RESUMO
Partial hand amputation is by far the most common type of amputation worldwide. Nevertheless, regardless of their potential clinical and socioeconomic impact, battery-powered partial hand prostheses, namely, powered digits, have modestly progressed so far, and very few clinical solutions are available today. Here, we present a mechanical architecture, an alternative to state-of-the-art solutions, which exploits a high efficiency, non-back drivable mechanical transmission based on a face-gear pair and a miniaturized clutch. We took inspiration from the synergetic prehension approach proposed by Childress for whole hand amputation. The finger was equipped with a myoelectric controller and a tactile sensor able to provide users with discrete event sensory feedback. Measured speed (90°/s) and force (6.5 N) of the newly dubbed S-Finger proved comparable with those of clinically available prostheses. The design demonstrated to be compact and rugged enough to undergo a clinical viability test with two partial hand amputees, fitted with custom three-fingered research prostheses using the S-Finger. The subjects successfully completed several dexterity tests and gave relevant feedback for the development of a second-generation device. These results contribute to the increasing research endeavors in the field of partial hand amputation.
Assuntos
Retroalimentação Sensorial , Dedos , Próteses e Implantes , Tato , Amputados , Fenômenos Biomecânicos , Eletromiografia , Mãos , Força da Mão , Humanos , Desenho de PróteseRESUMO
This paper proposes the use of an instrumented object for the study of the human grasping strategies. The proposed object is able to measure the grasping forces by means of three Force Sensitive Resistor (FSR) sensors and triaxial acceleration through an accelerometer. The object orientation angles (roll and pitch) can be estimated from the accelerometer output in quasi-static condition, whereas slippage events can be detected through the Power Spectrum Density (PSD) computation of the acceleration on at least one of the three axes. An experimental session on 7 healthy subjects has been performed; each subject used the instrumented object to perform 8 tripod grasp trials. All the sensory information, i.e. applied forces, object orientation and slippage, have been analyzed in order to evaluate the grasping strategies of the different subjects.
Assuntos
Fenômenos Biomecânicos/fisiologia , Engenharia Biomédica/instrumentação , Força da Mão/fisiologia , Acelerometria/instrumentação , Acelerometria/métodos , Engenharia Biomédica/métodos , Desenho de Equipamento , Dedos/fisiologia , HumanosRESUMO
The human hand is considered as the highest example of dexterous system capable of interacting with different objects and adapting its manipulation abilities to them. The control of poliarticulated prosthetic hands represents one important research challenge, typically aiming at replicating the manipulation capabilities of the natural hand. For this reason, this paper wants to propose a bio-inspired learning architecture based on parallel force/position control for prosthetic hands, capable of learning cyclic manipulation capabilities. To this purpose, it is focused on the control of a commercial biomechatronic hand (the IH2 hand) including the main features of recent poliarticulated prosthetic hands. The training phase of the hand was carried out in simulation, the parallel force/position control was tested in simulation whereas preliminary tests were performed on the real IH2 hand. The results obtained in simulation and on the real hand provide an important evidence of the applicability of the bio-inspired neural control to real biomechatronic hand with the typical features of a hand prosthesis.
Assuntos
Mãos/fisiologia , Robótica , Humanos , Desenho de PróteseRESUMO
This paper presents the design and realization of an instrumented object for force analysis during grasping. The object, with spherical shape, has been constructed with three contact areas in order to allow performing a tripod grasp. Force Sensing Resistor (FSR) sensors have been employed for normal force measurements, while an accelerometer has been used for slip detection. An electronic board for data acquisition has been embedded into the object, so that only the cables for power supply exit from it. Validation tests have been carried out for: (i) comparing the force measurements with a ground truth; (ii) assessing the capability of the accelerometer to detect slippage for different roughness values; (iii) evaluating object performance in grasp trials performed by a human subject.
Assuntos
Força da Mão , Robótica , Aceleração , Fenômenos Biomecânicos , Calibragem , Desenho de Equipamento , Articulações dos Dedos , Força da Mão/fisiologia , Humanos , Sistemas Homem-Máquina , Monitorização Ambulatorial/métodos , Movimento , Reprodutibilidade dos Testes , Interface Usuário-ComputadorRESUMO
OBJECTIVE: Obesity is associated with high insulin and glucagon plasma levels. Enhanced ß-cell function and ß-cell expansion are responsible for insulin hypersecretion. It is unknown whether hyperglucagonemia is due to α-cell hypersecretion or to an increase in α-cell mass. In this study, we investigated the dynamics of the ß-cell and α-cell function and mass in pancreas of obese normoglycemic baboons. METHODS: Pancreatic ß- and α-cell volumes were measured in 51 normoglycemic baboons divided into six groups according to overweight severity or duration. Islets morphometric parameters were correlated to overweight and to diverse metabolic and laboratory parameters. RESULTS: Relative α-cell volume (RαV) and relative islet α-cell volume (RIαV) increased significantly with both overweight duration and severity. Conversely, in spite of the induction of insulin resistance, overweight produced only modest effects on relative ß-cell volume (RßV) and relative islet ß-cell volume (RIßV). Of note, RIßV did not increase neither with overweight duration nor with overweight severity, supposedly because of the concomitant, greater increase in RIαV. Baboons' body weights correlated with serum levels of interleukin-6 and tumor necrosis factor-α soluble receptors, demonstrating that overweight induces abnormal activation of the signaling of two cytokines known to impact differently ß- and α-cell viability and replication. CONCLUSION: In conclusion, overweight and insulin resistance induce in baboons a significant increase in α-cell volumes (RαV, RIαV), whereas have minimal effects on the ß cells. This study suggests that an increase in the α-cell mass may precede the loss of ß cells and the transition to overt hyperglycemia and diabetes.
Assuntos
Células Secretoras de Glucagon/metabolismo , Resistência à Insulina , Células Secretoras de Insulina/metabolismo , Obesidade/metabolismo , Animais , Glicemia/metabolismo , Peso Corporal , Proliferação de Células , Feminino , Hiperglicemia/metabolismo , Imuno-Histoquímica , Interleucina-6/metabolismo , Masculino , Obesidade/fisiopatologia , Papio , Estado Pré-Diabético/metabolismo , Índice de Gravidade de Doença , Fatores de Tempo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: Study of endocrine pathology in animal models is critical to understanding endocrine pathology in humans. METHODS: We evaluated 434 endocrine-related diagnoses from 4619 baboon necropsies, established the incidence of spontaneous endocrine pathology, and analyzed the clinical and biochemical data associated with the individual cases. RESULTS: The most common diagnoses in descending order, were pancreatic islet cell amyloidosis (n = 259), ovarian cysts (n = 50), pituitary adenoma (n = 37), pancreatic islet cell adenoma (n = 20), granulosa cell tumor (n = 15), thyroid adenoma (n = 11), adrenal hyperplasia (n = 10), thyroid carcinoma (n = 8), and pheochromocytoma (n = 6). The incidence of pancreatic islet cell amyloidosis progressively increased with age. Pheochromocytomas were associated with renal and heart failure. The incidence of pancreatic islet cell amyloidosis and adrenal pathology was similar to humans; the incidence of pituitary adenoma and thyroid pathology was lower than in humans. CONCLUSIONS: Endocrine disease in baboons is common and shares clinical and biochemical characteristics with endocrine disease in humans.
Assuntos
Doenças do Sistema Endócrino/veterinária , Doenças dos Macacos/epidemiologia , Papio , Animais , Comorbidade , Doenças do Sistema Endócrino/epidemiologia , Feminino , Humanos , Incidência , MasculinoRESUMO
The quality of human islets is one of the factors decisive for the success of human islet transplantation. Several parameters have been proposed to characterize islet quality, but none of them has been able to predict the fate of a transplant. The aim of our study was to correlate a panel of in vitro parameters for islet viability with their in vivo function after transplantation in nude mice. Islets were obtained after enzymatic digestion of a human pancreas; they were purified from exocrine tissue using a continuous-density gradient. Two aliquots of islets (1000 and 2000 islets) were transplanted under the kidney capsule of diabetic nude mice. The animals were followed for 1 month with repeated measurements of blood glucose and body weight. One month after transplantation, mice were killed and their graft harvested for histologic analysis. In parallel we studied in vitro islet viability with propidium iodide and fura-2, their insulin content, their purity, and their insulin response to glucose upon static incubation. Ten islet preparations were transplanted: 3 out of 10 preparations did not restore normoglycemia; 4 out of 10 normalized glycemia only in mice receiving 2000 islets, and 3 out of 10 fully restore normoglycemia in all mice. The purity of preparations (R(2) = 0.63 and 0.85, respectively, with 1000 and 2000 islets) and the insulin content (R(2) = 0.75 with 2000 IE) correlated with transplant success. These data show that purity of islet preparations and their insulin content should be useful parameters for the selection of islet preparations for transplant purposes.
Assuntos
Diabetes Mellitus Experimental/cirurgia , Etanol/análogos & derivados , Sobrevivência de Enxerto/fisiologia , Transplante das Ilhotas Pancreáticas/métodos , Animais , Glicemia , Diabetes Mellitus Experimental/sangue , Humanos , Camundongos , Camundongos Nus , Modelos Animais , Valor Preditivo dos Testes , Ensaio de Cápsula Sub-Renal , Transplante HeterólogoRESUMO
Islet transplantation was proposed more than 10 years ago as treatment for normalising glucose homeostasis in type 1 diabetic patients. Since the beginning it has aroused great interest among diabetic patients being an easy procedure, burdened by minor complications: islet transplantation in fact consists on a transhepatic percutaneous injection under local anaesthesia. The initial clinical outcomes not came up to expectations, being low the insulin independence rate and the long term graft function in recipients. Recently, thanks to the introduction of new immunosuppression strategies, clinical data greatly improved: insulin independence was reached in all recipients and maintained in more than 70% of them 2 years from the transplant. The need of an immunosuppression therapy limits the indication of islet transplantation to diabetic patients already immunosuppressed for a previous organ transplant or to patients with brittle diabetes, that is not controlled also with the new strategies of insulin treatment, with a poor quality of life and an increased rate of diabetic complications. Other problems are represented by the progressive decrease of graft function during long term follow up, and by the low number of organ donors that limits the number of transplantation feasible per year.
Assuntos
Diabetes Mellitus Tipo 1/cirurgia , Transplante das Ilhotas Pancreáticas , Humanos , Ilhotas Pancreáticas/fisiologia , Transplante das Ilhotas Pancreáticas/fisiologia , Falha de TratamentoRESUMO
Voltage-operated calcium channels play crucial roles in stimulus-secretion coupling in pancreatic beta cells. A growing body of evidence indicates that these channels in beta cells are heterogeneous. In particular, not all the high-threshold calcium channels expressed belong to the best known L-type. In rat insulinoma cells, for example, L, N, and P/Q-type channels are present, while in human beta cells L-type and P/Q-type dominate. Where present, N-type and P/Q-type channels participate, alongside with the dominant L-type, in the control of sugar- or depolarization-induced hormone release. Distinct biophysical properties and selective modulation of the channel subtypes are likely to play important physiological roles. T-type channels are involved in beta cell apoptosis, while calcium channel autoantibodies recognizing high-threshold channels in beta cells, have been described both in neurological and diabetic patients. Subtype-selective calcium channel drugs have the potential for being beneficial in beta cell pathological states.
Assuntos
Canais de Cálcio/classificação , Canais de Cálcio/metabolismo , Membrana Celular/metabolismo , Diabetes Mellitus/metabolismo , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Doenças Neuromusculares/metabolismo , Animais , Canais de Cálcio/química , Glucose/metabolismo , Humanos , Ilhotas Pancreáticas/patologia , Potenciais da Membrana , Distribuição TecidualRESUMO
Despite the considerable interest for islet and pancreas transplantation, remarkably little is known about the direct effects of immunosuppressive drugs on human beta-cell function. We measured different insulin secretory parameters and insulin gene expression of human islets cultured for 5 days in the presence of mycophenolate mofetil (MMF), cyclosporin A (CsA), tacrolimus (FK506) or a mixture of 3 cytokines. Basal insulin release after exposure to cytokines and FK506 was significantly higher than in control islets. Responsiveness to an acute glucose stimulus did not differ significantly between control and treated islets. However, absolute incremental insulin responses (delta-AUCs) of islets exposed to cytokines or FK506 were significantly higher compared to islets exposed to CsA or MMF, mainly because of the higher basal release. Indeed, maximal over basal release (stimulation index, SI) tended to be lower in islets exposed to FK506 than in control islets. Insulin gene expression was significantly reduced only in islets exposed to CsA. FK506 was, among those tested, the immunosuppressive drug that most profoundly altered the normal insulin secretory pattern of human beta-cells, whereas CsA was the only inhibiting insulin gene expression. Although the abnormalities induced by the immunosoppressive drugs utilized in this study were modest, these in vitro data are consistent with the reported in vivo diabetogenicity of CsA and FK506 and point to MMF as the ideal immunosuppressive agent from a pancreatic beta-cell point of view.
Assuntos
Imunossupressores/farmacologia , Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Adulto , Ciclosporina/farmacologia , Expressão Gênica/efeitos dos fármacos , Humanos , Técnicas In Vitro , Insulina/genética , Secreção de Insulina , Pessoa de Meia-Idade , Ácido Micofenólico/análogos & derivados , Ácido Micofenólico/farmacologia , RNA Mensageiro/metabolismo , Tacrolimo/farmacologiaRESUMO
The insulin signaling cascade was investigated in rat myocardium in vivo in the presence of streptozocin (STZ)-induced diabetes and after diabetes treatment by islet transplantation under the kidney capsule. The levels of insulin-stimulated tyrosine phosphorylation of the insulin receptor beta-subunit, insulin receptor substrate (IRS)-2, and p52(Shc) were increased in diabetic compared with control heart, whereas tyrosine phosphorylation of IRS-1 was unchanged. The amount of the p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase) and the level of PI 3-kinase activity associated with IRS-2 were also elevated in diabetes, whereas no changes in IRS-1-associated PI 3-kinase were observed. Insulin-induced phosphorylation of Akt on Thr-308 was increased fivefold in diabetic heart, whereas Akt phosphorylation on Ser-473 was normal. In contrast with Akt phosphorylation, insulin-induced phosphorylation of glycogen synthase kinase (GSK)-3, a major cellular substrate of Akt, was markedly reduced in diabetes. In islet-transplanted rats, the majority of the alterations in insulin-signaling proteins found in diabetic rats were normalized, but insulin stimulation of IRS-2 tyrosine phosphorylation and association with PI 3-kinase was blunted. In conclusion, in the diabetic heart, 1) IRS-1, IRS-2, and p52(Shc) are differently altered, 2) the levels of Akt phosphorylation on Ser-473 and Thr-308, respectively, are not coordinately regulated, and 3) the increased activity of proximal-signaling proteins (i.e., IRS-2 and PI 3-kinase) is not propagated distally to GSK-3. Islet transplantation under the kidney capsule is a potentially effective therapy to correct several diabetes-induced abnormalities of insulin signaling in cardiac muscle but does not restore the responsiveness of all signaling reactions to insulin.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/cirurgia , Insulina/metabolismo , Transplante das Ilhotas Pancreáticas , Proteínas Serina-Treonina Quinases , Transdução de Sinais , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Quinase 3 da Glicogênio Sintase , Quinases da Glicogênio Sintase , Proteínas Substratos do Receptor de Insulina , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Miocárdio/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Fosfosserina/metabolismo , Fosfotirosina/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Ratos , Ratos Endogâmicos Lew , Receptor de Insulina/metabolismo , Proteínas Adaptadoras da Sinalização Shc , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de SrcRESUMO
1alpha,25-dihydroxyvitamin D3, the active form of vitamin D3, and mycophenolate mofetil, a selective inhibitor of T and B cell proliferation, modulate APC function and induce dendritic cells (DCs) with a tolerogenic phenotype. Here we show that a short treatment with these agents induces tolerance to fully mismatched mouse islet allografts that is stable to challenge with donor-type spleen cells and allows acceptance of donor-type vascularized heart grafts. Peritransplant macrophages and DCs from tolerant mice express down-regulated CD40, CD80, and CD86 costimulatory molecules. In addition, DCs from the graft area of tolerant mice secrete, upon stimulation with CD4+ cells, 10-fold lower levels of IL-12 compared with DCs from acutely rejecting mice, and induce a CD4+ T cell response characterized by selective abrogation of IFN-gamma production. CD4+ but not CD8+ or class II+ cells from tolerant mice, transferred into naive syngeneic recipients, prevent rejection of donor-type islet grafts. Graft acceptance is associated with impaired development of IFN-gamma-producing type 1 CD4+ and CD8+ cells and an increased percentage of CD4+CD25+ regulatory cells expressing CD152 in the spleen and in the transplant-draining lymph node. Transfer of CD4+CD25+ cells from tolerant but not naive mice protects 100% of the syngeneic recipients from islet allograft rejection. These results demonstrate that a short treatment with immunosuppressive agents, such as 1alpha,25-dihydroxyvitamin D3/mycophenolate mofetil, induces tolerance to islet allografts associated with an increased frequency of CD4+CD25+ regulatory cells that can adoptively transfer transplantation tolerance.
Assuntos
Transferência Adotiva , Calcitriol/administração & dosagem , Imunossupressores/administração & dosagem , Transplante das Ilhotas Pancreáticas/imunologia , Ácido Micofenólico/análogos & derivados , Ácido Micofenólico/administração & dosagem , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/transplante , Tolerância ao Transplante/imunologia , Administração Oral , Animais , Antígenos CD/biossíntese , Antígeno B7-1/biossíntese , Antígeno B7-2 , Antígenos CD4/biossíntese , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/transplante , Antígenos CD40/biossíntese , Movimento Celular/imunologia , Células Cultivadas , Regulação para Baixo/imunologia , Quimioterapia Combinada , Feminino , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/prevenção & controle , Transplante das Ilhotas Pancreáticas/patologia , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Receptores de Interleucina-2/biossíntese , Tolerância ao Transplante/efeitos dos fármacosRESUMO
BACKGROUND: The mechanisms of increased neointimal hyperplasia after coronary interventions in diabetic patients are still unknown. METHODS AND RESULTS: Glucose and insulin effects on in vitro vascular smooth muscle cell (VSMC) proliferation and migration were assessed. The effect of balloon injury on neointimal hyperplasia was studied in streptozotocin-induced diabetic rats with or without adjunct insulin therapy. To study the effect of balloon injury in nondiabetic rats with hyperinsulinemia, pancreatic islets were transplanted under the kidney capsule in normal rats. Glucose did not increase VSMC proliferation and migration in vitro. In contrast, insulin induced a significant increase in VSMC proliferation and migration in cell cultures. Furthermore, in VSMC culture, insulin increased MAPK activation. A reduction in neointimal hyperplasia was consistently documented after vascular injury in hyperglycemic streptozotocin-induced diabetic rats. Insulin therapy significantly increased neointimal hyperplasia in these rats. This effect of hyperinsulinemia was totally abolished by transfection on the arterial wall of the N17H-ras-negative mutant gene. Finally, after experimental balloon angioplasty in hyperinsulinemic nondiabetic islet-transplanted rats, a significant increase in neointimal hyperplasia was observed. CONCLUSIONS: In rats with streptozotocin-induced diabetes, balloon injury was not associated with an increase in neointimal formation. Exogenous insulin administration in diabetic rats and islet transplantation in nondiabetic rats increased both blood insulin levels and neointimal hyperplasia after balloon injury. Hyperinsulinemia through activation of the ras/MAPK pathway, rather than hyperglycemia per se, seems to be of crucial importance in determining the exaggerated neointimal hyperplasia after balloon angioplasty in diabetic animals.
Assuntos
Angioplastia com Balão , Doenças das Artérias Carótidas/patologia , Diabetes Mellitus Experimental/patologia , Hiperinsulinismo/patologia , Hiperplasia/patologia , Transplante das Ilhotas Pancreáticas , Túnica Íntima/patologia , Angioplastia com Balão/efeitos adversos , Animais , Glicemia , Doenças das Artérias Carótidas/etiologia , Doenças das Artérias Carótidas/genética , Divisão Celular/efeitos dos fármacos , Divisão Celular/genética , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/metabolismo , Modelos Animais de Doenças , Glucose/farmacologia , Hiperinsulinismo/induzido quimicamente , Hiperinsulinismo/metabolismo , Hiperplasia/etiologia , Hiperplasia/genética , Insulina/sangue , Insulina/farmacologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/enzimologia , Mutagênese Sítio-Dirigida , Ratos , Ratos Endogâmicos F344 , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Estreptozocina , Transfecção , Túnica Íntima/metabolismo , Proteínas ras/antagonistas & inibidores , Proteínas ras/genéticaRESUMO
Type 2 diabetes is characterized by insulin resistance and inadequate insulin secretion. In the advanced stages of the disease, beta-cell dysfunction worsens and insulin therapy may be necessary to achieve satisfactory metabolic control. Studies in autopsies found decreased beta-cell mass in pancreas of people with type 2 diabetes. Apoptosis, a constitutive program of cell death modulated by the Bcl family genes, has been implicated in loss of beta-cells in animal models of type 2 diabetes. In this study, we compared the effect of 5 days' culture in high glucose concentration (16.7 mmol/l) versus normal glucose levels (5.5 mmol/l) or hyperosmolar control (mannitol 11 mmol/l plus glucose 5 mmol/l) on the survival of human pancreatic islets. Apoptosis, analyzed by flow cytometry and electron and immunofluorescence microscopy, was increased in islets cultured in high glucose (HG5) as compared with normal glucose (NG5) or hyperosmolar control (NG5+MAN5). We also analyzed by reverse transcriptase-polymerase chain reaction and Western blotting the expression of the Bcl family genes in human islets cultured in normal glucose or high glucose. The antiapoptotic gene Bcl-2 was unaffected by glucose change, whereas Bcl-xl was reduced upon treatment with HG5. On the other hand, proapoptotic genes Bad, Bid, and Bik were overexpressed in the islets maintained in HG5. To define the pancreatic localization of Bcl proteins, we performed confocal immunofluorescence analysis on human pancreas. Bad and Bid were specifically expressed in beta-cells, and Bid was also expressed, although at low levels, in the exocrine pancreas. Bik and Bcl-xl were expressed in other endocrine islet cells as well as in the exocrine pancreas. These data suggest that in human islets, high glucose may modulate the balance of proapoptotic and antiapoptotic Bcl proteins toward apoptosis, thus favoring beta-cell death.
Assuntos
Apoptose , Glucose/administração & dosagem , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/fisiologia , Apoptose/genética , Células Cultivadas , Relação Dose-Resposta a Droga , Imunofluorescência , Regulação da Expressão Gênica/fisiologia , Glucose/farmacologia , Humanos , Proto-Oncogenes/fisiologia , Distribuição Tecidual , Transcrição Gênica/fisiologiaRESUMO
In recent years increasing attention has been given to apoptosis for its role in pathologic, organogenetic and homeostatic phenomena. Acridine orange (AO), Hoechst 33342 (HO) and propidium iodide (PI) are among the most used fluorescent dyes used to analyse cell culture viability. In fact, they respectively show specificity for living, apoptotic and late apoptosis/necrosis states. We explored whether HO, AO and PI can be used on prefixed monolayers of three commonly used cell lines. Here we mainly describe the metachromatic effects obtained by fluorescence microscopy with double and triple dye combinations. Furthermore, we propose an easy staining method in which a balanced sequential treatment with HO, AO and PI allows identification of different viability states onto fixed cells by using a long-pass FITC filter. This method extends the spectrum of suitable applications for these dyes in fluorescence viability detection onto previously fixed (prefixed) samples.
Assuntos
Laranja de Acridina , Benzimidazóis , Sobrevivência Celular , Corantes Fluorescentes , Propídio , Animais , Apoptose , Núcleo Celular/ultraestrutura , Cricetinae , Citoplasma/ultraestrutura , Camundongos , Microscopia de Fluorescência , Necrose , Ratos , Coloração e Rotulagem/métodos , Fixação de TecidosAssuntos
Calcitriol/farmacologia , Células Dendríticas/efeitos dos fármacos , Tolerância ao Transplante/efeitos dos fármacos , Animais , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/imunologia , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/prevenção & controle , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Tolerância ao Transplante/imunologiaAssuntos
Calcitriol/farmacologia , Rejeição de Enxerto/prevenção & controle , Imunossupressores/farmacologia , Transplante das Ilhotas Pancreáticas/imunologia , Ácido Micofenólico/farmacologia , Tolerância ao Transplante/efeitos dos fármacos , Animais , Quimioterapia Combinada , Sobrevivência de Enxerto , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Ácido Micofenólico/análogos & derivados , Fatores de TempoRESUMO
This contribution concerns the use of a supervisory expert system based on fuzzy logic for the parameter adjusting of myoelectric prostheses. The prosthesis system is an artificial arm produced by INAIL in Vigorso (Bologna, Italy), which is equipped with an on-board actuation system. The expert system guides patients through an interactive session whose aim is to test the prosthesis functionality and, when necessary, to self-adjust the parameters.
Assuntos
Membros Artificiais , Engenharia Biomédica , Lógica Fuzzy , Fenômenos Biomecânicos , Processamento Eletrônico de Dados , Eletrofisiologia , Desenho de Equipamento , Humanos , Músculo Esquelético/fisiologia , Processamento de Sinais Assistido por Computador , Interface Usuário-ComputadorRESUMO
We report a case of long-term (>4 yr) successful intrahepatic islet transplantation into a type 1 diabetic patient chronically immunosuppressed for a prior kidney graft. The exogenous insulin requirement decreased progressively after transplantation, and insulin treatment was withdrawn at 6 months. Glycosylated hemoglobin levels were in the normal range at 1 and 2 yr (5.3%) and increased slightly above the upper normal limit at 3 and 4 yr (6.3% and 6.4%). Fasting C peptide levels remained stable during the entire follow-up, but the proinsulin to insulin ratios increased dramatically at yr 3. Glycemic levels after an oral glucose tolerance test showed a diabetic profile at 1 yr, a normal profile at 2 yr, and an impaired glucose tolerance profile at 3 yr. Intravenous glucose tolerance test-induced first phase insulin release, present at 1 and 2 yr, disappeared at 3 yr. Diabetes-related autoantibodies (islet cell antibodies, glutamic acid decarboxylase antibodies, and tyrosine phosphatase-like protein antibodies) were undetectable before transplantation and remained so during the entire follow-up. The patient died of myocardial infarction 50 months after transplantation while she was still in good metabolic control (glycosylated hemoglobin, <6.8%) in the absence of exogenous insulin administration. The autoptic liver showed well granulated islets, richly vascularized and without evidence of lympho-mononuclear cell infiltration. The morphometrically extrapolated intrahepatic beta-cell mass was 99.9 mg. In conclusion, this successful islet graft showed a bell-shaped clinical effect, maximal at 2 yr after transplantation, followed by a slow progressive decline. The absence of allo- and autoreactivities against the transplanted islets points to a nonimmune-mediated beta-cell loss as the cause of graft functional deterioration.
Assuntos
Transplante de Células/fisiologia , Diabetes Mellitus Tipo 1/terapia , Transplante das Ilhotas Pancreáticas/fisiologia , Ilhotas Pancreáticas/fisiologia , Adulto , Glicemia/metabolismo , Peptídeo C/metabolismo , Feminino , Teste de Tolerância a Glucose , Humanos , Insulina/sangue , Insulina/metabolismo , Secreção de Insulina , Fígado/patologia , Proinsulina/sangue , Proinsulina/metabolismoRESUMO
Successful beta-cell replacement therapy in insulin-dependent (type I) diabetes is hindered by the scarcity of human donor tissue and by the recurrence of autoimmune destruction of transplanted beta cells. Availability of non-beta cells, capable of releasing insulin and escaping autoimmune recognition, would therefore be important for diabetes cell therapy. We developed rat pituitary GH3 cells stably transfected with a furin-cleavable human proinsulin cDNA linked to the rat PRL promoter. Two clones (InsGH3/clone 1 and 7) were characterized in vitro with regard to basal and stimulated insulin release and proinsulin transgene expression. Mature insulin secretion was obtained in both clones, accounting for about 40% of total released (pro)insulin-like products. Immunocytochemistry of InsGH3 cells showed a cytoplasmic granular insulin staining that colocalized with secretogranin II (SGII) immunoreactivity. InsGH3 cells/clone 7 contained and released in vitro significantly more insulin than clone 1. Secretagogue-stimulated insulin secretion was observed in both InsGH3 clones either under static or dynamic conditions, indicating that insulin was targeted also to the regulated secretory pathway. Proinsulin mRNA levels were elevated in InsGH3 cells, being significantly higher than in betaTC3 cells. Moreover, proinsulin gene expression increased in response to various stimuli, thereby showing the regulation of the transfected gene at the transcriptional level. In conclusion, these data point to InsGH3 cells as a potential beta-cell surrogate even though additional engineering is required to instruct them to release insulin in response to physiologic stimulations.