cryotop, when compared with other vitrification methods, seemed to be safe and could increase the viability, post-warming maturation and maturation-gene expression rates of sheep COC.
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Diferenciação Celular/fisiologia , Criopreservação/métodos , Células do Cúmulo/fisiologia , Expressão Gênica/fisiologia , Oócitos/fisiologia , Oogênese/fisiologia , Receptores de Ativinas/genética , Receptores de Ativinas/metabolismo , Animais , Proteína Morfogenética Óssea 15/genética , Proteína Morfogenética Óssea 15/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Comunicação Celular/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Crioprotetores/farmacologia , Células do Cúmulo/citologia , Células do Cúmulo/efeitos dos fármacos , Feminino , Fator 9 de Diferenciação de Crescimento/genética , Fator 9 de Diferenciação de Crescimento/metabolismo , Técnicas In Vitro , Nitrogênio/farmacologia , Oócitos/citologia , Oócitos/efeitos dos fármacos , OvinosRESUMO
The title compound, [Tl(2)(C(3)H(10)NO(9)P(3))](n), a Tl(I) organic-inorganic hybrid complex, was synthesized by the reaction of nitrilo-tris(methyl-enephospho-nic acid) with thallium(I) nitrate. There are two types of Tl(+) ions in the complex, with coordination numbers of eight and seven and with stereochemically active and inactive lone-pair electrons, respectively. In the crystal, the doubly deprotonated ligands form two-dimensional hydrogen-bonded layers through O-Hâ¯O hydrogen bonds. The NH group is involved in a trifurcated intra-molecular hydrogen bond. Coordination of the phospho-nate ligands to the Tl(+) ions creates a three-dimensional structure.
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OBJECTIVES: Fetal microchimerism is the persistence of allogeneic cell population that transfer from the fetus to the mother. The aim of this study was to evaluate the presence of fetal microchimerism in the pancreas of the mouse with acute pancreatitis (AP). MATERIALS AND METHODS: In this experimental study, female wild-type mice were mated with male EGFP+. AP model was obtained by injection of caerulein two days after delivery. Sixty mice were divided into 3 groups: the virgin pancreatitis-induced animals, pregnant pancreatitis-induced animals mated with transgenic EGFP mice, and pregnant sham animals. To prove pancreatitis induction, the blood amylase and lipase were assessed; and pancreas was removed from a subpopulation of each group for histopathological examinations after 6 hr. The remaining mice were kept for 3 weeks and histopathological exanimation, immunohistochemistry, and PCR were performed. RESULTS: EGFP+ cells were found in acini and around the blood vessels in the pancreas of pregnant pancreatitis-induced animals. They differentiated to acinar, adipocyte-like, and mesenchymal-like cells. PCR showed that 20% of the pregnant pancreatitis-induced animals were EGFP+. The histopathological study showed improvement in pancreatitis scores in the mice with history of pregnancy. CONCLUSION: It seems that pregnancy has a beneficial impact on caerulein-induced pancreatitis and improves the pancreatitis score in mouse.
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Burn injuries are one of the most common sources of trauma globally that comprise a significant drain on long-term personal and healthcare cost. Large surface area burn wounds are difficult to manage and may result in significant physiologic and psychologic sequelae. The goal of burn wound healing research is to fully repair and restore skin's original structure and functionality while minimizing problems such as hypertrophic scarring and contracture. One of the ways this can be achieved is through augmentation of the skin's natural healing process using the regenerative capability of stem cells. In this review, the authors highlight some recent developments in treatment of burn wounds employing stem cells. We compare and contrast the benefits and drawbacks to various sources of stem cells and techniques of delivery into damaged tissues that have been the focus of established and ongoing research, and avenues of exploration this burgeoning arena offers for the future.
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Queimaduras/terapia , Transplante de Células-Tronco , Células-Tronco/fisiologia , HumanosRESUMO
Acute and chronic wounds contribute to increased morbidity and mortality in affected people and impose significant financial burdens on healthcare systems. For these challenging wounds, acellular dermal matrices (ADMs) have been used as a biological wound coverage. Unlike engineered dermal matrices, ADMs are prepared through the removal of cells from skin, while preserving the extracellular matrix structure and function. In this study, our primary objective was to develop a detergent-free method for decellularization of the skin to mitigate chemical stress on matrix molecules. Then, we performed a set of in vitro and in vivo experiments to compare this method with nonionic and anionic detergent methods. All decellularization methods satisfactorily removed cells and supported fibroblast growth and migration in vitro. Sulfated glycosaminoglycan content was reduced significantly (p < 0.05) only in the ionic detergent treatment group. In contrast to the detergent-free method, all detergent-based methods significantly reduced scaffold mechanical strength and elastin content (p < 0.05). Three weeks after transplantation, the results showed reepithelialization, angiogenesis, and migration of host cell into scaffolds with no induction of immunogenic reaction in all ADM groups tested. In our study, the detergent-free method showed better preservation of matrix composition and biomechanical properties, but after transplantation, all methods of ADM preparation resulted in equally biofunctional matrices as wound coverage.
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Detergentes/química , Pele/citologia , Derme Acelular , Animais , Movimento Celular/fisiologia , Matriz Extracelular/química , Glicosaminoglicanos/química , Camundongos , Cicatrização/fisiologiaRESUMO
Human embryonic stem cells (hESCs) have enormous potential as a source of cells for cell replacement therapies and as a model for early human development. In this study we examined the differentiating potential of hESCs into hepatocytes in two- and three-dimensional (2D and 3D) culture systems. Embryoid bodies (EBs) were inserted into a collagen scaffold 3D culture system or cultured on collagen-coated dishes and stimulated with exogenous growth factors to induce hepatic histogenesis. Immunofluorescence analysis revealed the expression of albumin (ALB) and cytokeratin-18 (CK-18). The differentiated cells in 2D and 3D culture system displayed several characteristics of hepatocytes, including expression of transthyretin, alpha-1-antitrypsin, cytokeratin 8, 18, 19, tryptophan-2,3-dioxygenase, tyrosine aminotransferase, glucose-6-phosphatase (G6P), cytochrome P450 subunits 7a1 and secretion of alpha-fetoprotein (AFP) and ALB and production of urea. In 3D culture, ALB and G6P were detected earlier and higher levels of urea and AFP were produced, when compared with 2D culture. Electron microscopy of differentiated hESCs showed hepatocyte-like ultrastructure, including glycogon granules, well-developed Golgi apparatuses, rough and smooth endoplasmic reticuli and intercellular canaliculi. The differentiation of hESCs into hepatocyte-like cells within 3D collagen scaffolds containing exogenous growth factors, gives rise to cells displaying morphological features, gene expression patterns and metabolic activities characteristic of hepatocytes and may provide a source of differentiated cells for treatment of liver diseases.
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Diferenciação Celular , Hepatócitos/citologia , Células-Tronco/citologia , Técnicas de Cultura de Células , Linhagem Celular , Hepatócitos/metabolismo , Hepatócitos/fisiologia , Hepatócitos/ultraestrutura , Humanos , Imuno-Histoquímica , Dados de Sequência Molecular , Células-Tronco/fisiologiaRESUMO
Our aim was to evaluate the oocyte maturation rate and follicular genes expression pattern during in-vitro culture of vitrified mouse pre-antral follicles. Middle sized pre-antral follicles were isolated mechanically from the ovaries of pre-pubertal mice and distributed in vitrification and control groups. In the vitrification group, follicles were washed in equilibration and vitrification solutions and then were immersed in liquid nitrogen after loading on cryotop tips. After warming in descending concentrations of sucrose solutions, fresh and vitrified-warmed follicles were cultured for 13 days. Follicles survival rate and follicular genes expression were assessed during in vitro culture. Finally, at the end of the culture period oocytes maturation rate were compared in both groups. In the vitrification group, follicles survival rate was lower significantly comparing to the control group (P < 0.05), whereas oocytes maturation rate were similar. Although at the beginning of the culture period, expression of some genes such as Gdf9, Bmp15, Tgfß1 and BmprII were higher in the vitrification group (P < 0.05), during the rest of the culture period expression pattern of all follicular genes were similar in both groups. In conclusion, survival rate of cryotop vitrified pre-antral follicles reduced during culture period while oocytes maturation and follicular genes expression did not show any noticeable alteration.
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Oogênese , Folículo Ovariano/citologia , Folículo Ovariano/embriologia , Técnicas de Cultura de Tecidos/métodos , Animais , Sobrevivência Celular , Criopreservação , Meios de Cultura , Feminino , Perfilação da Expressão Gênica , Camundongos , Oogênese/genética , Folículo Ovariano/metabolismo , VitrificaçãoRESUMO
The genes conferring pathogenicity in Streptomyces turgidiscabies, a pathogen causing common scab of potato, are grouped together on a pathogenicity island (PAI), which has been found to be mobile and appears to transfer and disseminate like an integrative and conjugative element (ICE). However, in Streptomyces scabiei, another common scab-inducing species, the pathogenicity genes are clustered in two regions: the toxicogenic region (TR) and the colonization region. The S. scabiei 87.22 genome was analysed to investigate the potential mobility of the TR. Attachment sites (att), short homologous sequences that delineate ICEs, were identified at both extremities of the TR. An internal att site was also found, suggesting that the TR has a composite structure (TR1 and TR2). Thaxtomin biosynthetic genes, essential for pathogenicity, were found in TR1, whereas candidate genes with known functions in recombination, replication and conjugal transfer were found in TR2. Excision of the TR1 or TR2 subregions alone, or of the entire TR region, was observed, although the excision frequency of TR was low. However, the excision frequency was considerably increased in the presence of either mitomycin C or Streptomyces coelicolor cells. A composite TR structure was not observed in all S. scabiei and Streptomyces acidiscabies strains tested. Of the ten strains analysed, seven lacked TR2 and no TR excision event could be detected in these strains, thus suggesting the implication of TR2 in the mobilization of S. scabieiâ TR.