Critical role of metabotropic glutamate receptor 4 in bone marrow-derived dendritic cells in the Th17 cell differentiation and the melanogenesis of B16 cells

Vitiligo is an acquired pigmentary disorder resulting from selective destruction of melanocytes. Emerging studies have suggested that T helper cell 17 (Th17) is potentially implicated in vitiligo development and progression. It was recently discovered that metabotropic glutamate receptor 4 (mGluR4) can modulate Th17-mediated adaptive immunity. However, the influence of mGluR4 on melanogenesis of melanocytes has yet to be elucidated. In the present study, we primarily cultured mouse bone marrow-derived dendritic cells (BMDC) and then knocked down and over-expressed mGluR4 using transfection. Transduced BMDC were co-cultured with CD4+ T cells and the expression of Th17-related cytokines were measured. The morphology and melanogenesis of B16 cells were observed after being treated with co-culture medium of CD4+ T cells and transduced BMDC. We found that mGluR4 knockdown did not affect the co-stimulatory CD80 and CD86 upregulation after lipopolysaccharide stimulation but did increase the expression of Th17-related cytokines, and further down-regulated the expression of microphthalmia-associated transcription factor (MITF) and the downstream genes, decreased melanin production, and destroyed the morphology of B16 cells. Conversely, over-expression of mGluR4 reduced the expression of CD80 and CD86, suppressed the production of Th17-related cytokines, increased the expression of MITF, and did not destroy the morphology of B16 cells. Our study confirmed that mGluR4 modulated the Th17 cell polarization and resulted in the alteration of melanogenesis and morphology of B16 cells. Collectively, these findings suggest mGluR4 might be a potent target involved in the immune pathogenesis of vitiligo.

Abnormal expression of b10 cell frequencies: possible relation to pathogenesis and disease severity of aplastic anemia

SUMMARY OBJECTIVE: Aplastic anemia (AA) is an immune-mediated disease that destroys hematopoietic cells through activated T lymphocytes. B lymphocyte-mediated humoral immunity also plays an important role in the pathogenesis of AA. Regulatory B cell (Breg) subpopulation, which is defined as "B10", secretes interleukin 10 (IL-10). The objective of our experiment was to investigate whether the scale-down proportion of B10 cells in AA patients may play a key role in the pathogenesis. METHODS: A total of 38 AA patients (14 SAA patients and 24 NSAA patients) and 20 healthy control subjects were included. All subjects did not suffer from autoimmune diseases or any other diseases affecting the immune system, such as infectious diseases. Bone marrow mononuclear cells (PBMCs) were isolated and analyzed by Flow cytometry (FCM) and Immunofluorescence double-labeling assay. The relationship between the relative proportions of B10 and ProB10 and their associations to AA, as well as disease severity, were assessed by common clinical indicators and then examined. RESULTS: Our analyses revealed AA patients had significantly lower proportions of peripheral B10 and B10pro compared to healthy controls. SAA patients had a substantially lower percentage of B10 cells and B10pro cells compared to NSAA patients. In addition, B10 cells and B10pro cells were negatively correlated with absolute neutrophil counts, hemoglobin levels and platelet, and absolute reticulocyte counts in AA patients. CONCLUSIONS: The present study attempted to elucidate the potential role of the scale-down proportion of B10 cells in the pathogenesis of AA.

RESUMO OBJETIVO: A anemia aplástica (AA) é uma doença imunomediada que destrói células hematopoiéticas por meio dos linfócitos T ativados. A imunidade humoral mediada por linfócitos B também desempenha um papel importante na patogênese da AA. A subpopulação de células B reguladoras (Breg), que é definida como "B10", secreta interleucina 10 (IL-10). No experimento, investigou-se se a proporção reduzida de células B10 nos pacientes de AA pode desempenhar um papel-chave na patogênese. MÉTODOS: Um total de 38 pacientes de AA (14 pacientes de anemia aplástica grave e 24 pacientes de anemia aplástica não grave) e 20 indivíduos de controle saudáveis foram incluídos. Todos os indivíduos não sofriam de doenças autoimunes ou de quaisquer outras doenças que afetam o sistema imunológico, tais como doenças contagiosas. As células mononucleares da medula óssea (PBMCs) eram isoladas e analisadas por citometria de fluxo (FCM) e ensaio de dupla marcação por imunofluorescência. A relação entre as proporções relativas de células B10 e as células ProB10 e as suas associações à AA, assim como a gravidade da doença avaliada por indicadores clínicos comuns, foram examinadas. RESULTADOS: Nossas análises revelaram que os pacientes de AA têm proporções significativamente menores de células B10 e células ProB10 periféricas em comparação com indivíduos de controle saudáveis. Os pacientes de anemia aplástica grave tiveram uma percentagem substancialmente menor de células B10 e células B10pro em comparação com pacientes de anemia aplástica não grave. Além disso, as células B10 e B10pro foram negativamente correlacionadas com contagens absolutas de neutrófilos, níveis de hemoglobina e plaquetas e contagem de reticulócitos absolutos nos pacientes de AA. CONCLUSÕES: Além disso, o estudo presente tentou elucidar o papel imunorregulatório potencial das células B10 na patogênese da AA e fornecer uma nova estratégia para a aplicação de imunoterapia baseada na célula B para tratar a AA no futuro.

Effect of ProRoot MTA® and Biodentine® on osteoclastic differentiation and activity of mouse bone marrow macrophages

Abstract Objectives This investigation aimed to assess the differentiation inhibitory effects of ProRoot MTA® (PMTA) and Biodentine® (BIOD) on osteoclasts originated from murine bone marrow macrophages (BMMs) and compare these effects with those of alendronate (ALD). Materials and Methods Mouse BMMs were cultured to differentiate into osteoclasts with macrophage colony-stimulating factor and receptor activator of NF-κB (RANKL), treated with lipopolysaccharide. After application with PMTA, BIOD, or ALD, cell toxicities were examined using WST-1 assay kit, and RANKL-induced osteoclast differentiation and activities were determined by resorption pit formation assay and tartrate-resistant acid phosphate (TRAP) staining. The mRNA levels of osteoclast activity-related genes were detected with quantitative real time polymerase chain reaction. Expressions of molecular signaling pathways were assessed by western blot. All data were statistically analyzed with one-way ANOVA and Tukey's post-hoc test (p<0.05). Results Mouse BMMs applied with PMTA, BIOD, or ALD showed highly reduced levels of TRAP-positive osteoclasts. The BIOD treated specimens suppressed mRNA expressions of cathepsin K, TRAP, and c-Fos. Nonetheless, it showed a lower effect than PMTA or ALD applications. Compared with ALD, PMTA and BIOD decreased RANKL-mediated phosphorylation of ERK1/2 and IκBα. Conclusions PMTA and BIOD showed the inhibitory effect on osteoclast differentiation and activities similar to that of ALD through IκB phosphorylation and suppression of ERK signaling pathways.

Toxicity of synthetic flavorings, nature identical and artificial, to hematopoietic tissue cells of rodents / Toxicidade de aromatizantes alimentares sintéticos idênticos aos naturais e artificiais a células de tecido hematopoiético de roedores

Abstract The goal of this study was to analyze cytotoxicity, genotoxicity and mutagenicity to bone marrow cells of mice of nature identical synthetic flavorings, passion fruit and strawberry, and artificial synthetic flavorings, vanilla, chocolate, tutti-frutti and cookie, at doses 0.5; 1.0; 2.0; 5.0 and 10.0 mL/kg. The additives were given to the animals by gavage in a single daily application for seven days. Data were subjected to analysis of variance (ANOVA) followed by post Tukey's post hoc test, p <0.05. Animals treated with 2.0; 5.0 and 10.0 mL/Kg of flavorings chocolate, strawberry and cookie, and 5.0 and 10.0 mL/Kg of flavorings vanilla and passion fruit died on the fifth and sixth day of the experiment, respectively. The doses 0.5 and 1.0 mL/Kg of the six additives significantly reduced erythropoiesis in the examined tissue. Also, treatments 0.5 and 1.0 mL/Kg of chocolate, and 1.0 mL/Kg of strawberry and biscuit induced the formation of micronuclei in the bone marrow erythrocytes, at a significant frequency. Therefore, under the study conditions, the six microingredients analyzed were cytotoxic and genotoxic, and additives strawberry, chocolate and cookie were also mutagenic in at least one of the evaluated doses.

Resumo Os aromatizantes são essenciais para a indústria na confecção de alimentos industrializados. Porém, pouco se sabe sobre o potencial tóxico desses microingredientes alimentares. Dessa forma, objetivou-se neste trabalho analisar, em células de medula óssea de camundongos, a citotoxicidade, genotoxicidade e mutagenicidade de aromatizantes alimentares sintéticos idênticos ao natural, de maracujá e morango, e artificiais, de baunilha, chocolate, tutti-frutti e biscoito, nas doses 0,5; 1,0; 2,0; 5,0 e 10,0 mL/Kg. Os aditivos foram administrados aos animais via gavagem em aplicação diária única durante sete dias. Os dados obtidos foram submetidos ao procedimento estatístico ANOVA com pós teste de Tukey, com p < 0.05. Os animais tratados com 2,0; 5,0 e 10,0 mL/Kg dos aromatizantes de chocolate, morango e biscoito, e 5,0 e 10,0 mL/Kg dos aromatizantes de baunilha e maracujá vieram a óbito no quinto e sexto dia de experimento, respectivamente. As doses 0,5 e 1,0 mL/Kg dos seis aditivos reduziram significativamente a eritropoiese do tecido analisado. Ainda, os tratamentos 0,5 e 1,0 mL/kg de chocolate, e 1,0 mL/Kg de morango e biscoito induziram a formação de micronúcleos aos eritrócitos de medula em frequência significante. Portanto, nas condições de estudo estabelecidas, os seis microingredientes analisados foram citotóxico e genotóxicos, e os aditivos de morango, chocolate e biscoito também foram mutagênicos em pelo menos uma das doses avaliadas.

Reduction of doxorubicin-induced genotoxicity by Handroanthus impetiginosus in mouse bone marrow revealed by micronucleus assay / Redução da genotoxicidade induzida pela doxorrubicina por Handroanthus impetiginosus na medula óssea de camundongos revelada pelo ensaio do micronúcleo

Abstract Handroanthus impetiginosus has long been used in traditional medicine and various studies have determined the presence of bioactive chemical compounds and potential phytotherapeutics. In this study, the genotoxicity of the lyophilized tincture of H. impetiginosus bark (THI) was evaluated in mouse bone marrow using micronucleus assays. The interaction between THI and genotoxic effects induced by the chemotherapeutic agent, doxorubicin (DXR), was also analyzed. Experimental groups were evaluated 24 to 48 h after treatment with N-nitroso-N-ethylurea (NEU; 50 mg/kg), DXR (5 mg/kg), sodium chloride (NaCl; 150 mM), and THI (0.5-2 g/kg). Antigenotoxic assays were carried out using THI (0.5 g/kg) in combination with NEU or DXR. Analysis of the micronucleated polychromatic erythrocytes (MNPCEs) indicated no significant differences between treatment doses of THI (0.5-2 g/kg) and NaCl. Polychromatic erythrocyte (PCE) to normochromatic erythrocyte (NCE) ratios did not indicate any statistical differences between DXR and THI or NaCl, but there were differences between THI and NaCl. A significant reduction in MNPCEs and PCE/NCE ratios was observed when THI was administered in combination with DXR. This study suggested the absence of THI genotoxicity that was dose-, time-, and gender-independent and the presence of moderate systemic toxicity that was dose-independent, but time- and gender-dependent. The combination of THI and DXR also suggested antigenotoxic effects, indicating that THI reduced genotoxic effects induced by chemotherapeutic agents.

Resumo Handroanthus impetiginosus tem sido usada durante um longo período pela medicina tradicional e vários estudos têm demonstrados a presença de compostos químicos e potencial fitoterapêutico. Esta pesquisa avaliou a genotoxicidade da tintura da casca liofilizada de H. impetiginosus (THI) usando o ensaio do micronúcleo em medula óssea de camundongos. A interação entre THI e os efeitos genotóxicos induzidos pelo quimioterápico doxorrubicina (DXR) também foram analisados. Grupos experimentais foram analisados a 24-48 h após o tratamento com N-Nitroso-N-etiluréia (NEU; 50 mg/kg), DXR (5 mg/kg), NaCl (150 mM) e THI (0,5-2 g/kg). O ensaio antigenotóxico foi conduzido utilizando THI (0,5 g/kg) em combinação com NEU ou DXR. A análise de eritrócitos policromáticos micronucleados (EPCMNs) não mostrou diferenças significativas entre as doses de tratamento (0,5-2 g/kg) e NaCl. As proporções de eritrócitos policromáticos (EPC)/eritrócitos normocromáticos (ENC) não revelaram diferenças estatísticas entre DXR e THI ou NaCl, porém houve diferenças entre THI e NaCl. Uma redução significativa em EPCMNs e na razão EPC/ENC foi observada quando THI foi administrado em combinação com DXR. Essa pesquisa sugere ausência de genotoxicidade de THI, dose-, tempo- e sexo-independente, e moderada toxicidade sistêmica dose-independente, mas tempo- e sexo-dependente. A associação do THI e DXR também sugere efeitos antigenotóxicos. Por conseguinte, THI pode reduzir os efeitos genotóxicos induzidos pelo quimioterapêutico.

Isolation and characterization of mesenchymal progenitors derived from the bone marrow of goats native from northeastern Brazil

PURPOSE: To characterize bone marrow progenitors cells grown in vitro, using native goats from northeastern Brazil as animal model. METHODS: Ten northeastern Brazil native goats of both genders were used from the Piauí Federal University Agricultural Science Center's (UFPI) - Goat Farming Sector. Bone marrow aspirates where taken from the tibial ridge and seeded on culture plates for isolation, expansion and Flow Cytometry (expression markers - Oct-3/4, PCNA, Ck-Pan, Vimentina, Nanog). RESULTS: Progenitor cells showed colonies characterized by the presence of cell pellets with fibroblastoid morphology. Cell confluence was taken after 14 days culture and the non-adherent mononuclear cell progressive reduction. After the first passage, 94.36% cell viability was observed, starting from 4.6 x 106 cell/mL initially seeded. Cells that went through flow cytometry showed positive expression for Oct-3/4, PCNA, Ck-Pan, Vimentina, and Nanog. CONCLUSIONS: Bone marrow progenitor isolated of native goats from northeastern Brazil showed expression markers also seen in embryonic stem cells (Oct-3/4, Nanog), markers of cell proliferation (PCNA) and markers for mesenchymal cells (Vimentina and Ck-pan), which associated to morphological and culture growth features, suggest the existence of a mesenchymal stem cell (MSC) population in the goat bone marrow stromal cells studied. .

Brazilian minipig as a large-animal model for basic research and stem cell-based tissue engineering. Characterization and in vitro differentiation of bone marrow-derived mesenchymal stem cells

Stem cell-based regenerative medicine is one of the most intensively researched medical issues. Pre-clinical studies in a large-animal model, especially in swine or miniature pigs, are highly relevant to human applications. Mesenchymal stem cells (MSCs) have been isolated and expanded from different sources. Objective: This study aimed at isolating and characterizing, for the first time, bone marrow-derived MSCs (BM-MSCs) from a Brazilian minipig (BR1). Also, this aimed to validate a new large-animal model for stem cell-based tissue engineering. Material and Methods: Bone marrow (BM) was aspirated from the posterior iliac crest of twelve adult male BR1 under general anesthesia. MSCs were selected by plastic-adherence as originally described by Friedenstein. Cell morphology, surface marker expression, and cellular differentiation were examined. The immunophenotypic profile was determined by flow cytometry. The differentiation potential was assessed by cytological staining and by RT-PCR. Results: MSCs were present in all minipig BM samples. These cells showed fibroblastic morphology and were positive for the surface markers CD90 (88.6%), CD29 (89.8%), CD44 (86.9%) and negative for CD34 (1.61%), CD45 (1.83%), CD14 (1.77%) and MHC-II (2.69%). MSCs were differentiated into adipocytes, osteoblasts, and chondroblasts as demonstrated by the presence of lipidic-rich vacuoles, the mineralized extracellular matrix, and the great presence of glycosaminoglycans, respectively. The higher gene expression of adipocyte fatty-acid binding protein (AP2), alkaline phosphatase (ALP) and collagen type 2 (COLII) also confirmed the trilineage differentiation (p<0.001, p<0.001, p=0.031; respectively). Conclusions: The isolation, cultivation, and differentiation of BM-MSCs from BR1 makes this animal eligible as a useful large-animal model for stem cell-based studies in Brazil. .

Low-level laser irradiation induces in vitro proliferation of mesenchymal stem cells / Laser de baixa intensidade induz à proliferação in vitro de células-tronco mesenquimais

Objective : To evaluate the effect of low-level laser irradiation on the proliferation and possible nuclear morphological changes of mouse mesenchymal stem cells. Methods : Mesenchymal stem cells derived from bone marrow and adipose tissue were submitted to two applications (T0 and T48 hours) of low-level laser irradiation (660nm; doses of 0.5 and 1.0J/cm2). The trypan blue assay was used to evaluate cell viability, and growth curves were used to analyze proliferation at zero, 24, 48, and 72 hours. Nuclear alterations were evaluated by staining with DAPI (4’-6-diamidino-2-phenylindole) at 72 hours. Results : Bone marrow-derived mesenchymal stem cells responded to laser therapy in a dose-dependent manner. Higher cell growth was observed when the cells were irradiated with a dose of 1.0J/cm2, especially after 24 hours (p<0.01). Adipose-derived mesenchymal stem cells responded better to a dose of 1.0J/cm2, but higher cell proliferation was observed after 48 hours (p<0.05) and 72 hours (p<0.01). Neither nuclear alterations nor a significant change in cell viability was detected in the studied groups. Conclusion : Low-level laser irradiation stimulated the proliferation of mouse mesenchymal stem cells without causing nuclear alterations. The biostimulation of mesenchymal stem cells using laser therapy might be an important tool for regenerative therapy and tissue engineering. .

Objetivo : Avaliar o efeito da terapia com laser de baixa intensidade sobre a proliferação e as possíveis alterações morfológicas nucleares em células-tronco mesenquimais de camundongos. Métodos : Células-tronco mesenquimais derivadas da medula óssea e do tecido adiposo foram submetidas a duas aplicações (T0 e T48 horas) de laser de baixa intensidade (660nm; doses de 0,5 e 1,0J/cm2). O ensaio de azul de tripan foi utilizado para a avaliação da viabilidade celular, e curvas de crescimento foram usadas para avaliar a proliferação das células em zero, 24, 48, e 72 horas. Alterações nucleares foram avaliadas por coloração com DAPI (4-6-diamidino-2-fenilindolo) em 72 horas. Resultados : As células-tronco mesenquimais derivadas da medula óssea responderam a terapia com laser de forma dose-dependente. Um maior crescimento celular foi observado quando as células foram irradiadas com dose de 1,0J/cm2, especialmente depois de 24 horas (p<0,01). As células-tronco mesenquimais derivadas do tecido adiposo responderam melhor à dose de 1,0J/cm2, com maior proliferação após 48 (p<0,05) e 72 horas (p<0,01). Nem alterações nucleares nem a mudança significativa na viabilidade celular foi detectada nos grupos estudados. Conclusão : Laser de baixa intensidade estimulou a proliferação de células-tronco mesenquimais sem causar alterações nucleares. A bioestimulação de células-tronco mesenquimais por laserterapia pode ser uma ferramenta importante para a terapia regenerativa e a engenharia tecidual. .

Culture of equine bone marrow mononuclear fraction and adipose tissue-derived stromal vascular fraction cells in different media / Cultivo de células da fração mononuclear da medulla óssea e da fração vascular stromal do tecido adipose de equinos em diferentes meios

The objective of this study was to evaluate the culture of equine bone marrow mononuclear fraction and adipose tissue - derived stromal vascular fraction cells in two different cell culture media. Five adult horses were submitted to bone marrow aspiration from the sternum, and then from the adipose tissue of the gluteal region near the base of the tail. Mononuclear fraction and stromal vascular fraction were isolated from the samples and cultivated in DMEM medium supplemented with 10% fetal bovine serum or in AIM-V medium. The cultures were observed once a week with an inverted microscope, to perform a qualitative analysis of the morphology of the cells as well as the general appearance of the cell culture. Colony-forming units (CFU) were counted on days 5, 15 and 25 of cell culture. During the first week of culture, differences were observed between the samples from the same source maintained in different culture media. The number of colonies was significantly higher in samples of bone marrow in relation to samples of adipose tissue.

O objetivo deste estudo foi avaliar o cultivo de células da fração mononuclear da medula óssea e da fração vascular estromal do tecido adiposo de equinos em dois diferentes meios. Cinco cavalos foram submetidos à aspiração da medula óssea do esterno e à coleta de tecido adiposo da região glútea, próxima à inserção da cauda. A fração mononuclear e a fração vascular estromal foram obtidas das amostras e cultivadas em meio DMEM suplementado com soro fetal bovino a 10% ou em meio AIM-V. As culturas foram observadas uma vez por semana com um microscópio de luz invertida, com o intuito de se realizar uma análise qualitativa das características morfológicas das células, bem como do aspecto geral do cultivo celular. As unidades formadoras de colônia (CFU) foram contadas nos dias 5, 15 e 25 do cultivo celular. Durante a primeira semana, foram observadas diferenças entre amostras obtidas de mesma origem mantidas em diferentes meios. O número de colônias foi significativamente maior nas amostras de medula óssea em relação às amostras de tecido adiposo.

Osteogenesis induced in goat bone marrow progenitor cells by recombinant adenovirus coexpressing bone morphogenetic protein 2 and basic fibroblast growth factor

Bone morphogenetic protein 2 (BMP2) and basic fibroblast growth factor (bFGF) have been shown to exhibit a synergistic effect to promote bone repair and healing. In this study, we constructed a novel adenovirus with high coexpression of BMP2 and bFGF and evaluated its effect on osteogenic differentiation of goat bone marrow progenitor cells (BMPCs). Recombinant adenovirus Ad-BMP2-bFGF was constructed by using the T2A sequence. BMPCs were isolated from goats by density gradient centrifugation and adherent cell culture, and were then infected with Ad-BMP2-bFGF or Ad-BMP2. Expression of BMP2 and bFGF was detected by ELISA, and alkaline phosphatase (ALP) activity was detected by an ALP assay kit. In addition, von Kossa staining and immunocytochemical staining of collagen II were performed on BMPCs 21 days after infection. There was a high coexpression of BMP2 and bFGF in BMPCs infected with Ad-BMP2-bFGF. Twenty-one days after infection, ALP activity was significantly higher in BMPCs infected with Ad-BMP2-bFGF than in those infected with Ad-BMP2. Larger and more mineralized calcium nodules, as well as stronger collagen II staining, were observed in BMPCs infected with Ad-BMP2-bFGF than in those infected with Ad-BMP2. In summary, we developed a novel adenovirus vector Ad-BMP2-bFGF for simultaneous high coexpression of BMP2 and bFGF, which could induce BMPCs to differentiate efficiently into osteoblasts.

Overexpression of hsa-miR-125b during osteoblastic differentiation does not influence levels of Runx2, osteopontin, and ALPL gene expression

Multipotent mesenchymal stromal cells (MSCs) were first isolated from bone marrow and then from various adult tissues including placenta, cord blood, deciduous teeth, and amniotic fluid. MSCs are defined or characterized by their ability to adhere to plastic, to express specific surface antigens, and to differentiate into osteogenic, chondrogenic, adipogenic, and myogenic lineages. Although the molecular mechanisms that control MSC proliferation and differentiation are not well understood, the involvement of microRNAs has been reported. In the present study, we investigated the role of miR-125b during osteoblastic differentiation in humans. We found that miR-125b increased during osteoblastic differentiation, as well as Runx2 and ALPL genes. To study whether the gain or loss of miR-125b function influenced osteoblastic differentiation, we transfected MSCs with pre-miR-125b or anti-miR-125b and cultured the transfected cells in an osteoblastic differentiation medium. After transfection, no change was observed in osteoblastic differentiation, and Runx2, OPN, and ALPL gene expression were not changed. These results suggest that the gain or loss of miR-125b function does not influence levels of Runx2, OPN, and ALPL during osteoblastic differentiation.

Triiodotironina não aumenta a diferenciação osteogênica reduzida pela idade de células-tronco mesenquimais da medula óssea de ratas / Triiodothyronine does not increase osteogenic differentiation reduced by age in bone marrow mesenchymal stem cells of female rats

OBJETIVO: Avaliar se a adição de T3 aumenta o potencial osteogênico das células-tronco mesenquimais da medula óssea (CTM-MO) de ratas adultas normais comparado ao de ratas jovens. MATERIAIS E MÉTODOS: CTM-MO foram cultivadas em meio osteogênico e separadas em seis grupos: 1) CTM-MO de ratas jovens; 2) CTM-MO de ratas adultas; 3, 4, 5 e 6) CTM-MO de ratas adultas com T3 nas concentrações de 0,01; 1; 100 e 1000 nM, respectivamente. Foram avaliados: atividade da fosfatase alcalina, conversão do dimetiltiazol (MTT) e síntese de colágeno aos sete, 14 e 21 dias e celularidade e número de nódulos de mineralização aos 21 dias de diferenciação. RESULTADOS: T3 reduziu significativamente a conversão do MTT, a atividade da fosfatase alcalina, a síntese de colágeno e a formação dos nódulos de mineralização em pelo menos uma das doses e dos períodos estudados (p < 0,05). Os valores foram menores quando comparados aos das CTM-MO de ratas jovens e adultas sem T3 (p < 0,05). CONCLUSÃO: T3 apresenta efeitos negativos sobre os fatores envolvidos na diferenciação osteogênica das CTM-MO de ratas adultas.

OBJECTIVE: To examine if triiodothyronine (T3) increases osteogenic differentiation in bone marrow mesenchymal stem cells (BMMSCs) of adult rats compared with young rats. MATERIALS AND METHODS: BMMSCs were cultured in osteogenic medium and distributed into six groups: 1) BMMSCs of young rats; 2) BMMSCs of adult rats; 3, 4, 5 and 6) BMMSCs of adult rats with T3 (0.01, 1, 100 to 1000 nM). We analyzed alkaline phosphatase activity, dimethylthiazol (MTT) conversion, and collagen synthesis at 7, 14, and 21 days, and percentage of cells per field and number of mineralized nodules at 21 days of differentiation. RESULTS: T3 reduced MTT conversion, alkaline phosphatase activity, collagen synthesis, and the synthesis of mineralizalized nodules in at least one of the doses and periods studied (p < 0.05). Values were lower when compared with young and adult rats BMMSCs (p < 0.05) without T3. CONCLUSION: T3 has a negative effect on the factors involved in osteogenic differentiation of BMMSC from adult rats.

Evaluación de la proliferación de células madres mesenquimales estimuladas con diferentes intensidades de ultrasonido de baja intensidad / Evaluation of mesenchymal stem cell proliferation using different low intensity pulsed ultrasound intensities

Las células madres mesenquimales (CMM) pueden ser afectadas en su capacidad de proliferar in vitro bajo estimulación física o bioquímica, siendo esta una capacidad esencial para un adecuado cultivo celular. Un método de estimulación física que ha demostrado ser eficiente en este sentido es el Ultrasonido pulsátil de baja intensidad (USBI), aplicado en intensidades iguales o inferiores a 100 mW/cm2, habitualmente entre 30 y 50 mW/cm2. El objetivo de esta investigación fue determinar el nivel de intensidad de ultrasonido pulsátil de baja intensidad óptimo entre 30 y 50 mW/cm2 para estimular la proliferación de CMM de médula ósea de ratas Sprague Dawley, in vitro. CMM (1x106cls/kg) de medula ósea de rata Sprague-Dawley fueron cultivadas (alfa-MEM, 20 por ciento FBS y 1 por ciento antibiotico) y estimuladas con USBI (0,02 milisegundos), por 20 minutos dos veces al día por 10 días con intensidades de 0, 30 y 50 mW/cm2 (grupos A [control], B, C respectivamente). Se contabilizó el número de células en el cultivo y se evaluó morfología celular en microscopio óptico. Se utilizaron tests de ANOVA on Ranks y Bonferroni. Los cultivos estimulados con USBI presentaron mayores recuentos celulares, y se observaron diferencias estadísticamente significativas entre los grupos A y C (p<0,05). Se observaron diferencias morfológicas entre células de grupos estimulados con USBI y el control. La estimulación de las CMM en cultivos bidimensionales con USBI influencia cambios en la morfología celular y se concluye que 50mW/cm2 es la intensidad óptima dentro de las evaluadas para producir aumento en la proliferación celular (p<0.05).

Mesenchymal stem cells (MSCs) can be affected in their capabilities to proliferate in vitro under physical and/or biochemical stimulation. The aim of this study was to select an optimal intensity level for low intensity pulsed ultrasound (LIPUS) stimulation of Sprague-Dawley bone marrow MSCs proliferation in vitro. Bone marrow MSCs of Sprague-Dawley rats where cultured (a-MEM, 20 percent FBS and 1 percent antibiotic) and stimulated with LIPUS (0,02 milisec), for 20 minutes twice daily for 10 days, at intensities of 0 (control), 30 and 50mW/cm2 (groups A, B, C). Cellular count and morphological evaluation were performed. ANOVA and Bonferroni tests were performed. LIPUS-stimulated cultures displayed greater cellular counts, and significant differences were observed between groups A and C (p<0,05). Morphological differences were observed between cells from LIPUS-stimulated and control groups. An intensity of 50mW/cm2 elicits increased cellular proliferation (p<0.05). Stimulation of MSCs cultures with LIPUS influences cellular morphology.

Co-culture of chondrocytes and bone marrow mesenchymal stem cells in vitro enhances the expression of cartilaginous extracellular matrix components

Chondrocytes and bone marrow mesenchymal stem cells (BMSCs) are frequently used as seed cells in cartilage tissue engineering. In the present study, we determined if the co-culture of rabbit articular chondrocytes and BMSCs in vitro promotes the expression of cartilaginous extracellular matrix and, if so, what is the optimal ratio of the two cell types. Cultures of rabbit articular chondrocytes and BMSCs were expanded in vitro and then cultured individually or at a chondrocyte:BMSC ratio of 4:1, 2:1, 1:1, 1:2, 1:4 for 21 days and cultured in DMEM/F12. BMSCs were cultured in chondrogenic induction medium. Quantitative real-time RT-PCR and Western blot were used to evaluate gene expression. In the co-cultures, type II collagen and aggrecan expression increased on days 14 and 21. At the mRNA level, the expression of type II collagen and aggrecan on day 21 was much higher in the 4:1, 2:1, and 1:1 groups than in either the articular chondrocyte group or the induced BMSC group, and the best ratio of co-culture groups seems to be 2:1. Also on day 21, the expression of type II collagen and aggrecan proteins in the 2:1 group was much higher than in all other groups. The results demonstrate that the co-culture of rabbit chondrocytes and rabbit BMSCs at defined ratios can promote the expression of cartilaginous extracellular matrix. The optimal cell ratio appears to be 2:1 (chondrocytes:BMSCs). This approach has potential applications in cartilage tissue engineering since it provides a protocol for maintaining and promoting seed-cell differentiation and function.

Avaliação do método da imunofenotipagem por citometria de fluxo no diagnóstico das doenças displásicas (SMD, LMA-relacionada à mielodisplasia, LMMC e LMMJ) em adultos e crianças / Evaluation of the method of immunophenotyping by flow cytometry in the diagnosis of dysplastic disease (MDS, AML-related to myelodysplasia,CMML and JMML) in adults and children

As síndromes mielodisplásicas (SMD) se caracterizam por terem uma hematopoese displásica, citopenias e pelo risco de progressão para leucemia mielóide aguda. O diagnóstico baseia-se na clínica e nos achados citomorfológicos da medula óssea (MO) e citogenéticos. Na fase inicial ou quando a MO é hipocelular o diagnóstico é difícil e a citogenética frequentemente é normal. A imunofenotipagem (IMF) tem sido cada vez mais utilizada nos casos de SMD em adultos e pouco explorada na SMD pediátrica. Os nossos objetivos foram: estudar os casos de SMD e doenças correlatas (LMA relacioanda à SMD: LMA-rMD; leucemia mielomonocítica crônica: LMMC e leucemia mielomonocítica juvenil: LMMJ) em adultos e crianças, associando os dados clínicos e laboratoriais aos obtidos pela IMF, que utilizou um painel de anticorpos monoclonais para as várias linhagens medulares. No período compreendido entre 2000 e 2010 foram estudados 87 pacientes (64 adultos e 23 crianças) oriundos do HUPE/UERJ e IPPMG/UFRJ e 46 controles (23 adultos e 20 crianças). Todos os doentes realizaram mielograma, biópsia óssea, citogenética, citoquímica e estudo imunofenotípico. Segundo os critérios da OMS 50 adultos foram classificados como SMD, 11 como LMA-rMD e 3 LMMC. Entre as crianças 18 eram SMD, 2 LMA e 3 LMMJ. Os pacientes adultos com SMD foram divididos em alto risco (n=9; AREB-1 e AREB-2) e baixo risco (n=41; CRDU, CRDM, CRDM-SA, SMD-N e SMD-5q). As crianças com SMD em CR (n=16) e AREB (n=2). Anormalidades clonais recorrentes foram encontradas em 22 pacientes adultos e em 7 crianças. Na análise de IMF foi utilizada a metodologia da curva ROC para a determinação dos valores de ponto de corte a fim de identificar os resultados anormais dos anticorpos monoclonais nos pacientes e nos controles, permitindo determinar a sensibilidade e especificidade desses em cada linhagem. A IMF foi adequada para a análise em todos os pacientes e 3 ou mais anormalidades foram encontradas. A associação da IMF...

Myelodysplastic syndrome (MDS) is characterized by having a dysplastic hematopoiesis, cytopenias and risk of progression to acute myeloid leukemia. The diagnosis is based on clinical and cytomorphologic findings in bone marrow (BM) and cytogenetics. In the initial phase of when the BM is hypocellular, diagnosis is difficult and often with normal karyotype. The flow cytometry immunophenotyping (FCI) analysis has been broadly used in adult MDS cases but is rarely in pediatric MDS. The objectives of this work were: to study MDS cases and correlated diseases (AML with myelodysplasia-related changes; chronic myelomonocytic leukemia - CMML and juvenite myelomonocytic leukemia - JMML) and to correlate laboratorial data to FCI using a panel of monoclonal antibodies for the various marrow lineages in both adult and children. In the period between 2000 and 2010, 87 patients were studied (64 adults and 23 children) coming from HUPE/UERJ and IPPMG/UFRJ and 46 controls (26 adults and 20 children). All patients were submitted to myelogram, bone marrow biopsy, cytogenetic, cytochemistry and immunophenotypic study. According to WHO criteria 50 adults were classified MDS, 12 AML and 3 CMML. Among the children there were 18 MDS, 2 AML, and 3 JMML. MDS adult patients were subdivided into high risk (n=9; RAEB-1 and RAEB-2) and low risk (n=41; RCUD, RCMD-RS, MDS-U and MDS-5q). MDS children were classified as RCC (n=16) and RAEB (n=2). Clonal abnormalities were found in 22 (35%) adult patients and 7 (30%) children. In the analysis of FCI methodology ROC curve was used for determination of cut off abnormalities at monoclonal antibodies in patients and controls which allowed to estimate the sensitivity and specificity of each strain. The FCI was suitable for analysis in all patients and 3 or more abnormalities were found. The association of the FCI increased the sensitivity of morphological analysis in the erythroid lineage from 70 to 97% in adults and from 59 to 86% in children...

Possible mechanisms of retinal function recovery with the use of cell therapy with bone marrow-derived stem cells / Possíveis mecanismos de recuperação da função da retina com uso de terapia celular com células tronco derivadas da medula óssea

Bone marrow has been proposed as a potential source of stem cells for regenerative medicine. In the eye, degeneration of neural cells in the retina is a hallmark of such widespread ocular diseases as age-related macular degeneration (AMD) and retinitis pigmentosa. Bone marrow is an ideal tissue for studying stem cells mainly because of its accessibility. Furthermore, there are a number of well-defined mouse models and cell surface markers that allow effective study of hematopoiesis in healthy and injured mice. Because of these characteristics and the experience of bone marrow transplantation in the treatment of hematological disease such as leukemia, bone marrow-derived stem cells have also become a major tool in regenerative medicine. Those cells may be able to restore the retina function through different mechanisms: A) cellular differentiation, B) paracrine effect, and C) retinal pigment epithelium repair. In this review, we described these possible mechanisms of recovery of retinal function with the use of cell therapy with bone marrow-derived stem cells.

As células tronco derivadas da medula óssea têm sido propostas como uma fonte em potencial de células para medicina regenerativa. No olho, a degeneração de células neurais da retina são a marca de doenças difusas, como a degeneração macular relacionada com a idade (DMRI) e a retinose pigmentar. A medula óssea é um tecido ideal para estudar as células tronco por causa da sua acessibilidade. Devido a estas características e a experiência do transplante de medula óssea no tratamento de doenças hematológicas, como as leucemias, as célulastronco derivadas da medula óssea têm se tornado a maior ferramenta na medicina regenerativa. Essas células podem ser capazes de restaurar a função da retina através dos seguintes mecanismos: A) diferenciação celular; B) efeito parácrino; C) reparo do epitélio pigmentado da retina. Nesta revisão nós descrevemos os possíveis mecanismos de recuperação da função da retina com uso de terapia celular com células tronco derivadas da medula óssea.

Chondrogenic differentiation of bone marrow mesenchymal stem cells: first successful Latin-American report / Diferenciación condrogénica de células madres mesenquimáticas de médula osea: primer reporte exitoso latinoamericano

Osteoarthritis is the more frequent cause of disability in adult people and it is associated to cartilage degeneration of affected joints. This cartilage has a limited ability to repair. Several treatments have been tested including the use of Mesenchymal Stem Cells. These cells are an attractive source for cartilage repair because of their availability to chodrogenic differentiation by progressing sequentially through the expression of cartilage specific extracelullar matrix molecules, as in the embryologic human development. The aim is to obtain, culture and differentiate rabbit Bone Marrow derived Mesenchymal Stem Cells in vitro to chondral lineage. By differential centrifugation the mononuclear cell level was obtained from rabbit bone marrow samples. This level was cultured until 70 percent confluence. Chondrogenic differentiation was performed in an aggregate culture system with TGF-b1. Sample quantity, culture efficiency, confluence time of cultures and differentiation quality were all evaluated. An average sample of 14.5 ml per side was obtained, culture efficiency was 80 percent, and average confluence time (70 percent) was 18 days. Differentiation culture had an 80 percent efficiency and optimal differentiation quality. Rabbit Bone Marrow derived Mesenchymal Stem Cells culture is a reproducible technique and by the use of an adequate methodology chondrogenic cells can be obtained in vitro. This model permits the study of chondral differentiation process and could have direct clinical application. This is the first successful Latin-American report in Mesenchymal Stem Cells culture and chondrogenic differentiation.

La osteoartritis es la causa más frecuente de discapacidad en personas adultas y se asocia a la degeneración de los cartílagos de las articulaciones afectadas. Este cartílago tiene una capacidad limitada a la reparación. Muchos tratamientos han sido probados, incluyendo el uso de células madre mesenquimáticas. Estas células son una fuente atractiva para la reparación del cartílago debido a su disponibilidad a la diferenciación condrogénica progresando de forma secuencial a través de la expresión de moléculas de la matriz del cartílago específicos extracelular, como en el desarrollo humano embrionario. El objetivo es obtener, el cultivo y la diferenciación células madre mesenquimáticas de conejo procedentes de médula ósea al linaje condral in vitro. Por centrifugación diferencial a nivel de células mononucleares se obtuvieron muestras de médula ósea de conejo. Este nivel se cultivó hasta su confluencia al 70 por ciento. La diferenciación condrógenica se realizó en un sistema de cultivo de agregado con el TGF-b1. La cantidad de muestra, la eficiencia cultura, el tiempo confluencia de cultivos y la calidad de la diferenciación fueron evaluadas. Una muestra media de 14,5 ml por lado fue obtenida, la eficiencia de cultivo fue del 80 por ciento, y el tiempo promedio de confluencia (70 por ciento) fue de 18 días. En la diferenciación del cultivo se obtuvo una eficiencia del 80 por ciento y con una calidad óptima de diferenciación. El cultivo de células madre mesenquimáticas derivadas de médula ósea de Conejo es una técnica reproducible y con el uso de una metodología adecuada células condrogénica se pueden obtener in vitro. Este modelo permite el estudio del proceso de diferenciación condral y podría tener una aplicación clínica directa. Este es el primer informe de éxito de América Latina en el cultivo de células madre mesenquimáticas y diferenciación condrogénica.

Utilidad de la biopsia de médula ósea en estadificación de pacientes con cáncer de mama / Usefulness of bone marrows biopsy in the breast cancer patients staging

El objetivo del presente estudio es determinar la utilidad de la biopsia de médula ósea en la estadificación de pacientes con cáncer de mama. Estudio prospectivo, descriptivo y con diseño transversal. La población estuvo representada por los nuevos casos diagnosticados como cáncer de mama en la consulta de cirugía oncológica y patología mamaria, la muestra quedó conformada por 18 casos. Los datos obtenidos se clasificaron y procesaron en una matriz de datos, se analizaron mediante técnica de estadística simple descriptiva o “tabulación simple porcentual” apoyadas en medias de dispersión (frecuencias relativas). La mayoría de los casos se estadificaron en etapas clínicas IIA y IIB. La totalidad de los casos incluidos en esta investigación resultaron negativos para micrometástasis en médula ósea, lo cual podría predecir que se tratan de pacientes con buen pronóstico. Ante la falla como factor pronóstico del estado ganglionar se especula que la detección de células tumorales tempranas puede predecir el curso de la enfermedad mejor que el estado de los ganglios axilares. Actualmente la determinación genética ha tenido un impacto mayor como factor pronóstico que los estudios de estadificación convencionales. Ahora bien la determinación de células tumorales en la médula podría establecerse como parte del protocolo de estadificación en los diversos centros de referencia oncológico, y convertirse al igual que la determinación del perfil genético, como un factor pronóstico y predictivo importante en las pacientes con cáncer de mama y etapa clínicas tempranas.

The objective of the present study is to determine the usefulness and importance of bone marrows biopsy in the breast cancer patients staging. To such end, a cross sectional, prospective, and descriptive study was performed. The population was represented by new diagnosed cases of breast cancer view and evaluated in the consult of surgery oncology y mammalian pathology of our institution, formed the sample by 18 cases. The data obtained were processed and classified in an adequate data matrix; the results once separated were analyzed by the simple descriptive statistic technique or percentile simple tabulation, supported by the dispersion measures (the relative frequencies). Most of the cases were classified in IIA and IIB clinical stages. All included cases in this research were negative to bone marrow´s micro metastases, and which can predict good prognosis in these patients. At lymph node staging failure as a prognostic factor we can be speculate that the early tumor cell detection can predict the course of the disease better than the staging of axillaries lymph nodes. Actually the genetic determination has a better impact as a prognostic factor that the conventional staging studies. The determination of bone marrow´s tumor cell can be establish as a very important part of the staging protocol in the different especial oncologic reference centers, and became as same as genetic profile determination in an important prognostic and predictive factor in breast cancer patients at early stages.

Cyclosporin A-treated Dendritic Cells may affect the outcome of organ transplantation by decreasing CD4+CD25+ regulatory T cell proliferation

One of the mechanisms for generation of tolerance involves immature dendritic cells (DCs) and a subpopulation of regulatory CD4+ CD25+ T lymphocytes (T REG). The purpose of this work was to analyze how Cyclosporine A (CsA), a widely used immunosuppressive drug, may affect T REG proliferation. Purified and activated murine DCs obtained from bone marrow precursors differentiated with rGMCSF were co-cultured with purified CFSE-labeled T REG from OTII mice, and their phenotype and proliferation analyzed by flow cytometry. Our data indicate that DCs differentiated in the presence of CsA show an altered phenotype, with a lower expression of MHC-II and a lower activating capacity. Additionally, these CsA-treated DCs show decreased production of IL-2 and IL-12 and increased IL-10 secretion when stimulated with LPS, indicating an effect on the polarization of the immune response. Interestingly, CsA-treated DCs show an anti-tolerogenic effect since they reduce the proliferation of T REG cells from 72 to 47 percent. Further inhibition to a 24 percent of T REG proliferation was obtained as a direct effect of CsA on T REG. In conclusion, the anti-tolerogenic effect of CsA should be considered in the planning of immunosuppression in the context of clinical transplantation.

Bone resorptive activity of osteoclast-like cells generated in vitro by PEG-induced macrophage fusion

Normal bone remodeling is maintained by a balance between osteoclast and osteoblast activity, whereas defects in osteoclast activity affecting such balance result in metabolic bone disease. Macrophage-macrophage fusion leading to multinucleated osteoclasts being formed is still not well understood. Here we present PEG-induced fusion of macrophages from both U937/A and J774 cell lines and the induced differentiation and activation of osteoclast-like cells according to the expression of osteoclast markers such as tartrate resistant acid phosphatase (TRAP) and bone resorptive activity. PEG-induced macrophage fusion, during the non-confuent stage, signifcantly increased the osteoclastogenic activity of macrophages from cell lines compared to that of spontaneous cell fusion in the absence of PEG (polyethylene glycol). The results shown in this work provide evidence that cell fusion per se induces osteoclast-like activity. PEG-fused macrophage differential response to pretreatment with osteoclastogenic factors was also examined in terms of its ability to form TRAP positive multinucleated cells (TPMNC) and its resorptive activity on bovine cortical bone slices. Our work has also led to a relatively simple method regarding those previously reported involving cell co-cultures. Multinucleated osteoclast-like cells obtained by PEG-induced fusion of macrophages from cell lines could represent a suitable system for conducting biochemical studies related to basic macrophage fusion mechanisms, bone-resorption activity and the experimental search for bone disease therapeutic alternatives.