RESUMEN
BACKGROUND: Diabetes mellitus is a metabolic disorder characterized by hyperglycaemia resulting from uncontrolled glucose regulation. Reactive oxygen species are recognized as one link between hyperglycaemia and diabetic complications. Studies have shown that diabetes mellitus is associated with decreases in antioxidant potential and increased formation of free radicals leading to oxidative stress. The present study was undertaken because an unequivocal demonstration that control of hyperglycaemia can reduce oxidative stress is still lacking. METHODS: In the present study, we investigated oxidative stress profile of normal, streptozotocin-induced diabetic, insulin-treated and untreated diabetic animals. On the one hand, oxidative damage caused to lipids, proteins and DNA was measured. On other hand, antioxidant defense was measured in terms of specific activities of antioxidant enzymes (AOEs) and antioxidant molecules. RESULTS: It was observed that the damage to lipids, proteins and DNA caused by free radicals increased in diabetic animals compared with that in controls. In diabetic animals not treated with insulin, damage to all biological molecules increased further significantly (p ≤ 0.005). Changes in AOEs from different tissues were complex depicting a varied AOE level in different tissues. Insulin treatment significantly improved the oxidative stress profile in all tissues studies. CONCLUSIONS: The control of hyperglycaemia improves oxidative stress profile, that is, the ability of cells to cope up with oxidative stress.
Asunto(s)
Diabetes Mellitus Experimental/metabolismo , Hiperglucemia/fisiopatología , Estrés Oxidativo/fisiología , Animales , Antioxidantes/metabolismo , Glucemia/metabolismo , Daño del ADN , Diabetes Mellitus Experimental/inducido químicamente , Femenino , Técnicas In Vitro , Peroxidación de Lípido , Masculino , Ratones , Oxidación-Reducción , Carbonilación Proteica/fisiología , Especies Reactivas de Oxígeno/metabolismo , EstreptozocinaRESUMEN
Cardiac myocytes are the first cells to differentiate during the development of a vertebrate embryo. A wide variety of molecules take part in various steps in this process. While exploring biologically active molecules from marine sources, we found that a constituent of perivitelline fluid from embryos of the Indian horseshoe crab can enhance growth and differentiation of chick embryonic heart. We have purified the factor and identified the cardiac promoting molecule to be a novel lectin. We show that this molecule influences cardiac development by increasing the number of cells constituting the heart and by modulating the expression of several cardiac development regulatory genes in chick embryos. Using mouse embryonic stem cells we show that the cardiac myocyte-enhancing capacity of this molecule extends to mammals and its effects can be blocked using methylated sugars. This molecule may prove to be an important tool in the study of cardiomyocyte differentiation.
Asunto(s)
Embrión no Mamífero/metabolismo , Corazón/embriología , Cangrejos Herradura/embriología , Lectinas/farmacología , Organogénesis/efectos de los fármacos , Vertebrados/embriología , Membrana Vitelina/metabolismo , Animales , Proteínas Portadoras/metabolismo , Recuento de Células , Diferenciación Celular/efectos de los fármacos , Fraccionamiento Químico , Pollos , Células Madre Embrionarias/citología , Células Madre Embrionarias/efectos de los fármacos , Gastrulación/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Corazón/efectos de los fármacos , Hematopoyesis/efectos de los fármacos , Ratones , Proteínas Musculares/metabolismo , Miocardio/citología , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Neovascularización Fisiológica/efectos de los fármacos , Especificidad de Órganos/efectos de los fármacosRESUMEN
The pancreatic ductal stem cells are known to differentiate into islets of Langerhans; however, their yield is limited and the islet population is not defined. Therefore, the aims of the present study were to improvise a methodology for obtaining large numbers of islets in vitro and to characterize their morphological and functional status for islet cell banking and transplantation. Pancreatic ductal epithelial cell cultures were set in serum-free medium. Monolayers of epithelial cells in culture gave rise to islet-like clusters within 3-4 weeks. The identity of neoislets was confirmed by dithizone staining and analysis of the gene expression for endocrine markers by reverse transcriptase-polymerase chain reaction (RT-PCR). The islet population obtained was analysed by image analysis and insulin secretion in response to secretagogues. The cellular extracts from neoislets were immunoreactive to anti-insulin antibody and expressed insulin, glucagon, GLUT-2, PDX-1 and Reg-1 genes. The islets generated within 3-4 weeks exhibited a mixed population of large- and small-sized islets with clear cut dichotomy in the pattern of their insulin secretion in response to L-arginine and glucose. These neoislets maintained their structural and functional integrity on cryopreservation and transplantation indicating their suitability for islet cell banking. Thus, the present study describes an improved method for obtaining a constant supply of large numbers of islets from pancreatic ductal stem cell cultures. The newly generated islets undergo functional maturation indicating their suitability for transplantation.
Asunto(s)
Proteínas de Homeodominio , Islotes Pancreáticos/citología , Conductos Pancreáticos/citología , Células Madre/citología , Animales , Arginina/farmacología , Biomarcadores/análisis , Proteínas de Unión al Calcio/análisis , Técnicas de Cultivo de Célula , Diferenciación Celular , Criopreservación , Células Epiteliales/citología , Glucagón/análisis , Glucosa/farmacología , Transportador de Glucosa de Tipo 2 , Insulina/análisis , Insulina/metabolismo , Secreción de Insulina , Trasplante de Islotes Pancreáticos , Litostatina , Ratones , Ratones Endogámicos BALB C , Modelos Animales , Proteínas de Transporte de Monosacáridos/análisis , Bancos de Tejidos , Transactivadores/análisisRESUMEN
We previously showed that CD28 is expressed on human peripheral blood neutrophils and plays an important role in CXCR-1 expression and IL-8-induced neutrophil migration. In this work we demonstrate that Leishmania major infection of macrophages results in parasite dose-dependent IL-8 secretion in vitro and in IL-8-directed neutrophil migration, as blocked by both anti-IL-8 and anti-IL-8R Abs, toward the L. major-infected macrophages. In the neutrophil-macrophage cocultures, both CTLA4-Ig, a fusion protein that blocks CD28-CD80/CD86 interaction, and a neutralizing anti-IFN-gamma Ab inhibit the anti-leishmanial function of neutrophils, suggesting that the neutrophil-macrophage interaction via CD28-CD80/CD86 plays an important role in the IFN-gamma-dependent restriction of the parasite growth. Cross-linking of neutrophil-expressed CD28 by monoclonal anti-CD28 Ab or B7.1-Ig or B7.2-Ig results in phosphatidylinositol 3-kinase association with CD28 and in wortmannin-sensitive but cyclosporin A-resistant induction and secretion of IFN-gamma. Whereas the neutrophils secrete IFN-gamma with CD28 signal alone, the T cells do not secrete the cytokine in detectable amounts with the same signal. Thus, neutrophil-expressed CD28 modulates not only the granulocyte migration but also induction and secretion of IFN-gamma at the site of infection where it migrates from the circulation.
Asunto(s)
Antígeno B7-1/fisiología , Antígenos CD28/fisiología , Inhibidores de Crecimiento/inmunología , Interferón gamma/metabolismo , Leishmania major/crecimiento & desarrollo , Macrófagos/inmunología , Neutrófilos/inmunología , Fosfatidilinositol 3-Quinasas/fisiología , Androstadienos/farmacología , Animales , Antígenos CD/fisiología , Antígeno B7-2 , Antígenos CD28/biosíntesis , Comunicación Celular/inmunología , Células Cultivadas , Técnicas de Cocultivo , Ciclosporina/farmacología , Relación Dosis-Respuesta Inmunológica , Inhibidores Enzimáticos/farmacología , Inhibidores de Crecimiento/metabolismo , Interacciones Huésped-Parásitos/inmunología , Humanos , Interferón gamma/antagonistas & inhibidores , Interferón gamma/biosíntesis , Interferón gamma/genética , Interleucina-8/metabolismo , Interleucina-8/fisiología , Leishmania major/inmunología , Macrófagos/metabolismo , Macrófagos/parasitología , Glicoproteínas de Membrana/fisiología , Infiltración Neutrófila/inmunología , Neutrófilos/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Transducción de Señal/inmunología , Transcripción Genética/inmunología , WortmaninaAsunto(s)
Hipoglucemiantes/farmacología , Administración Oral , Animales , Hipoglucemiantes/administración & dosificación , Técnicas In Vitro , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
A dot-enzyme-linked immunosorbent assay (dot-ELISA) using soluble adult-guineaworm antigen was developed for immunodiagnosis of Dracunculus medinensis infection. The test was found to be specific and sensitive when compared with ELISA. The dot-ELISA described here was found to be time-saving and easy to perform, and its possible use as a diagnostic and epidemiological tool under field conditions is therefore discussed.
Asunto(s)
Dracunculiasis/diagnóstico , Animales , Antígenos Helmínticos/inmunología , Dracunculus/inmunología , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Pruebas SerológicasRESUMEN
A sandwich enzyme-linked immunosorbent assay (s-ELISA) is developed for detecting circulatory antigens in individuals infected with Wuchereria bancrofti in an endemic area using antibody (Ig) against excretory-secretory-metabolic antigens of the microfilariae raised in rabbit (anti-mf-ESM) and labelled with alkaline phosphatase (ESM-Ig-conjugate). An optical density reading of a sample greater than 0.075 (after subtracting the background) was taken as positive in the s-ELISA. When homologous (WbmfESM) and heterologous (sonicated antigens of human and model intestinal helminths-Ascaris lumbricoides. Trichuris muris, Necator americanus and Strongyloides ratti) antigens were spiked at 2.5, 5, and 7.5 microgram/well, rabbit ESM-Ig-conjugate reacted specifically with the samples containing homologous antigens. Amongst 21 sera in five different categories of non-endemic group, only four (two in helminth-ve and two in mixed intestinal helminthic group) were found to be positive. Out of 19 sera from endemic residents, three of 7 endemic normals (ENS), all microfilaraemics (mf+) (n = 7) and 4 out of 5 elephantoid patient sera were positive. This preliminary data show that rabbit mfESM-Ig-conjugate is efficient in detecting sera samples containing antigenic components of microfilariae. This assay was found to be discriminatory in detecting individuals carrying current infection. This test requires further validation with larger number of samples and it may prove of value for detecting lymphatic filarial infection.
Asunto(s)
Antígenos Helmínticos/sangre , Filariasis Linfática/diagnóstico , Ensayo de Inmunoadsorción Enzimática , Wuchereria bancrofti/inmunología , Animales , Humanos , Microfilarias/inmunología , Valor Predictivo de las PruebasRESUMEN
Monoclonal antibodies (mAbs) have been prepared against excretory-secretory-metabolic (ESM) antigens of microfilariae (mf) of Wuchereria bancrofti (WbmfESM) and against third stage larvae (L3) of Brugia malayi (BmL3), and purified from ascites fluids with ammonium sulphate. Both antibodies were of the IgM type and did not react with phosphorycholine. The mAb against BmL3 (F46) reacted in ELISA with antigens of L3 of B. malayi, B. pahangi and W. bancrofti and of adults of B. malayi. The mAb raised against wbmfESM (F32) resembled F46 in this respect, though with a lower titer towards the antigens, and in addition reacted with the ESM-antigens of mf and of L3 of W. bancrofti. F46 was able to detect L3 antigens of filarial parasites in spiked serum samples with a detection limit of 8-16 ng in absolute amount. The antibody was found to label the cuticular portion of L3 and adults of the lymphatic parasites, and not the epicuticular surface, in immunoelectron microscopic studies. The antibody recognized a 36 kDa component of the beta-mercaptoethanol extracts of B. pahangi-adults in Western blot analysis.
Asunto(s)
Antígenos Helmínticos , Brugia/inmunología , Wuchereria bancrofti/inmunología , Animales , Anticuerpos Antihelmínticos , Anticuerpos Monoclonales , Especificidad de Anticuerpos , Brugia/ultraestructura , Larva/inmunología , Microfilarias/inmunología , Microscopía Inmunoelectrónica , Peso Molecular , Proteínas/inmunología , Wuchereria bancrofti/ultraestructuraRESUMEN
Three types of in vitro released excretory, secretory and metabolic antigens of Wuchereria bancrofti third larval stage (L3ESM) are evaluated in ELISA test to detect infected individuals in the endemic area. A total of 104 reference sera are used to predict the sensitivity of these antigens. None of L3 ESM antigens, although homologous in nature, did not identify correctly the categorised reference sera. This study clearly indicated a need for defined antigens to detect W. bancrofti infection early in the endemic residents.
Asunto(s)
Anticuerpos Antihelmínticos/análisis , Antígenos Helmínticos/inmunología , Filariasis Linfática/diagnóstico , Filariasis/diagnóstico , Wuchereria bancrofti/inmunología , Wuchereria/inmunología , Animales , Ensayo de Inmunoadsorción Enzimática , Humanos , Sueros Inmunes/inmunología , Valor Predictivo de las PruebasRESUMEN
Individuals residing in an area endemic to Wuchereria bancrofti infection were broadly categorised as endemic normals (EN), microfilaraemics (mf + ve) and elephantoids i.e., chronic lymphatic filariasis (EL). The immune status of these three groups was examined in terms of (i) specific antibody levels; (ii) ability to induce antibody dependent cellular cytotoxicity (ADCC) to microfilariae; and (iii) ability to recognise different microfilarial antigens by immunoblotting. All three groups of endemic residents were indistinguishable in their antibody levels as measured by ELISA with B. malayi microfilarial antigen. Many endemic normal sera and most elephantoid sera exerted strong cytotoxicity against W. bancrofti microfilariae whereas none of the mf + ve sera had any such activity. Immunoblotting studies revealed that a protein with mol. wt of 79 KDa was the only one among the proteins of B. malayi microfilarial extracts that was consistently recognised by sera from all endemic residents. Endemic normal sera and elephantoid sera, which exerted maximum cytotoxicity, together specifically recognised three proteins with molecular weights 25, 58 and 68 KDa and these three proteins could be among the candidate antigens that induce resistance to filarial infection.
Asunto(s)
Anticuerpos Antihelmínticos/inmunología , Antígenos Helmínticos/inmunología , Brugia/inmunología , Filariasis Linfática/inmunología , Filariasis/inmunología , Wuchereria bancrofti/inmunología , Wuchereria/inmunología , Animales , Citotoxicidad Celular Dependiente de Anticuerpos , Humanos , ImmunoblottingRESUMEN
BALB/C mice were immunized with heavy or low infection of live B. malayi microfilariae or immunised with different fractions of the microfilarial antigens. Antibody levels and antibody dependent macrophage mediated cytotoxicity to B. malayi microfilariae were determined in the immunized sera. Antigens responsible for induction of antibodies were recognised in B. malayi microfilarial extract by immunoblotting. Appearance of cytotoxic antibodies correlates with recognition of certain common antigens in microfilarial extract such as 45, 54, 62, 66 and 76 KDa mol. wt. proteins.
Asunto(s)
Antígenos Helmínticos/análisis , Brugia/inmunología , Animales , Anticuerpos Antihelmínticos/análisis , Formación de Anticuerpos , Citotoxicidad Celular Dependiente de Anticuerpos , Inmunización , Immunoblotting , Ratones , Ratones Endogámicos BALB C , Microfilarias/inmunologíaRESUMEN
Monoclonal antibodies were produced following immunization of mice with live infective larvae of Brugia malayi. One of these, 46.08.76, is an antibody that promotes adherence of mouse peritoneal macrophages and human peripheral blood leucocytes to the infective larvae of B. malayi and Wuchereria bancrofti, respectively, and kills them. Fresh normal serum, as a source of complement, augments this effect. The same monoclonal antibody conferred 89% protection to jirds (Meriones unguiculatus) against challenge infection of B. malayi stage-three larvae. This monoclonal antibody recognizes antigens of 80,000, 67,000, 52,000 and 36,000 MW proteins present among the antigens of larvae, as detected by an immunoblotting technique. The antibody also reacts with antigens of infective larvae of Litomosoides carinii, Dipetalonema viteae and B. pahangi, but to a smaller extent.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Filariasis/inmunología , Animales , Especificidad de Anticuerpos , Citotoxicidad Celular Dependiente de Anticuerpos , Antígenos Helmínticos/análisis , Brugia/inmunología , Reacciones Cruzadas , Gerbillinae , Inmunización Pasiva , Ratones , Ratones Endogámicos BALB C , Microfilarias/inmunología , Peso MolecularRESUMEN
The binding of 10 different lectins to the surface of microfilariae of Wuchereria bancrofti has been investigated. Wheat germ agglutinin (WGA) and Helix pomatia lectin (HPA) bound specifically to the sheathed microfilariae indicating the presence of N-acetyl-D-glucosamine and N-acetyl-D-galactosamine respectively on the surface. Exsheathed microfilariae did not react with any of the lectins. Treatment of sheathed microfilariae with proteases resulted in increased binding of WGA and HPA. Such treated microfilariae showed a weak binding of Concanavalin A (Con A), and lectins of lentil (LCH) and of Limulus polyphemus (LPA). Sheathed microfilariae incubated with sera of people living in endemic zones of filariasis but with no apparent evidence of infection (endemic normals), or with sera of chronic elephantiasis patients, or with their respective gamma globulin fractions, bound Con A and LCH. These lectins bound weakly to exsheathed microfilariae under the same conditions. Binding was due to the mannose components of the specific immunoglobulins of the sera which coated the microfilariae. However, microfilariae when incubated with sera or their globulin fractions from non-endemic normals (NEN), or from microfilarial carriers, did not bind Con A and LCH, suggesting that specific immunoglobulins were neither present in NEN sera nor in significant amounts in sera of microfilarial carriers.
Asunto(s)
Filarioidea/metabolismo , Lectinas/metabolismo , Microfilarias/metabolismo , Wuchereria bancrofti/metabolismo , Wuchereria/metabolismo , Acetilgalactosamina/farmacología , Animales , Sueros Inmunes/farmacología , Péptido Hidrolasas/farmacología , Wuchereria bancrofti/efectos de los fármacosRESUMEN
Albino rat macrophages and neutrophils, in the presence of fresh normal rat serum as a source of complement, adhered to and promoted killing of Brugia malayi infective larvae in vitro. Eosinophils, by themselves, were marginally cytotoxic at a high cell-target ratio but promoted cytotoxicity when mixed with macrophages. Eosinophil culture supernatants enhanced the macrophage mediated killing of infective larvae. The complement of fresh normal rat serum was found to act by the alternate pathway. Fresh normal rat serum depleted of alternate pathway complement activity by treatment with zymosan A, or of Factor B by heating at 50 C for 20 min, or of Factor D by passing through Sephadex G75 column, failed to promote cell adherence to the parasite. C3 molecules were detected on the surface of infective larvae by immunofluorescence. There was a significant consumption of complement when Brugia malayi infective larvae were incubated in fresh normal rat serum. Albino rat cells were more potent in inducing cytotoxicity to infective larvae in vitro than those from jird or Mastomys natalensis, which may reflect the greater resistance offered by the rat to B. malayi infection. There was much less cellular infiltration on introduction of Brugia malayi infective larvae into the peritoneal cavity of jirds compared to rats and Mastomys natalensis indicating the greater susceptibility of jirds to intraperitoneally induced infections.