RESUMO
Dentistry and orthopedics are undergoing a revolution in order to provide more reliable, comfortable and long-lasting implants to patients. Titanium (Ti) and titanium alloys have been used in dental implants and total hip arthroplasty due to their excellent biocompatibility. However, Ti-based implants in human body suffer surface degradation (corrosion and wear) resulting in the release of metallic ions and solid wear debris (mainly titanium dioxide) leading to peri-implant inflammatory reactions. Unfortunately, our current understanding of the biological interactions with titanium dioxide nanoparticles is still very limited. Taking this into consideration, this study focuses on the internalization of titanium dioxide nanoparticles on primary bone cells, exploring the events occurring at the nano-bio interface. For the first time, we report the selective binding of calcium (Ca), phosphorous (P) and proteins from cell culture medium to anatase nanoparticles that are extremely important for nanoparticle internalization and bone cells survival. In the intricate biological environment, anatase nanoparticles form bio-complexes (mixture of proteins and ions) which act as a kind of 'Trojan-horse' internalization by cells. Furthermore, anatase nanoparticles-induced modifications on cell behavior (viability and internalization) could be understand in detail. The results presented in this report can inspire new strategies for the use of titanium dioxide nanoparticles in several regeneration therapies.
Assuntos
Endocitose , Nanopartículas Metálicas/química , Osteoblastos/metabolismo , Titânio/metabolismo , Cálcio/metabolismo , Sobrevivência Celular , Células Cultivadas , Citoplasma/metabolismo , Citoplasma/ultraestrutura , Humanos , Nanopartículas Metálicas/ultraestrutura , Microscopia Eletrônica , Osteoblastos/citologia , Osteoblastos/ultraestrutura , Tamanho da Partícula , Fósforo/metabolismo , Ligação Proteica , Titânio/química , Difração de Raios XRESUMO
Peripheral-nerve injuries are a common clinical problem and often result in long-term functional deficits. Reconstruction of peripheral-nerve defects is currently undertaken with nerve autografts. However, there is a limited availability of nerves that can be sacrificed and the functional recovery is never 100% satisfactory. We have previously shown that gene therapy with vascular endothelial growth factor (VEGF) significantly improved nerve regeneration, neuronal survival, and muscle activity. Our hypothesis is that granulocyte colony-stimulating factor (G-CSF) synergizes with VEGF to improve the functional outcome after sciatic nerve transection. The left sciatic nerves and the adjacent muscle groups of adult mice were exposed, and 50 or 100 µg (in 50 µl PBS) of VEGF and/or G-CSF genes was injected locally, just below the sciatic nerve, and transferred by electroporation. The sciatic nerves were transected and placed in an empty polycaprolactone (PCL) nerve guide, leaving a 3-mm gap to challenge nerve regeneration. After 6 weeks, the mice were perfused and the sciatic nerve, the dorsal root ganglion (DRG), the spinal cord and the gastrocnemius muscle were processed for light and transmission electron microscopy. Treated animals showed significant improvement in functional and histological analyses compared with the control group. However, the best results were obtained with the G-CSF+VEGF-treated animals: quantitative analysis of regenerated nerves showed a significant increase in the number of myelinated fibers and blood vessels, and the number of neurons in the DRG and motoneurons in the spinal cord was significantly higher. Motor function also showed that functional recovery occurred earlier in animals receiving G-CSF+VEGF-treatment. The gastrocnemius muscle showed an increase in weight and in the levels of creatine phosphokinase, suggesting an improvement of reinnervation and muscle activity. These results suggest that these two factors acted synergistically and optimized the nerve repair potential, improving regeneration after a transection lesion.
Assuntos
Fator Estimulador de Colônias de Granulócitos/uso terapêutico , Regeneração Nervosa/fisiologia , Recuperação de Função Fisiológica/fisiologia , Neuropatia Ciática/terapia , Fator A de Crescimento do Endotélio Vascular/uso terapêutico , Análise de Variância , Animais , Creatina Quinase/metabolismo , Modelos Animais de Doenças , Feminino , Gânglios Espinais/metabolismo , Gânglios Espinais/patologia , Gânglios Espinais/ultraestrutura , Fator Estimulador de Colônias de Granulócitos/genética , Humanos , Locomoção/genética , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Músculo Esquelético/ultraestrutura , Regeneração Nervosa/genética , Recuperação de Função Fisiológica/genética , Neuropatia Ciática/patologia , Medula Espinal/patologia , Medula Espinal/ultraestrutura , Transplante , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Galectin-3 (gal-3) is a ß-galactoside binding protein present in multivalent complexes with an extracellular matrix and with cell surface glycoconjugates. In this context, it can deliver a variety of intracellular signals to modulate cell activation, differentiation and survival. In the hematopoietic system, it was demonstrated that gal-3 is expressed in myeloid cells and surrounding stromal cells. Furthermore, exogenous and surface gal-3 drive the proliferation of myeloblasts in a granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent manner. Here, we investigated whether gal-3 regulates the formation of myeloid bone marrow compartments by studying galectin-3(-/-) mice (gal-3(-/-)) in the C57BL/6 background. The bone marrow histology of gal-3(-/-) mice was significantly modified and the myeloid compartments drastically disturbed, in comparison with wild-type (WT) animals. In the absence of gal-3, we found reduced cell density and diaphyseal disorders containing increased trabecular projections into the marrow cavity. Moreover, myeloid cells presented limited capacity to differentiate into mature myeloid cell populations in gal-3(-/-) mice and the number of hematopoietic multipotent progenitors was increased relative to WT animals. In addition, bone marrow stromal cells of these mice had reduced levels of GM-CSF gene expression. Taken together, our data suggest that gal-3 interferes with hematopoiesis, controlling both precursors and stromal cells and favors terminal differentiation of myeloid progenitors rather than proliferation.
Assuntos
Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Galectina 3/deficiência , Animais , Diferenciação Celular , Galectina 3/genética , Galectina 3/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Endogamia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Estromais/citologia , Células Estromais/metabolismoRESUMO
Cell cultures are often used to study bone mineralization; however, not all systems achieve a bone-like matrix formation. In this study, the mineralized matrix assembled in F-OST osteoblast cultures was analyzed, with the aim of establishing a novel model for bone mineralization. The ultrastructure of the cultures was investigated using scanning electron microscopy, atomic force microscopy, and transmission electron microscopy (TEM). The mineral phase was characterized using conventional and high-resolution TEM, energy-dispersive X-ray spectroscopy, X-ray diffraction, Fourier transform infrared spectroscopy, and solid-state (31)P and (1)H nuclear magnetic resonance. F-OST osteoblast cultures presented a clear nodular mineralization pattern. The chief features of the mineralizing nodules were globular accretions ranging from about 100 nm to 1.5 µm in diameter, loaded with needle-shaped crystallites. Accretions seemed to bud from the cell membrane, increase in size, and coalesce into larger ones. Arrays of loosely packed, randomly oriented collagen fibrils were seen along with the accretions. Mineralized fibrils were often observed, sometimes in close association with accretions. The mineral phase was characterized as a poorly crystalline hydroxyapatite. The Ca/P atomic ratio was 1.49 ± 0.06. The presence of OH was evident. The lattice parameters were a = 9.435 Å and c = 6.860 Å. The average crystallite size was 20 nm long and 10 nm wide. Carbonate substitutions were seen in phosphate and OH sites. Water was also found within the apatitic core. In conclusion, F-OST osteoblast cultures produce a bone-like matrix and may provide a good model for bone mineralization studies.
Assuntos
Matriz Óssea/ultraestrutura , Osteoblastos/citologia , Animais , Matriz Óssea/química , Calcificação Fisiológica , Células Cultivadas , Durapatita/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão , Osteoblastos/metabolismo , Osteoblastos/ultraestrutura , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Gangliosides have been extensively described to be involved in the proliferation and differentiation of various cell types, such including hematopoietic cells. Our previous studies on murine models of stroma-mediated myelopoiesis have shown that gangliosides are required for optimal capacity of stromal cells to support proliferation of myeloid precursor cells, being shed to the supernatant and selectively incorporated into myeloid cell membranes. Here we describe the effect of gangliosides on the specific granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced proliferation. For that, we used the monocytic FDC-P1 cell line, which is dependent upon GM-CSF for survival and proliferation. Cells were cultured in the presence of GM-CSF and exogenous gangliosides (GM3, GD1a or GM1) or in the absence of endogenous ganglioside synthesis by the use of a ceramide-synthase inhibitor, D-PDMP. We observed that exogenous addition of GD1a enhanced the GM-CSF-induced proliferation of the FDC-P1 cells. Also, we detected an increase in the expression of the α isoform of the GM-CSF receptor (GMRα) as well as of the transcription factor C/EBPα. On the contrary, inhibition of glucosylceramide synthesis was accompanied by a decrease in cell proliferation, which was restored upon the addition of exogenous GD1a. We also show a co-localization of GD1a and GMR by immunocytochemistry. Taken together, our results suggest for the first time that ganglioside GD1a play a role on the modulation of GM-CSF-mediated proliferative response, which might be of great interest not only in hematopoiesis, but also in other immunological processes, Alzheimer disease, alveolar proteinosis and wherever GM-CSF exerts its effects.
Assuntos
Gangliosídeos/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Animais , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Densitometria , Imunofluorescência , Gangliosídeo G(M3)/farmacologia , Gangliosídeos/biossíntese , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Microscopia Confocal , Morfolinas/farmacologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genética , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Solubilidade/efeitos dos fármacosRESUMO
AIMS: Recent studies have emphasized the beneficial effects of the vascular endothelial growth factor (VEGF) on neurone survival and Schwann cell proliferation. VEGF is a potent angiogenic factor, and angiogenesis has long been recognized as an important and necessary step during tissue repair. Here, we investigated the effects of VEGF on sciatic nerve regeneration. METHODS: Using light and electron microscopy, we evaluated sciatic nerve regeneration after transection and VEGF gene therapy. We examined the survival of the neurones in the dorsal root ganglia and in lumbar 4 segment of spinal cord. We also evaluated the functional recovery using the sciatic functional index and gastrocnemius muscle weight. In addition, we evaluated the VEGF expression by immunohistochemistry. RESULTS: Fluorescein isothiocyanate-dextran (FITC-dextran) fluorescence of nerves and muscles revealed intense staining in the VEGF-treated group. Quantitative analysis showed that the numbers of myelinated fibres and blood vessels were significantly higher in VEGF-treated animals. VEGF also increased the survival of neurone cell bodies in dorsal root ganglia and in spinal cord. The sciatic functional index and gastrocnemius muscle weight reached significantly higher values in VEGF-treated animals. CONCLUSION: We demonstrate a positive relationship between increased vascularization and enhanced nerve regeneration, indicating that VEGF administration can support and enhance the growth of regenerating nerve fibres, probably through a combination of angiogenic, neurotrophic and neuroprotective effects.
Assuntos
Terapia Genética/métodos , Regeneração Nervosa/fisiologia , Traumatismos dos Nervos Periféricos/terapia , Recuperação de Função Fisiológica/fisiologia , Nervo Isquiático/fisiologia , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Feminino , Camundongos , Músculo Esquelético/inervação , Músculo Esquelético/fisiologia , Traumatismos dos Nervos Periféricos/fisiopatologiaRESUMO
Chronic myeloid leukemia (CML) is rare in the pediatric population, accounting for 2-3 percent of childhood leukemia cases, with an annual incidence of one case per million children. The low toxicity profile of imatinib mesylate has led to its approval as a front-line therapy in children for whom interferon treatment has failed or who have relapsed after allogeneic transplantation. We describe the positive responses of 2 children (case 1 - from a 7-year-old male since May 2005; case 2 - from a 5-year-old female since June 2006) with Philadelphia-positive chromosome CML treated with imatinib (300 mg/day, orally) for up to 28 months, as evaluated by morphological, cytogenetic, and molecular approaches. Our patients are alive, are in the chronic phase, and are in continuous morphological complete remission.
Assuntos
Criança , Pré-Escolar , Feminino , Humanos , Masculino , Antineoplásicos/uso terapêutico , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Piperazinas/uso terapêutico , Pirimidinas/uso terapêutico , Neoplasia Residual , Resultado do TratamentoRESUMO
Chronic myeloid leukemia (CML) is rare in the pediatric population, accounting for 2-3% of childhood leukemia cases, with an annual incidence of one case per million children. The low toxicity profile of imatinib mesylate has led to its approval as a front-line therapy in children for whom interferon treatment has failed or who have relapsed after allogeneic transplantation. We describe the positive responses of 2 children (case 1 - from a 7-year-old male since May 2005; case 2 - from a 5-year-old female since June 2006) with Philadelphia-positive chromosome CML treated with imatinib (300 mg/day, orally) for up to 28 months, as evaluated by morphological, cytogenetic, and molecular approaches. Our patients are alive, are in the chronic phase, and are in continuous morphological complete remission.
Assuntos
Antineoplásicos/uso terapêutico , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Piperazinas/uso terapêutico , Pirimidinas/uso terapêutico , Benzamidas , Criança , Pré-Escolar , Feminino , Humanos , Mesilato de Imatinib , Masculino , Neoplasia Residual , Resultado do TratamentoRESUMO
B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most common malignancy in children. The Wnt signaling pathway has been found to be extensively involved in cancer onset and progression but its role in BCP-ALL remains controversial. We evaluate the role of the Wnt pathway in maintenance of BCP-ALL cells and resistance to chemotherapy. Gene expression profile revealed that BCP-ALL cells are potentially sensitive to modulation of Wnt pathway. Nalm-16 and Nalm-6 cell lines displayed low levels of canonical activation, as reflected by the virtually complete absence of total beta-catenin in Nalm-6 and the beta-catenin cell membrane distribution in Nalm-16 cell line. Canonical activation with Wnt3a induced nuclear beta-catenin translocation and led to BCP-ALL cell death. Lithium chloride (LiCl) also induced a cytotoxic effect on leukemic cells. In contrast, both Wnt5a and Dkk-1 increased Nalm-16 cell survival. Also, Wnt3a enhanced the in vitro sensitivity of Nalm-16 to etoposide (VP-16) while treatment with canonical antagonists protected leukemic cells from chemotherapy-induced cell death. Overall, our results suggest that canonical activation of the Wnt pathway may exerts a tumor suppressive effect, thus its inhibition may support BCP-ALL cell survival.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Etoposídeo/farmacologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamento farmacológico , Proteínas Wnt/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras B/fisiopatologia , Transporte Proteico , Transdução de Sinais , beta Catenina/metabolismoRESUMO
BACKGROUND AND OBJECTIVE: Hereditary gingival fibromatosis (HGF) is a benign disorder manifested by fibrous enlargement of keratinized gingiva. Evidence exists concerning the role of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in mediating normal and pathological processes, including HGF. Nevertheless, there are few and contradictory results on the analysis of MMPs and TIMPs transcripts in this pathology. MATERIAL AND METHODS: We studied the expression of the transcripts encoding MMP-1, -2 and -9 and TIMP-1 and -2 in gingival biopsies, obtained from three cases of HGF within a family, by semi-quantitative reverse transcriptase-polymerase chain reaction analysis. Samples were also processed for gelatin zymography. RESULTS: Except for MMP-9, most transcripts presented a higher level of expression in biopsies from HGF patients compared with control subjects. Accordingly, MMP-9 gelatinase activity was detected at low and similar levels among samples. Moreover, MMP-2 enzymatic activity was not detected at all. CONCLUSION: The mRNA expression of MMP-1 and -2 and TIMP-1 and -2 does not explain the gingival overgrowth presented in these cases. In addition, it is suggested that the gene expression of those molecules in the course of HGF is regulated at the translational or post-translational level.
Assuntos
Fibromatose Gengival/genética , Metaloproteinases da Matriz/análise , Inibidores Teciduais de Metaloproteinases/análise , Biópsia , Precursores Enzimáticos/análise , Precursores Enzimáticos/genética , Fibromatose Gengival/enzimologia , Regulação Enzimológica da Expressão Gênica/genética , Gengiva/enzimologia , Gengiva/patologia , Humanos , Metaloproteinase 1 da Matriz/análise , Metaloproteinase 1 da Matriz/genética , Metaloproteinase 2 da Matriz/análise , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 9 da Matriz/análise , Metaloproteinase 9 da Matriz/genética , Metaloproteinases da Matriz/genética , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Inibidor Tecidual de Metaloproteinase-1/análise , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-2/análise , Inibidor Tecidual de Metaloproteinase-2/genética , Inibidores Teciduais de Metaloproteinases/genética , Transcrição Gênica/genéticaRESUMO
The parasite Trichomonas vaginalis causes one of the most common non-viral sexually transmitted infections in humans. Mycoplasmas are frequently found with trichomonads but the consequences of this association are not yet known. In the present study, the effects of T. vaginalis harboring M. hominis on human vaginal epithelial cells and on MDCK cells are described. The results were analyzed by light, scanning and transmission electron microscopy, as well as using cell viability assays. There was an increase in the cytopathic effects on the epithelial cells infected with T. vaginalis associated with M. hominis compared to T. vaginalis alone. The epithelial cells exhibited an increase in the intercellular spaces, a lesser viability, and increased destruction provoked by the infected T. vaginalis. In addition, the trichomonads presented a higher amoeboid transformation rate and an intense phagocytic activity, characteristics of higher virulence behavior.
Assuntos
Células Epiteliais/parasitologia , Mycoplasma hominis/isolamento & purificação , Trichomonas vaginalis/microbiologia , Trichomonas vaginalis/patogenicidade , Animais , Antibacterianos/farmacologia , Células Cultivadas , Células Epiteliais/ultraestrutura , Feminino , Humanos , Testes de Sensibilidade Microbiana , Vagina/citologiaRESUMO
Galectin-3 (gal-3), a beta-galactoside-binding animal lectin, plays a role in cell-cell and cell-extracellular matrix interactions. Extracellular gal-3 modulates cell migration and adhesion in several physiological and pathological processes. Gal-3 is highly expressed in activated macrophages. Schistosoma mansoni eggs display a large amount of gal-3 ligands on their surface and elicit a well-characterized, macrophage-dependent, granulomatous, inflammatory reaction. Here, we have investigated the acute and chronic phases of S. mansoni infection in wild-type and gal-3(-/-) mice. In the absence of gal-3, chronic-phase granulomas were smaller in diameter, displaying thinner collagen fibers with a loose orientation. Schistosoma-infected gal-3(-/-) mice had remarkable changes in the monocyte/macrophage, eosinophil, and B lymphocyte subpopulations as compared with the infected wild-type mice. We observed a reduction of macrophage number, an increase in eosinophil absolute number, and a decrease in B lymphocyte subpopulation (B220(+/high) cells) in the periphery during the evolution of the disease in gal-3(-/-) mice. B lymphopenia was followed by an increase of plasma cell number in bone marrow, spleen, and mesenteric lymph nodes of the infected gal-3(-/-) mice. The plasma IgG and IgE levels also increased in these mice. Gal-3 plays a role in the organization, collagen distribution, and mobilization of inflammatory cells to chronic-phase granulomas, niches for extramedullary myelopoiesis, besides interfering with monocyte-to-macrophage and B cell-to-plasma cell differentiation.
Assuntos
Diferenciação Celular , Galectina 3/genética , Linfonodos/citologia , Esquistossomose/imunologia , Doença Aguda , Animais , Linfócitos B/citologia , Linfócitos B/fisiologia , Contagem de Células , Doença Crônica , Cruzamentos Genéticos , Eosinófilos/citologia , Eosinófilos/fisiologia , Feminino , Granuloma/etiologia , Granuloma/patologia , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Imuno-Histoquímica , Cinética , Fígado/patologia , Linfonodos/fisiologia , Linfopenia , Macrófagos/citologia , Macrófagos/fisiologia , Masculino , Mesentério/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/citologia , Monócitos/fisiologia , Plasmócitos/citologia , Schistosoma mansoni/imunologia , Schistosoma mansoni/patogenicidade , Esquistossomose/metabolismoRESUMO
Protein-energy malnutrition (PEM) decreases resistance to infection by impairing a number of physiological processes, including haematopoiesis. The aim of this study was to evaluate the microanatomical aspects of bone marrow (BM) in mice that were subjected to PEM, in particular, with respect to the components of the local extracellular matrix and the proliferative activity of haematopoietic cells. For this, histological, histochemical, immunohistochemical and ultrastructural techniques were used. Two-month old male Swiss mice were fed with a low-protein diet containing 4% protein and control mice fed a 20% protein diet. When the experimental group had attained a 25% loss of their original body weight, we collected the different biological samples. Malnourished mice had presented severe BM atrophy as well as a reduction in proliferating cell nuclear antigen and gelatinous degeneration. The malnourished mice had more fibronectin accretion in paratrabecular and endosteal regions and more laminin deposition in perisinusal sites than controls. Endosteal cell activation and hyperplasia were found, suggesting their participation in the process. Additionally, we have observed a decrease in the capacity of malnourished haematopoietic stroma to support the growth of haematopoietic stem cells (CD34+) in vitro. These findings point to a structural impairment of the haematopoietic microenvironments in mice with PEM, possibly hampering the interactions between cells and cellular signalling.
Assuntos
Medula Óssea/patologia , Hematopoese/fisiologia , Desnutrição Proteico-Calórica/patologia , Animais , Medula Óssea/ultraestrutura , Matriz Extracelular/patologia , Matriz Extracelular/ultraestrutura , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Desnutrição Proteico-Calórica/complicaçõesRESUMO
BACKGROUND/AIMS: Oxidative stress plays an important role in liver fibrosis. Under pathological conditions, hepatic stellate cells (HSC) undergo an activation process, developing a myofibroblast-like phenotype from the lipocyte phenotype. In this study, we determined the levels of oxidative stress and proliferation in different activation states of an experimental model of mouse HSC, the GRX cell line. These cells can be induced in vitro to display a more activated state or a quiescent phenotype. METHODS/RESULTS: We observed increased oxidative damage and higher levels of reactive oxygen species, measured by thiobarbituric acid reactive species and 2',7'-dichlorofluorescein diacetate, respectively, and diminished catalase activity in activated cells. Activation decreased proliferation and increased the number of cells in G2/M. Antioxidants N-acetylcysteine and Trolox varied in their capacity to correct the oxidative stress and proliferation status. CONCLUSIONS: The differences in physiological functions of stellate cell phenotypes suggest a relationship between oxidative stress levels and activation state.
Assuntos
Cirrose Hepática/fisiopatologia , Fígado/citologia , Fígado/fisiologia , Estresse Oxidativo/fisiologia , Acetilcisteína/farmacologia , Animais , Antioxidantes/farmacologia , Catalase/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Cromanos/farmacologia , Citocinas/farmacologia , Fígado/efeitos dos fármacos , Cirrose Hepática/patologia , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Fenótipo , Espécies Reativas de Oxigênio/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Vitamina E/farmacologiaRESUMO
Cell fate decisions are governed by a complex interplay between cell-autonomous signals and stimuli from the surrounding tissue. In vivo cells are connected to their neighbors and to the extracellular matrix forming a complex three-dimensional (3-D) microenvironment that is not reproduced in conventional in vitro systems. A large body of evidence indicates that mechanical tension applied to the cytoskeleton controls cell proliferation, differentiation and migration, suggesting that 3-D in vitro culture systems that mimic the in vivo situation would reveal biological subtleties. In hematopoietic tissues, the microenvironment plays a crucial role in stem and progenitor cell survival, differentiation, proliferation, and migration. In adults, hematopoiesis takes place inside the bone marrow cavity where hematopoietic cells are intimately associated with a specialized three 3-D scaffold of stromal cell surfaces and extracellular matrix that comprise specific niches. The relationship between hematopoietic cells and their niches is highly dynamic. Under steady-state conditions, hematopoietic cells migrate within the marrow cavity and circulate in the bloodstream. The mechanisms underlying hematopoietic stem/progenitor cell homing and mobilization have been studied in animal models, since conventional two-dimensional (2-D) bone marrow cell cultures do not reproduce the complex 3-D environment. In this review, we will highlight some of the mechanisms controlling hematopoietic cell migration and 3-D culture systems.
Assuntos
Animais , Humanos , Células da Medula Óssea/citologia , Técnicas de Cultura de Células/métodos , Movimento Celular/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Esferoides Celulares/fisiologia , Células Estromais/fisiologiaRESUMO
Cell fate decisions are governed by a complex interplay between cell-autonomous signals and stimuli from the surrounding tissue. In vivo cells are connected to their neighbors and to the extracellular matrix forming a complex three-dimensional (3-D) microenvironment that is not reproduced in conventional in vitro systems. A large body of evidence indicates that mechanical tension applied to the cytoskeleton controls cell proliferation, differentiation and migration, suggesting that 3-D in vitro culture systems that mimic the in vivo situation would reveal biological subtleties. In hematopoietic tissues, the microenvironment plays a crucial role in stem and progenitor cell survival, differentiation, proliferation, and migration. In adults, hematopoiesis takes place inside the bone marrow cavity where hematopoietic cells are intimately associated with a specialized three 3-D scaffold of stromal cell surfaces and extracellular matrix that comprise specific niches. The relationship between hematopoietic cells and their niches is highly dynamic. Under steady-state conditions, hematopoietic cells migrate within the marrow cavity and circulate in the bloodstream. The mechanisms underlying hematopoietic stem/progenitor cell homing and mobilization have been studied in animal models, since conventional two-dimensional (2-D) bone marrow cell cultures do not reproduce the complex 3-D environment. In this review, we will highlight some of the mechanisms controlling hematopoietic cell migration and 3-D culture systems.
Assuntos
Células da Medula Óssea/citologia , Técnicas de Cultura de Células/métodos , Movimento Celular/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Esferoides Celulares/fisiologia , Animais , Humanos , Células Estromais/fisiologiaRESUMO
Desmin is the main intermediate filament (IF) protein of muscle cells. In skeletal muscle, desmin IFs form a scaffold that interconnects the entire contractile apparatus with the subsarcolemmal cytoskeleton and cytoplasmic organelles. The interaction between desmin and the sarcolemma is mediated by a number of membrane proteins, many of which are Ca2+-sensitive. In the present study, we analyzed the effects of the Ca2+ chelator EGTA (1.75 mM) on the expression and distribution of desmin in C2C12 myoblasts grown in culture. We used indirect immunofluorescence microscopy and reverse transcription polymerase chain reaction (RT-PCR) to analyze desmin distribution and expression in C2C12 cells grown in the presence or absence of EGTA. Control C2C12 myoblasts showed a well-spread morphology after a few hours in culture and became bipolar when grown for 24 h in the presence of EGTA. Control C2C12 cells showed a dense network of desmin from the perinuclear region to the cell periphery, whereas EGTA-treated cells showed desmin aggregates in the cytoplasm. RT-PCR analysis revealed a down-regulation of desmin expression in EGTA-treated C2C12 cells compared to untreated cells. The present results suggest that extracellular Ca2+ availability plays a role in the regulation of desmin expression and in the spatial distribution of desmin IFs in myoblasts, and is involved in the generation and maintenance of myoblast cell shape.
Assuntos
Cálcio/metabolismo , Forma Celular/fisiologia , Desmina/metabolismo , Filamentos Intermediários/metabolismo , Músculo Esquelético/química , Mioblastos/fisiologia , Animais , Quelantes/farmacologia , Desmina/efeitos dos fármacos , Desmina/genética , Regulação para Baixo , Ácido Egtázico/farmacologia , Matriz Extracelular , Filamentos Intermediários/efeitos dos fármacos , Camundongos , Microscopia de Fluorescência , Coelhos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
In a previous study we monitored the distribution and phenotype expression of B1 cells during the evolution of experimental murine schistosomiasis mansoni and we proposed that the B1 cells were heterogeneous: a fraction which originated in the spleen and followed the migratory pathway to mesenteric ganglia, while the other was the resident peritoneal B1-cell pool. In the present study, we have addressed the question of whether these two B1-lymphocyte populations are involved in the production of the late Ig isotype IgE, which is present in high levels in schistosomal infection. Lymphocyte expression of surface markers and immunoglobulins were monitored by immunofluorescence flow cytometry. Both in the spleen and mesenteric ganglia, the B1 and B2 cells were induced to switch from IgM to IgE in the early Th2-dominated phase of the disease, with an increase of IgE in its later phases. Conversely, peritoneal B1-IgM+ switched to the remaining IgE+ present in high numbers in the peritoneal cavity throughout the disease. We correlated the efficient induction of the expression of late Ig isotypes by B1 cells with high levels of inflammatory cytokines due to the intense host response to the presence of worms and their eggs in the abdominal cavity. In conclusion, B1 cells have a different switch behavior from IgM to IgE indicating that these cell sub-populations depend on the microenvironment.
Assuntos
Linfócitos B/imunologia , Imunoglobulina E/metabolismo , Esquistossomose mansoni/imunologia , Animais , Antígenos de Superfície/imunologia , Linfócitos B/metabolismo , Modelos Animais de Doenças , Feminino , Citometria de Fluxo , Imunofluorescência , Imunoglobulina E/imunologia , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos C3H , Cavidade Peritoneal/citologiaRESUMO
Desmin is the main intermediate filament (IF) protein of muscle cells. In skeletal muscle, desmin IFs form a scaffold that interconnects the entire contractile apparatus with the subsarcolemmal cytoskeleton and cytoplasmic organelles. The interaction between desmin and the sarcolemma is mediated by a number of membrane proteins, many of which are Ca2+-sensitive. In the present study, we analyzed the effects of the Ca2+ chelator EGTA (1.75 mM) on the expression and distribution of desmin in C2C12 myoblasts grown in culture. We used indirect immunofluorescence microscopy and reverse transcription polymerase chain reaction (RT-PCR) to analyze desmin distribution and expression in C2C12 cells grown in the presence or absence of EGTA. Control C2C12 myoblasts showed a well-spread morphology after a few hours in culture and became bipolar when grown for 24 h in the presence of EGTA. Control C2C12 cells showed a dense network of desmin from the perinuclear region to the cell periphery, whereas EGTA-treated cells showed desmin aggregates in the cytoplasm. RT-PCR analysis revealed a down-regulation of desmin expression in EGTA-treated C2C12 cells compared to untreated cells. The present results suggest that extracellular Ca2+ availability plays a role in the regulation of desmin expression and in the spatial distribution of desmin IFs in myoblasts, and is involved in the generation and maintenance of myoblast cell shape.
Assuntos
Animais , Camundongos , Coelhos , Cálcio/metabolismo , Forma Celular/fisiologia , Desmina/metabolismo , Filamentos Intermediários/metabolismo , Músculo Esquelético/química , Mioblastos/fisiologia , Quelantes/farmacologia , Regulação para Baixo , Desmina/efeitos dos fármacos , Desmina/genética , Matriz Extracelular , Ácido Egtázico/farmacologia , Filamentos Intermediários/efeitos dos fármacos , Microscopia de Fluorescência , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
In a previous study we monitored the distribution and phenotype expression of B1 cells during the evolution of experimental murine schistosomiasis mansoni and we proposed that the B1 cells were heterogeneous: a fraction which originated in the spleen and followed the migratory pathway to mesenteric ganglia, while the other was the resident peritoneal B1-cell pool. In the present study, we have addressed the question of whether these two B1-lymphocyte populations are involved in the production of the late Ig isotype IgE, which is present in high levels in schistosomal infection. Lymphocyte expression of surface markers and immunoglobulins were monitored by immunofluorescence flow cytometry. Both in the spleen and mesenteric ganglia, the B1 and B2 cells were induced to switch from IgM to IgE in the early Th2-dominated phase of the disease, with an increase of IgE in its later phases. Conversely, peritoneal B1-IgM+ switched to the remaining IgE+ present in high numbers in the peritoneal cavity throughout the disease. We correlated the efficient induction of the expression of late Ig isotypes by B1 cells with high levels of inflammatory cytokines due to the intense host response to the presence of worms and their eggs in the abdominal cavity. In conclusion, B1 cells have a different switch behavior from IgM to IgE indicating that these cell sub-populations depend on the microenvironment.