RESUMEN
BACKGROUND: Immunological characterization of mycobacterial peptides may help not only in the preparation of a vaccine for leprosy but also in developing in vitro T-cell assays that could perhaps be used as an in vitro correlate for treatment outcome. The main goal of this study was to evaluate the use of Mycobacterium bovis recombinant 32-kDa protein (r32-kDa) antigen-stimulated T-cell assay as a surrogate marker for treatment outcome and monitor vitamin D receptor (VDR)-mediated anti-microbial responses during multidrug therapy (MDT) in leprosy. METHODS: Newly diagnosed tuberculoid and lepromatous leprosy patients were enrolled and followed up during their course of MDT at 6 and 12 months. IFN-γ, IL-10, IL-17 and IL-23 levels in culture supernatants and expression of VDR, TLR2, LL37 and DEFB in r32-kDa-stimulated PBMCs were measured. Controls comprised household contacts (HHCs) and healthy endemic subjects (HCs). RESULTS: Significant differences were observed in the levels of IFN-γ, IL-17, IL-23, VDR and anti-microbial peptides LL37 and DEFB after treatment and when compared with that of HHCs and HCs, respectively. CONCLUSIONS: These findings suggest that responses to r32-kDa antigen reflect an improved immunological and anti-microbial response in leprosy patients during therapy, thereby indicating its potential use as an immune correlate in the treatment of leprosy patients.
Asunto(s)
Antígenos Bacterianos/farmacología , Proteínas Bacterianas/farmacología , Citocinas/inmunología , Lepra/inmunología , Mycobacterium bovis , Linfocitos T/inmunología , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Péptidos Catiónicos Antimicrobianos , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Catelicidinas/inmunología , Femenino , Estudios de Seguimiento , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Humanos , Lepra/patología , Masculino , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/farmacología , Linfocitos T/patología , Receptor Toll-Like 2/inmunologíaRESUMEN
BACKGROUND: Vitamin D Receptor (VDR) is a transacting transcription factor which mediates immunomodulatory function and plays a key role in innate and adaptive immune responses through its ligand and polymorphisms in VDR gene may affect its regulatory function. OBJECTIVE: To investigate the association of three VDR gene polymorphisms (TaqI rs731236, FokI rs2228570 and ApaI rs7975232) with leprosy. METHODS: The study group includes 404 participants of which 222 were leprosy patients (paucibacillary=87, multibacillary=135) and 182 healthy controls. Genotyping was done using PCR-RFLP technique. Statistical analysis was performed using SNP Stats and PLINK software. RESULTS: The VDR FokI (rs2228570) ff genotype, ApaI (rs7975232) AA, Aa genotype and haplotype T-f-a, T-F-A were positively associated with leprosy when compared to healthy controls. CONCLUSION: The two variants at Fok and Apa positions in VDR gene are significantly associated with leprosy. Genotypes at FokI (ff), ApaI (aa) and haplotype (T-F-a, T-f-a) may contribute to the risk of developing leprosy by altering VDR phenotype/levels subsequently modulation of immune response.
Asunto(s)
Enzimas de Restricción del ADN/química , Predisposición Genética a la Enfermedad , Lepra/genética , Polimorfismo de Nucleótido Simple , Receptores de Calcitriol/genética , Alelos , Estudios de Casos y Controles , Femenino , Expresión Génica , Haplotipos , Humanos , Lepra/inmunología , Lepra/patología , Masculino , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Receptores de Calcitriol/inmunología , RiesgoRESUMEN
Leprosy is a chronic granulomatous infection caused by the obligate intracellular organism Mycobacterium leprae. TLR2 plays a key role when activated by M. leprae lipoproteins initiating protective responses which induce bacterial killing and therefore control of disease spread. Microsatellite polymorphisms in intron2 of TLR2 gene have been reported to be associated with development of clinical features of several infectious diseases. The study aims to evaluate the influence of GT microsatellite on the expression of TLR2 which could make humans prone to M. leprae infections. A total of 279 individuals were enrolled in the study, 88 were leprosy patients, 95 were house hold contacts (HHC) and 96 were healthy controls (HC). Genotyping was done using PCR-Sequencing method. TLR2 mRNA expression was analyzed by RT-PCR. IL-10 and IFN-γ levels were measured using ELISA in MLSA stimulated cell culture supernatants. Statistical analysis was performed using Chi-Square (χ(2)) test and t-tests. Allele/genotype of TLR2 microsatellite which includes longer GT repeats was associated with low TLR2 mRNA expression and high IL-10 production while that including shorter GT repeats was associated with high TLR2 mRNA expression and low IL-10 production. High IL10 producing allele of TLR2 microsatellite might predispose house hold contacts to leprosy.
Asunto(s)
Intrones , Lepra/genética , Repeticiones de Microsatélite , Polimorfismo Genético , Receptor Toll-Like 2/genética , Alelos , Estudios de Casos y Controles , Repeticiones de Dinucleótido , Expresión Génica , Frecuencia de los Genes , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Interferón gamma/biosíntesis , Interleucina-10/biosíntesis , Lepra/metabolismo , Leucocitos Mononucleares/metabolismo , ARN Mensajero/genéticaRESUMEN
Fragile X syndrome (FRAXA) is one of the most common forms of mental retardation. It is caused by the expansion of cytosine-guanine-guanine (CGG) repeats in the 5' untranslated region of the fragile X mental retardation 1 (FMR1) gene, located at Xq27.3. The number of CGG repeats in the FMR1 gene occurs in four distinct ranges: 2-50 (normal), 50-60 (gray zone), 60-200 (premutation), and > 200 (full mutation). When the number of CGG repeats exceeds 200, the gene becomes hypermethylated and transcriptionally silenced, which results in the loss of FMR protein and causes FRAXA. The key clinical features of FRAXA are mental retardation, macro-orchidism, long face, prominent jaw, connective tissue abnormalities, and behavioral problems. A modified 15-item checklist was used to assess the clinical features in 337 individuals (316 males and 21 females) who have mental retardation of unknown etiology. These patients were in institutions. Molecular diagnosis was performed using polymerase chain reaction and Southern blot analysis and revealed that 14 males were positive for FRAXA. Studies of the families of the affected males revealed an additional 11 affected males and 20 carrier females. Retrospective analysis of clinical features was performed in a total of 327 males and 41 females. Six clinical features were statistically significant in FRAXA individuals when compared to non-FRAXA individuals. These features were hyperactivity (p<0.05), poor eye contact (p<0.001), hyper extensibility of joints (p<0.001), large ears (p<0.001), macro-orchidism (p<0.001), and a family history of mental retardation (p<0.001). When a total score of 5 out of 15 was used as the threshold clinical score, 73.18% of the patients with total scores < 5 could be eliminated as FRAXA-negative patients, thereby improving the reliability of FRAXA testing using the clinical checklist.
Asunto(s)
Síndrome del Cromosoma X Frágil/diagnóstico , Síndrome del Cromosoma X Frágil/fisiopatología , Adolescente , Niño , Preescolar , Femenino , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/genética , Pruebas Genéticas , Humanos , India , Masculino , Factores de Riesgo , Índice de Severidad de la Enfermedad , Evaluación de la Tecnología Biomédica , Expansión de Repetición de Trinucleótido/genética , Adulto JovenRESUMEN
BACKGROUND: Leprosy is an infectious disease in which the susceptibility to the pathogen Mycobacterium leprae and the clinical manifestations are attributed to host immune cell response. Receptor mediated events and signalling in the immune cells are mediated by protein phosphorylation. The main signalling pathways and protein kinases known to be involved in the regulation of immune cells are cAMP dependent kinases, calcium/calmodulin dependent kinases, protein kinase C and mitogen activated protein kinases. The cumulative consequence of alterations in signalling pathways can be evaluated by intrinsic cellular protein phosphorylation by gamma-P32 ATP. The present study was designed to assess the protein phosphorylation in the immune cells of leprosy patients as compared with normal individuals. METHODOLOGY: Lymphocyte protein phosphorylation was conducted in 15 leprosy patients and 9 normal individuals. Protein phosphorylation of lymphocytes was carried out in the presence/absence of protein kinase modulators. The phosphorylation patterns were documented and analysed consequent to SDS-PAGE, staining, destaining, drying and autoradiography. RESULTS: The major phosphorylated proteins in lymphocytes were of molecular weights 20-22, 24-29, 30-35, 43, 46-50 and 66-68 kDa. In general, the major phosphorylated proteins were similar in the controls and in the patients. The phosphorylatability of these proteins varied with different modulators. Variations in the phosphorylation pattern were observed in 25% of the leprosy patients where there was a decrease of the 66 kDa protein and a decrease of 20-22 kDa protein phosphorylation. CONCLUSION: The observed alterations in the protein phosphorylation pattern could be due to alteration in kinases and/or their substrates or due to the effect of M. leprae on immune cells.
Asunto(s)
Lepra/inmunología , Fosfoproteínas/metabolismo , Linfocitos T/metabolismo , Autorradiografía , Estudios de Casos y Controles , Humanos , Fosforilación , Proteínas Quinasas/metabolismoRESUMEN
OBJECTIVES: In an analysis of enzymes in easily accessible tissues like blood cells, serum can provide a valuable information and a simple tool for disease and carrier detection. In the study presented we have analyzed calcineurin activity in Duchenne muscular dystrophy (DMD) and carrier sera and lymphocytes for its diagnostic value and its status in DMD pathology. DESIGN AND METHODS: We have monitored calcineurin activity in sera and lymphocytes of DMD, in carriers and in controls using colorimetric method by following the p-nitrophenol released in the presence and absence of Trifluoperazine (TFP), an inhibitor of calcineurin. RESULTS: Results showed a significant decrease in serum and lymphocyte calcineurin activity in DMD (p<0.001) without alteration in carriers compared to normal. CONCLUSION: Further studies are required to understand possible alterations mediated by calcineurin with reference to DMD lymphocytes as any alteration in phosphorylation/dephosphorylation pathway can disturb the normal functioning of these cells. The decreased calcineurin activity observed in DMD serum compared with controls could be further examined for its diagnostic utility.
Asunto(s)
Calcineurina/sangre , Distrofia Muscular de Duchenne/sangre , Adulto , Análisis de Varianza , Niño , Preescolar , Femenino , Humanos , Linfocitos/metabolismo , Masculino , Malondialdehído/sangre , Distrofia Muscular de Duchenne/diagnóstico , Superóxido Dismutasa/sangreRESUMEN
BACKGROUND: Calpain II is an calcium-dependent cysteine protease involved in essential regulatory or processing functions of the cell, mediated by physiological concentrations of Ca(2+). However, in an environment of abnormal intracellular calcium as in Duchenne muscular dystrophy (DMD), calpain is suggested to cause membrane alterations. METHODS: Twelve individuals with dystrophin gene deletion and an equal number of age and sex matched controls were chosen for the study. The expression pattern of calpain II (both at RNA and protein levels), its cellular location upon activation and its activity in lymphocytes were specifically assessed to know if our earlier report of increased calpain activity in DMD lymphocytes is a result of de novo synthesis or is due to basic defect in calcium handling. RESULTS: We found a significant increase in the expression, alteration in calpain II distribution and increased activity of this enzyme. CONCLUSION: Membrane abnormalities and altered signaling pathways observed in DMD lymphocytes may be due to increased association of calpain II onto membrane and cytosol.
Asunto(s)
Calpaína/genética , Calpaína/metabolismo , Regulación Enzimológica de la Expresión Génica/genética , Linfocitos/metabolismo , Distrofia Muscular de Duchenne/genética , Calpaína/aislamiento & purificación , Membrana Celular/enzimología , Citosol/enzimología , Perfilación de la Expresión Génica , Humanos , Linfocitos/enzimología , Distrofia Muscular de Duchenne/enzimología , Distrofia Muscular de Duchenne/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Fracciones Subcelulares/enzimologíaRESUMEN
Phospholipid-dependent, Ca(2+)-independent isoenzymes termed novel protein kinase C or nPKC, include PKC delta, epsilon, eta, theta and mu. Status and role of nPKC and PKC theta in Duchenne muscular dystrophic (DMD) condition is unknown. In the present study, we have shown that most of the nPKC isoforms are translocated to the membrane fraction of DMD tissue specimen. It is well established that translocation plays a key role in signal transduction by individual PKC isoforms. In our experiment, the increased association of nPKC isoform PKC theta to membrane was further confirmed by Western blot. Increased expression of PKC theta mRNA was identified by dot blot analysis. The above results suggest that, the alterations in nPKC location and increased expression of PKC theta observed is a result of modification of PKC-mediated signal transduction and cell function.