RESUMO
Deep second and third degree burns treatment requires fibroblasts, keratinocytes and other skin cells in order to grow new dermis and epidermis. Cells can proliferate, secrete growth factors and extracellular matrix required to repair the damaged tissue. Radiosterilized human amnion and radiosterilized pig skin have been used as natural origin skin dressings for burned patients. Adipose-derived mesenchymal stem cells can differentiate into fibroblasts and keratinocytes and improve wound-healing progress. These cells can stimulate vascular tissue formation, release growth factors, synthetize new extracellular matrix and immunoregulate other cells. In this study, we developed mesenchymal stem cells-cellularized skin substitutes based from radiosterilized human amnion or pig skin. Third-degree burns were induced in mice animal models to evaluate the effect of cellularized skin substitutes on burn wound healing. Mesenchymal phenotype was immunophenotypically confirmed by flow cytometry and cell viability was close to 100%. Skin recovery was evaluated in burned mice after seven and fourteen days post-coverage with cellularized and non-cellularized sustitutes. Histological techniques and immunofluorescence were used to evaluate re-epithelization and type I collagen deposition. We determined that cellularized-human amnion or cellularized-pig skin in combination with mesenchymal stem cells improve extracellular matrix deposition. Both cellularized constructs increase detection of type I collagen in newly formed mouse skin and can be potentially used as skin coverage for further clinical treatment of burned patients.
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Platelet rich plasma (PRP) is used to speed up tissue repair. Despite its widespread use, the therapeutic application of PRP generates controversies in clinical results due to the variability in methods of obtaining the different preparations and differences between the components of different types of PRP, so it's recommended to mention the type of platelet preparation used. In this article, we describe technical and biologics characteristics of our platelet product, and we compare them to different commercial preparations described in order to validate their clinical use. Our results determine that the preparation can be considered a platelet rich plasma with biological activity in vivo and in vitro, which supports its use as a valid therapeutic tool, alternative to products currently available in Regenerative Medicine. (AU)
Assuntos
Humanos , Medicina Regenerativa/tendências , Plasma Rico em Plaquetas , Terapia Baseada em Transplante de Células e TecidosRESUMO
We report the synthesis and in vitro evaluation of dual-function chitosan-silver nanoparticles (CTS-AgNPs) films with potential applications as wound dressings. We attempted to formulate nanocomposite films with appropriate AgNPs concentrations to simultaneously display antibacterial activity and suitability for cell culture. Nanocomposites were obtained by CTS-mediated in situ chemical reduction of AgNO3. Circular-shape AgNPs (sizes ca. 7-50â¯nm) well distributed within the CTS matrices were obtained in concentrations from 0.018 to 0.573â¯wt%. Efficacy (bacteriostatic and bactericidal properties) of CTS-AgNPs films to decrease planktonic and biofilm bacterial growth was AgNPs concentration- and bacteria strain-dependent. Films showed significant antibacterial activity against Gram-negative E. coli and P. aeruginosa and Gram-positive S. aureus. Antibacterial activity against S. epidermidis was moderated. Films suitability for cell culture was characterized using primary human fibroblasts (HF). HF displayed cell viability higher than 90% and the characteristic fusiform morphology of adhered fibroblast upon culture on films with AgNPs concentrationâ¯≤â¯0.036â¯wt%. HF cultured on these films also showed positive expression of tropoelastin, procollagen type I and Ki-67, characteristic proteins of extracellular matrix and proliferative cells, respectively. In vitro assays demonstrated that cytocompatibility/antibacterial properties decreased/increased as silver concentration increased, suggesting that CTS-AgNPS nanocomposite films with ≈0.04-0.20â¯wt% might be considered as potential temporary dual-function wound dressings.
Assuntos
Bandagens , Quitosana/farmacologia , Nanocompostos/química , Prata/farmacologia , Ferimentos e Lesões/terapia , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Bactérias/crescimento & desenvolvimento , Sobrevivência Celular/efeitos dos fármacos , Colágeno Tipo I/metabolismo , Contagem de Colônia Microbiana , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Humanos , Antígeno Ki-67/metabolismo , Masculino , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Nanocompostos/ultraestrutura , Espectrofotometria Ultravioleta , Tropoelastina/metabolismo , ÁguaRESUMO
PROBLEM ADDRESSED: Shiga toxin-producing Escherichia coli (STEC) are a group of bacteria responsible for food-associated diseases. Clinical features include a wide range of symptoms such as diarrhea, hemorrhagic colitis and the hemolytic uremic syndrome (HUS), a life-threatening condition. OBJECTIVE: Our group has observed that animals naturally colonized with STEC strains of unknown serotype were not efficiently colonized with E. coli O157:H7 after experimental infection. In order to assess the basis of the interference, three STEC strains were isolated from STEC persistently-colonized healthy cattle from a dairy farm in Buenos Aires, Argentina. METHODS AND RESULTS: The three isolated strains are E. coli O22:H8 and carry the stx1 and stx2d genes. The activatable activity of Stx2d was demonstrated in vitro. The three strains carry the adhesins iha, ehaA and lpfO113. E. coli O22:H8 formed stronger biofilms in abiotic surface than E. coli O157:H7 (eae+, stx2+) and displayed a more adherent phenotype in vitro towards HeLa cells. Furthermore, when both serotypes were cultured together O22:H8 could reduce O157:H7 adherence in vitro. When calves were intragastrically pre-challenged with 108 CFU of a mixture of the three STEC strains and two days later challenged with the same dose of the strain E. coli O157:H7 438/99, the shedding of the pathogen was significantly reduced. CONCLUSIONS: These results suggest that E. coli O22:H8, a serotype rarely associated with human illness, might compete with O157:H7 at the bovine recto-anal junction, making non-O157 carrying-calves less susceptible to O157:H7 colonization and shedding of the bacteria to the environment.
Assuntos
Aderência Bacteriana/fisiologia , Doenças dos Bovinos/microbiologia , Infecções por Escherichia coli/veterinária , Escherichia coli Shiga Toxigênica/fisiologia , Animais , Antibacterianos/farmacologia , Biofilmes , Bovinos , Chlorocebus aethiops , Farmacorresistência Bacteriana , Infecções por Escherichia coli/microbiologia , Escherichia coli O157 , Feminino , Regulação Bacteriana da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Escherichia coli Shiga Toxigênica/genética , Células Vero , Virulência , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
The external ear is composed of elastic cartilage. Microtia is a congenital malformation of the external ear that involves a small reduction in size or a complete absence. The aim of tissue engineering is to regenerate tissues and organs clinically implantable based on the utilization of cells and biomaterials. Remnants from microtia represent a source of cells for auricular reconstruction using tissue engineering. To examine the macromolecular architecture of microtia cartilage and behavior of chondrocytes, in order to enrich the knowledge of this type of cartilage as a cell reservoir. Auricular cartilage remnants were obtained from pediatric patients with microtia undergoing reconstructive procedures. Extracellular matrix composition was characterized using immunofluorescence and histological staining methods. Chondrocytes were isolated and expanded in vitro using a mechanical-enzymatic protocol. Chondrocyte phenotype was analyzed using qualitative PCR. Microtia cartilage preserves structural organization similar to healthy elastic cartilage. Extracellular matrix is composed of typical cartilage proteins such as type II collagen, elastin and proteoglycans. Chondrocytes displayed morphological features similar to chondrocytes derived from healthy cartilage, expressing SOX9, COL2 and ELN, thus preserving chondral phenotype. Cell viability was 94.6 % during in vitro expansion. Elastic cartilage from microtia has similar characteristics, both architectural and biochemical to healthy cartilage. We confirmed the suitability of microtia remnant as a reservoir of chondrocytes with potential to be expanded in vitro, maintaining phenotypical features and viability. Microtia remnants are an accessible source of autologous cells for auricular reconstruction using tissue engineering strategies.
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Condrócitos/patologia , Microtia Congênita/patologia , Cartilagem da Orelha/patologia , Procedimentos de Cirurgia Plástica/métodos , Engenharia Tecidual/métodos , Proliferação de Células , Separação Celular , Forma Celular , Criança , Pré-Escolar , Colágeno Tipo II/metabolismo , Elastina/metabolismo , Matriz Extracelular/metabolismo , Humanos , FenótipoRESUMO
Escherichia coli O157:H7 is the main causative agent of haemolytic uremic syndrome. Cattle are the main reservoir of these bacteria, and have been shown to develop immune response to colonization. Our aim was to investigate the faecal shedding pattern of E. coli O157:H7 in calves challenged intragastrically with either 10(8) or 10(10) CFU, as well as the ability of specific preexisting antibodies to reduce shedding of the pathogen. Shedding was analysed by direct counting as well as enrichment of rectoanal mucosal swabs. Statistical analysis was performed using a linear model for repeated measures with and without the inclusion of preexisting antibodies against the carboxy-terminal fraction of intimin-γ (γ-intimin C280) as a covariable. Results suggest that there is a statistical difference in the area under the shedding curves between both doses for 14 as well as 28 days after challenge (p = 0.0069 and 0.0209, resp.). This difference is increased when the prechallenge antibodies are taken into account (p = 0.0056 and 0.0185). We concluded that the bacterial dose influences shedding on calves experimentally challenged and that preexisting antibodies against E. coli O157:H7 γ-intimin C280 could partially reduce faecal excretion.
Assuntos
Derrame de Bactérias/imunologia , Infecções por Escherichia coli , Escherichia coli O157 , Proteínas de Escherichia coli/imunologia , Interações Hospedeiro-Patógeno/imunologia , Fosfoproteínas/imunologia , Animais , Anticorpos Antibacterianos/sangue , Bovinos , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/microbiologia , Modelos Animais de Doenças , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/veterinária , Escherichia coli O157/química , Escherichia coli O157/imunologia , Escherichia coli O157/patogenicidade , MasculinoRESUMO
Tissue engineering with the use of biodegradable and biocompatible scaffolds is an interesting option for ear repair. Chitosan-Polyvinyl alcohol-Epichlorohydrine hydrogel (CS-PVA-ECH) is biocompatible and displays appropriate mechanical properties to be used as a scaffold. The present work, studies the potential of CS-PVA-ECH scaffolds seeded with chondrocytes to develop elastic cartilage engineered-neotissues. Chondrocytes isolated from rabbit and swine elastic cartilage were independently cultured onto CS-PVA-ECH scaffolds for 20 days to form the appropriate constructs. Then, in vitro cell viability and morphology were evaluated by calcein AM and EthD-1 assays and Scanning Electron Microscopy (SEM) respectively, and the constructs were implanted in nu/nu mice for four months, in order to evaluate the neotissue formation. Histological analysis of the formed neotissues was performed by Safranin O, Toluidine blue (GAG's), Verhoeff-Van Gieson (elastic fibers), Masson's trichrome (collagen) and Von Kossa (Calcium salts) stains and SEM. Results indicate appropriate cell viability, seeded with rabbit or swine chondrocyte constructs; nevertheless, upon implantation the constructs developed neotissues with different characteristics depending on the animal species from which the seeded chondrocytes came from. Neotissues developed from swine chondrocytes were similar to auricular cartilage, while neotissues from rabbit chondrocytes were similar to hyaline cartilage and eventually they differentiate to bone. This result suggests that neotissue characteristics may be influenced by the animal species source of the chondrocytes isolated.
Assuntos
Materiais Biocompatíveis , Diferenciação Celular/fisiologia , Quitosana , Condrócitos/fisiologia , Cartilagem da Orelha/citologia , Cartilagem da Orelha/crescimento & desenvolvimento , Hidrogéis , Alicerces Teciduais , Animais , Etídio/análogos & derivados , Cartilagem Hialina/citologia , Cartilagem Hialina/crescimento & desenvolvimento , Camundongos , Osteogênese/fisiologia , Álcool de Polivinil , Coelhos , Sus scrofa , SuínosRESUMO
INTRODUCTION: Symptomatic or asymptomatic Shiga toxin producing Escherichia coli (STEC) infections during early pregnancy may cause maternal or fetal damage mediated by Shiga toxin type 2 (Stx2). The aim of this study is to elucidate the mechanisms responsible for early pregnancy loss in rats treated with Stx2. METHODS: Sprague Dawley pregnant rats were intraperitoneally injected at day 8 of gestation with a sublethal dose (0.5 ng of Stx2/g of total body weight, 250 µl) of purified Stx2. Control rats were injected with the same volume of PBS. The expression of globotriaosylceramide (Gb3) glycosphingolipid receptor for Stx2 was evaluated by thin-layer chromatography (TLC). Regions of hypoxia in decidual tissue were determined by pimonidazole immunohistochemistry and vascular endothelial growth factor (VEGF) expression by Western blot and immunohistochemistry. Tumor necrosis factor-alpha (TNF-α) levels in serum and decidual tissue were evaluated by ELISA. Serum progesterone levels were determined by RIA. RESULTS: Decidual tissue from both, control and Stx2-treated rats showed similar expression of Gb3 receptor. Intrauterine growth restriction was observed in Stx2-treated rats, associated with hypoxia and an increase of decidual TNF-α levels. Decrease of serum progesterone levels and decidual VEGF expression were also demonstrated. DISCUSSION: Our findings indicate that Stx2 reaches the uteroplacental unit, binds Gb3 and triggers damage in decidual tissue. Poor oxygen supply accompanied with damage in the uteroplacental unit and inflammation could be responsible for the early pregnancy loss. Decrease in the pregnancy protective factors, serum progesterone and local VEGF, may contribute to the pregnancy loss.
Assuntos
Aborto Espontâneo/patologia , Hipóxia/patologia , Inflamação/patologia , Toxina Shiga II , Aborto Espontâneo/induzido quimicamente , Aborto Espontâneo/metabolismo , Animais , Feminino , Hipóxia/metabolismo , Inflamação/metabolismo , Gravidez , Ratos , Ratos Sprague-Dawley , Triexosilceramidas/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Introducción: La deficiencia de vitamina B12 es una de las complicaciones del vegetarianismo y sus variantes. Los lactantes de madres vegetarianas tienen mayor riesgo de deficiencia y son más lábiles a sus efectos. Objetivo: Relatar un caso para alertar sobre la importancia de la sospecha de deficiencia de vitamina B12 en hijos de madres con hábito vegetariano. Caso clínico: Se presenta una lactante de 12 meses, hija de una mujer vegetariana de larga data, que se presenta con compromiso neurológico y hematológico por déficit de vitamina B12. Luego de un corto período de administración de cianocobalamina parenteral y apoyo enteral nutricional, evoluciona con una notoria mejoría clínica y de laboratorio, aunque aún presenta retraso del desarrollo residual. Conclusiones: La deficiencia de vitamina B12 no es frecuentemente sospechada por el pediatra en lactantes sanos. Sus manifestaciones clínicas pueden ser tan inespecíficas, como la apatía, el rechazo alimentario y el deterioro progresivo del desarrollo psicomotor. Una anamnesis nutricional breve realizada a la madre (con mayor énfasis en aquellas vegetarianas) para estimar sus reservas en el periodo previo, durante y después del parto pueden ser fundamentales para detectar el riesgo déficit de esta vitamina en el niño pequeño.
Introduction: Vitamin B12 deficiency is extremely common in strict vegetarians and their variants. Infants of vegetarian mothers have a higher risk of deficiency and are more prone to its effects. Objective: To report a case in order to warn people about the importance of suspected vitamin B12 deficiency in children of vegetarian mothers. Case report: A 12-month old infant, daughter of a longtime vegetarian woman, who presented neurological and hematological compromise due to vitamin B12 deficiency, is discussed. After a short period of parenteral administration of cyanocobalamin and enteral nutrition, the patient evolved with clinical and laboratory improvement, although she still had residual development delay. Conclusions: Vitamin B12 deficiency is often not suspected by the pediatrician in healthy infants. Clinical manifestations can be nonspecific, such as apathy, food refusal and progressive impairment of psychomotor development. A nutritional anamnesis performed on the mother (with great emphasis on those strict vegetarians) to estimate her reserves in the period prior to, during and after delivery can be critical to detect the risk of this vitamin deficiency in young children.
Assuntos
Humanos , Adulto , Feminino , Lactente , /complicações , /etiologia , Dieta Vegetariana/efeitos adversos , Anemia Megaloblástica/etiologia , /tratamento farmacológico , Deficiências do Desenvolvimento/etiologia , Nutrição Parenteral , /administração & dosagemRESUMO
Escherichia coli O157:H7 is responsible for severe intestinal disease and hemolytic uremic syndrome (HUS), a serious systemic complication which particularly affects children. Cattle are the primary reservoir for E. coli O157:H7 and the main source of infection for humans. In this study, we evaluated the ability of transferred maternal colostral antibodies against γ-Intimin C280 and EspB, to protect young weaned calves from E. coli O157:H7 infection. Hyperimmune colostra were obtained by immunization of pregnant cows with a mix of the mentioned antigens. All vaccinated cows mounted a significant IgG response against γ-Intimin C280, and EspB in sera and colostra. Colostrum-fed calves also exhibited high serum IgG titers against γ-Intimin C280 and EspB along with a rise in mucosal γ-Intimin C280-specific IgG antibodies at recto-anal junction and ileum. Additionally, 70 day-old calves received a challenge with E. coli O157:H7 but no reduction in total bacterial shedding or frequency of E. coli O157:H7 excretion from these calves was observed. Most tissue samples showed granulocyte focal infiltrations of the lamina propria and enterocyte erosion. In conclusion, up to the 70th day, the passively acquired γ-Intimin-C280 and EspB-IgG antibodies present in sera and recto-anal mucosa reached a titer insufficient to reduce EHEC O157 shedding and damages of experimentally inoculated young calves.
Assuntos
Adesinas Bacterianas/imunologia , Anticorpos Antibacterianos/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Doenças dos Bovinos/prevenção & controle , Colostro/imunologia , Infecções por Escherichia coli/veterinária , Proteínas de Escherichia coli/imunologia , Animais , Anticorpos Antibacterianos/sangue , Derrame de Bactérias , Bovinos , Doenças dos Bovinos/imunologia , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/prevenção & controle , Escherichia coli O157 , Vacinas contra Escherichia coli/administração & dosagem , Fezes/microbiologia , Feminino , Imunidade Materno-Adquirida , Imunidade nas Mucosas , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , GravidezRESUMO
INTRODUCTION: Vitamin B12 deficiency is extremely common in strict vegetarians and their variants. Infants of vegetarian mothers have a higher risk of deficiency and are more prone to its effects. OBJECTIVE: To report a case in order to warn people about the importance of suspected vitamin B12 deficiency in children of vegetarian mothers. CASE REPORT: A 12-month old infant, daughter of a longtime vegetarian woman, who presented neurological and hematological compromise due to vitamin B12 deficiency, is discussed. After a short period of parenteral administration of cyanocobalamin and enteral nutrition, the patient evolved with clinical and laboratory improvement, although she still had residual development delay. CONCLUSIONS: Vitamin B12 deficiency is often not suspected by the pediatrician in healthy infants. Clinical manifestations can be nonspecific, such as apathy, food refusal and progressive impairment of psychomotor development. A nutritional anamnesis performed on the mother (with great emphasis on those strict vegetarians) to estimate her reserves in the period prior to, during and after delivery can be critical to detect the risk of this vitamin deficiency in young children.
Assuntos
Deficiências do Desenvolvimento/etiologia , Dieta Vegetariana/efeitos adversos , Deficiência de Vitamina B 12/complicações , Vitamina B 12/administração & dosagem , Deficiências do Desenvolvimento/fisiopatologia , Nutrição Enteral/métodos , Feminino , Humanos , Lactente , Mães , Gravidez , Deficiência de Vitamina B 12/diagnóstico , Deficiência de Vitamina B 12/etiologiaRESUMO
Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a major cause of intestinal disease and hemolytic uremic syndrome, a serious systemic complication that particularly affects children. Cattle are primary reservoirs for EHEC O157:H7 and the main source of infection for humans. Vaccination of cattle with different combinations of bacterial virulence factors has shown efficacy in decreasing EHEC O157:H7 shedding. It is, therefore, important to demonstrate whether vaccination of pregnant cows with EHEC O157:H7 induces high titers of transferable antibodies to avoid early colonization of calves by the bacteria. In this study we evaluated the ability of EspA, EspB, the C-terminal fragment of 280 amino acids of γ-intimin (γ-intimin C280) and inactivated Shiga toxin (Stx) 2 proteins to induce specific antibodies in colostrum and their passive transference to colostrum-fed calves. Friesian pregnant cows immunized by the intramuscular route mounted significantly high serum and colostrum IgG responses against EspB and γ-intimin C280 that were efficiently transferred to their calves. Antibodies to EspB and γ-intimin C280 were detected in milk samples of vaccinated cows at d 40 postparturition. Significant Stx2-neutralizing titers were also observed in colostrum from Stx2-vaccinated cows and sera from colostrum-fed calves. The results presented showed that bovine colostrum with increased levels of antibodies against EHEC O157:H7 may be obtained by systemic immunization of pregnant cows, and that these specific antibodies are efficiently transferred to newborn calves by feeding colostrum. Hyperimmune colostrum and milk may be an alternative to protect calves from early colonization by EHEC O157:H7 and a possible key source of antibodies to block colonization and toxic activity of this bacterium.
Assuntos
Adesinas Bacterianas/farmacologia , Anticorpos Antibacterianos/imunologia , Proteínas da Membrana Bacteriana Externa/farmacologia , Bovinos/imunologia , Colostro/imunologia , Escherichia coli O157/imunologia , Proteínas de Escherichia coli/farmacologia , Imunidade Materno-Adquirida/imunologia , Toxina Shiga II/farmacologia , Vacinação/veterinária , Adesinas Bacterianas/imunologia , Animais , Animais Recém-Nascidos/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas de Escherichia coli/imunologia , Feminino , Gravidez , Toxina Shiga II/imunologiaRESUMO
Pets can be reservoirs of Shiga toxin-producing Escherichia coli (STEC) strains. The aim of this study was to examine nine strains belonging to several serotypes (O91:H21, O91:H16, O178:H19, O8:H19, O22:H8, O22:HNT, ONT:H8), previously recovered from cats or dogs. To this end, we assessed a set of additional virulence genes (stx(2) subtype, subAB, ehxA, eae and saa), cytotoxic activity, and genetic relationships with strains isolated from cattle, meat and humans using pulsed-field gel electrophoresis (PFGE). Most of the isolates carried the stx(2) and/or stx(2vh-b) sequences, while only the O91:H21 isolate presented the mucus-activatable stx(2d) variant, as confirmed by sequencing the genes of subunits A and B. All the strains showed cytotoxic activity in cultured cells. One of the two O178:H19, selected for its high level of cytotoxicity in Vero cells, showed the ability to cause functional alterations in the human colon mucosa in vitro. None of the strains possessed the subAB, eae or saa genes and only the strains belonging to serotype O8:H19 carried the ehxA gene. The isolates shared 90-100% similarity by PFGE to epidemiologically unrelated strains of the corresponding serotypes recovered from cattle, meat or humans. Our results demonstrate that dogs and cats may have a role in the infection of humans by STEC, probably serving as a vehicle for bovine strains in the cycle of human infection, and thus emphasize the health risks for owners and their families.
Assuntos
Gatos/microbiologia , Cães/microbiologia , Escherichia coli Shiga Toxigênica/classificação , Sequência de Aminoácidos , Animais , Argentina , Bovinos/microbiologia , Chlorocebus aethiops , Eletroforese em Gel de Campo Pulsado , Proteínas de Escherichia coli/genética , Fezes/microbiologia , Humanos , Carne/microbiologia , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Sorotipagem , Escherichia coli Shiga Toxigênica/patogenicidade , Células Vero , Fatores de Virulência/genéticaRESUMO
We studied five unrelated Mexican carriers of the Spanish (δß)(0)-thalassemia [(δß)(0)-thal] mutation to characterize the size of the deletion, the 5' and 3' breakpoints and the 5' ß-globin haplotype. Sequence analysis revealed the presence of an 89,548 bp deletion. The δ- and ß-globin genes, two olfactory receptor genes (OR51V1 and OR52A1) and two pseudogenes (OR52Z1P and OR51A1P) were deleted. The 5' breakpoint was located at the same position as previously reported, and the 3' breakpoint was situated 7.0 kb downstream of OR52A1 and 11.7 kb upstream of OR52A5. The Spanish (δß)(0)-thal allele was associated with the 5' haplotype 2 [- + + - +] in the studied patients. Because this mutation is relatively frequent in Spain, and the Mexican population contains a high level of Spanish genetic background, we propose that the mutation in both populations share a common ancestral origin.
Assuntos
Americanos Mexicanos/genética , Deleção de Sequência , Talassemia/genética , Globinas beta/genética , Globinas delta/genética , Alelos , Sequência de Bases , Pontos de Quebra do Cromossomo , Haplótipos , Heterozigoto , Humanos , México/epidemiologia , Dados de Sequência Molecular , Mutação , Reação em Cadeia da Polimerase , Pseudogenes , Receptores Odorantes/deficiência , Receptores Odorantes/genética , Análise de Sequência de DNA , Espanha/etnologia , Talassemia/etnologia , Globinas beta/deficiência , Globinas delta/deficiênciaRESUMO
Intracellular calcium levels ([Ca2+]i) and glucose uptake are central to cardiomyocyte physiology, yet connections between them have not been studied. We investigated whether insulin regulates [Ca2+]i in cultured cardiomyocytes, the participating mechanisms, and their influence on glucose uptake via SLC2 family of facilitative glucose transporter 4 (GLUT4). Primary neonatal rat cardiomyocytes were preloaded with the Ca2+ fluorescent dye fluo3-acetoxymethyl ester compound (AM) and visualized by confocal microscopy. Ca2+ transport pathways were selectively targeted by chemical and molecular inhibition. Glucose uptake was assessed using [3H]2-deoxyglucose, and surface GLUT4 levels were quantified in nonpermeabilized cardiomyocytes transfected with GLUT4-myc-enhanced green fluorescent protein. Insulin elicited a fast, two-component, transient increase in [Ca2+]i. Nifedipine and ryanodine prevented only the first component. The second one was reduced by inositol-1,4,5-trisphosphate (IP3)-receptor-selective inhibitors (xestospongin C, 2 amino-ethoxydiphenylborate), by type 2 IP3 receptor knockdown via small interfering RNA or by transfected Gßγ peptidic inhibitor ßARKct. Insulin-stimulated glucose uptake was prevented by bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid-AM, 2-amino-ethoxydiphenylborate, and ßARK-ct but not by nifedipine or ryanodine. Similarly, insulin-dependent exofacial exposure of GLUT4-myc-enhanced green fluorescent protein was inhibited by bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid-AM and xestospongin C but not by nifedipine. Phosphatidylinositol 3-kinase and Akt were also required for the second phase of Ca2+ release and GLUT4 translocation. Transfected dominant-negative phosphatidylinositol 3-kinase γ inhibited the latter. In conclusion, in primary neonatal cardiomyocytes, insulin induces an important component of Ca2+ release via IP3 receptor. This component signals to glucose uptake via GLUT4, revealing a so-far unrealized contribution of IP3-sensitive Ca2+ stores to insulin action. This pathway may influence cardiac metabolism in conditions yet to be explored in adult myocardium.
Assuntos
Transportador de Glucose Tipo 4/metabolismo , Glucose/farmacocinética , Receptores de Inositol 1,4,5-Trifosfato/fisiologia , Inositol 1,4,5-Trifosfato/fisiologia , Insulina/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Cálcio/metabolismo , Células Cultivadas , Glucose/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Inositol 1,4,5-Trifosfato/farmacologia , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Miócitos Cardíacos/metabolismo , Transporte Proteico/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Regulação para Cima/efeitos dos fármacosRESUMO
BACKGROUND/AIMS: In Argentina, hemolytic uremic syndrome (HUS) constitutes the most frequent cause of acute renal failure in children. The aim of our study was to analyze the early tubular response under the effect of Shiga toxin type 2 (Stx2) in a rat experimental model of HUS. METHODS: Adult male Sprague-Dawley rats were injected intraperitoneally with culture supernatant from recombinant Escherichia coli expressing Stx2. Functional, histological, immunohistochemical and Western blot studies were performed at 48 h postinoculation. RESULTS: Renal tubules showed the loss of the epithelial markers E-cadherin and ß-catenin, and an increase in transforming growth factor-ß1 expression. We detected the expression of α-smooth muscle actin in the interstitium and fibrosis in the periglomerular areas. CONCLUSION: Our results indicate that the early tubular response to the effects of Stx2 is related to an immunophenotype change of tubular cells and the presence of mild fibrosis in the interstitium.
Assuntos
Síndrome Hemolítico-Urêmica/patologia , Síndrome Hemolítico-Urêmica/fisiopatologia , Túbulos Renais/patologia , Actinas/análise , Animais , Caderinas/análise , Células Epiteliais/química , Células Epiteliais/efeitos dos fármacos , Imuno-Histoquímica , Túbulos Renais/química , Masculino , Modelos Animais , Ratos , Ratos Sprague-Dawley , Toxina Shiga II , Fator de Crescimento Transformador beta1/análise , beta Catenina/análiseRESUMO
Hyperosmotic stress promotes rapid and pronounced apoptosis in cultured cardiomyocytes. Here, we investigated if Ca(2+) signals contribute to this response. Exposure of cardiomyocytes to sorbitol [600 mosmol (kg water)(-1)] elicited large and oscillatory intracellular Ca(2+) concentration increases. These Ca(2+) signals were inhibited by nifedipine, Cd(2+), U73122, xestospongin C and ryanodine, suggesting contributions from both Ca(2+) influx through voltage dependent L-type Ca(2+) channels plus Ca(2+) release from intracellular stores mediated by IP(3) receptors and ryanodine receptors. Hyperosmotic stress also increased mitochondrial Ca(2+) levels, promoted mitochondrial depolarization, reduced intracellular ATP content, and activated the transcriptional factor cyclic AMP responsive element binding protein (CREB), determined by increased CREB phosphorylation and electrophoretic mobility shift assays. Incubation with 1 mM EGTA to decrease extracellular [Ca(2+)] prevented cardiomyocyte apoptosis induced by hyperosmotic stress, while overexpression of an adenoviral dominant negative form of CREB abolished the cardioprotection provided by 1 mM EGTA. These results suggest that hyperosmotic stress induced by sorbitol, by increasing Ca(2+) influx and raising intracellular Ca(2+) concentration, activates Ca(2+) release from stores and causes cell death through mitochondrial function collapse. In addition, the present results suggest that the Ca(2+) increase induced by hyperosmotic stress promotes cell survival by recruiting CREB-mediated signaling. Thus, the fate of cardiomyocytes under hyperosmotic stress will depend on the balance between Ca(2+)-induced survival and death pathways.
Assuntos
Apoptose/fisiologia , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Sobrevivência Celular , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/fisiologia , Sorbitol/farmacologia , Animais , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Humanos , Indicadores e Reagentes/farmacologia , Mitocôndrias/metabolismo , Miócitos Cardíacos/citologia , Pressão Osmótica , Ratos , Ratos Sprague-DawleyRESUMO
Infection associated with Shiga toxin-producing Escherichia coli (STEC) and subsequent Hemolytic-Uremic Syndrome (HUS) have become relevant in public health since STEC is considered as one of the most important emergent pathogens. STEC infection may either be asymptomatic or begin with watery diarrhea associated with hemorrhagic colitis and HUS. The major virulence factor of STEC is Shiga toxin type 1 or 2 (Stx1, Stx2) although strains that express only Stx2 are highly prevalent. Up to now, it has not been established whether STEC infection affect pregnant women. In this study, we evaluated the effect of Stx2 on maternal lethality, fetal status and delivery time by injecting Stx2 in rats in the late stage of pregnancy. Stx2 induced fetal resorption, placental abruption, intrauterine hemorrhage and fetal death at 1-2 days post-injection in a dose-dependent manner. With 2ng Stx2/g body weight, placentas and fetuses presented extensive necrotic areas, while uteri and kidneys showed normal histology. Immunolocalization of Stx2 was observed in placentas and fetuses. With 4 and 6ng Stx2/g body weight maternal death was also observed. Those rats that survived after Stx2-treatment were able to become pregnant and deliver normal pups at term. Our results show, for the first time, that the preterm labor with fetal death observed in treated rats may be a consequence of the action of Stx2 on the feto-maternal unit. Although there are no reports of Stx2 effects in human pregnancy, we speculate that STEC infections could be one of the causes not yet determined of fetal morbimortality.
Assuntos
Morte Fetal/induzido quimicamente , Nascimento Prematuro/induzido quimicamente , Toxina Shiga II/farmacologia , Feto Abortado/efeitos dos fármacos , Animais , Feminino , Idade Gestacional , Injeções Intraperitoneais , Masculino , Gravidez , Nascimento Prematuro/mortalidade , Ratos , Ratos Sprague-Dawley , Toxina Shiga II/administração & dosagem , Toxina Shiga II/metabolismo , Análise de Sobrevida , Útero/metabolismo , Útero/patologiaRESUMO
Introducción: la caracterización estudiantil es un proceso útil en la generación de información, pues permite diagnosticar, evaluar y retroalimentar la planeación y organización curricular. Métodos: el objetivo de esta investigación de corte descriptivo transversal fue identificar las características demográficas, socioeconómicas, académicas y profesionales, al igual que otras actividades extracurriculares de los estudiantes de los posgrados matriculados durante el semestre 2004-1. La población estuvo conformada por 48 estudiantes a quienes se les aplicó una encuesta con preguntas abiertas y cerradas. Los datos fueron procesados en el programa EPI-INFO. Resultados y conclusiones: muestran que el grupo de estudiantes son en su mayoría mujeres, solteras y con edades que oscilan entre los 28 y 30 años; un poco más de la mitad labora para su sostenimiento entre 6 y 10 horas semanales y ganan entre 1 y 2 SMLV mensuales, desempeñándose en el área clínica odontológica, sin presentar dificultades académicas por su actividad laboral, ni dificultades laborales por su labor académica. La información aquí presentada puede servir como insumo para el rediseño curricular y planeaciones futuras, así como de sustento para los procesos de acreditación que los posgrados ya han iniciado.
Assuntos
Currículo , EspecializaçãoRESUMO
En el presente trabajo hacemos una revisión y análisis descriptivo de 5 casos clínicos de pacientes operados por obesidad mórbida, que se presentaron en el Hospital Clínico de la Universidad de Chile en el período de junio del 2003 a septiembre del 2005 en los que se detectó fallas de algunos de estos procedimientos manifestadas por síntomas y reganancia de su peso.