RESUMEN
The overexpression of the prostate cancer antigen 3 (PCA3) gene is well-defined as a marker for prostate cancer (PCa) diagnosis. Although widely used in clinical research, PCA3 molecular mechanisms remain unknown. Herein we used phage display technology to identify putative molecules that bind to the promoter region of PCA3 gene and regulate its expression. The most frequent peptide PCA3p1 (80%) was similar to the Rho GTPase activating protein 21 (ARHGAP21) and its binding affinity was confirmed using Phage Bead ELISA. We showed that ARHGAP21 silencing in LNCaP prostate cancer cells decreased PCA3 and androgen receptor (AR) transcriptional levels and increased prune homolog 2 (PRUNE2) coding gene expression, indicating effective involvement of ARHGAP21 in androgen-dependent tumor pathway. Chromatin immunoprecipitation assay confirmed the interaction between PCA3 promoter region and ARHGAP21. This is the first study that described the role of ARHGAP21 in regulating the PCA3 gene under the androgenic pathway, standing out as a new mechanism of gene regulatory control during prostatic oncogenesis.
Asunto(s)
Antígenos de Neoplasias , Proteínas Activadoras de GTPasa , Regulación Neoplásica de la Expresión Génica , Neoplasias de la Próstata , Humanos , Masculino , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Regulación Neoplásica de la Expresión Génica/genética , Línea Celular Tumoral , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Regiones Promotoras Genéticas/genética , Inmunoprecipitación de Cromatina , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Ensayo de Inmunoadsorción EnzimáticaRESUMEN
The overexpression of the prostate cancer antigen 3 (PCA3) gene is well-defined as a marker for prostate cancer (PCa) diagnosis. Although widely used in clinical research, PCA3 molecular mechanisms remain unknown. Herein we used phage display technology to identify putative molecules that bind to the promoter region of PCA3 gene and regulate its expression. The most frequent peptide PCA3p1 (80%) was similar to the Rho GTPase activating protein 21 (ARHGAP21) and its binding affinity was confirmed using Phage Bead ELISA. We showed that ARHGAP21 silencing in LNCaP prostate cancer cells decreased PCA3 and androgen receptor (AR) transcriptional levels and increased prune homolog 2 (PRUNE2) coding gene expression, indicating effective involvement of ARHGAP21 in androgen-dependent tumor pathway. Chromatin immunoprecipitation assay confirmed the interaction between PCA3 promoter region and ARHGAP21. This is the first study that described the role of ARHGAP21 in regulating the PCA3 gene under the androgenic pathway, standing out as a new mechanism of gene regulatory control during prostatic oncogenesis.
RESUMEN
BACKGROUND: Metallothioneins (MTs) gene polymorphisms have been associated with the ability of free radical scavenging and detoxification of heavy metals leading to cancer development. Our aim was to revisit, in a Brazilian population, single-nucleotide polymorphisms (SNPs) of the MT gene family previously associated with oral squamous cell carcinoma (OSCC). MATERIAL AND METHODS: A case-control investigation with 28 OSCC patients and 45 controls was conducted, using conventional risk factors (tobacco use and alcohol consumption) as covariates. SNPs genotyping for rs8052334 (MT1B), rs964372 (MT1B), and rs1610216 (MT2A) was performed by PCR-RFLP, and SNPs for rs11076161 (MT1A) were analyzed by TaqMan assay. RESULTS: The only SNP associated with increased risk for OSCC was the MT-1A AA genotype (OR = 4.7; p = 0.01). We have also evidenced for the first time a significant linkage disequilibrium between the SNPs of MT-2A and MT-1A in this population with the highest frequency (30%) of the unfavorable haplotype G/A/C/T (rs1610216 / rs11076161 / rs964372 / rs8052334) of MT gene polymorphisms (OR = 6.2; p = 0.04). Interestingly, after removing the effects of conventional risk factors, we have uncovered the significance of the AA genotype of the rs11076161 with increased odds of 19-fold higher towards OSCC development. CONCLUSIONS: This is the first demonstration that a significant linkage disequilibrium among gene polymorphisms of the MT family may affect susceptibility to oral cancer, which is conditioned by the G/A/C/T haplotype (rs1610216/rs11076161/rs964372/ rs8052334) and the MT-1A gene polymorphism has a potential clinical utility for the OSCC risk assessment.
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Carcinoma de Células Escamosas , Neoplasias de Cabeza y Cuello , Neoplasias de la Boca , Brasil , Carcinoma de Células Escamosas/genética , Estudios de Casos y Controles , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Metalotioneína/genética , Neoplasias de la Boca/genética , Polimorfismo de Nucleótido Simple , Factores de Riesgo , Carcinoma de Células Escamosas de Cabeza y CuelloRESUMEN
AIMS: Diagnosis of leprosy, a chronic infection caused by Mycobacterium leprae, predominantly depends on clinical manifestations and histopathological analysis, hampering rapid and accurate diagnostics. Our aim was to increase accuracy of leprosy diagnosis by improving M. leprae's DNA detection based on polymerase chain reaction (PCR) technique using new specific primers for the RLEP repetitive sequence. METHODS AND RESULTS: The specific target region, RLEP, of M. leprae's genome was selected based on comparative genomics. After confirming the specificity of this region, using blastn analysis, primers were designed and tested for their in silico specificity. To evaluate the specificity and sensitivity of these primers in vitro, 184 blood samples from patients were used in qPCR. The new primer pair LYON1/LYON2 produced 91% positive samples, whereas the current primer pair LP1/LP2 produced 46%. Specificity and DNA detection limit test were carried out to compare the efficiency of the developed primer pair. The LYON1/LYON2 primer showed 100% specificity, whereas LP1/LP2 showed 64%. The DNA detection limit of LYON1/LYON2 was 10 copies of bacterial genomes per millilitre, whereas LP1/LP2 was 1000 copies of bacterial genomes per millilitre. CONCLUSIONS: In conclusion, the developed LYON1/LYON2 primer pair presented to be a specific and sensitive new molecular marker for the diagnosis of leprosy. SIGNIFICANCE AND IMPACT OF THE STUDY: The development of a specific primer pair for the detection of the M. leprae genome through qPCR technique contributes to a fast, sensitive and specific diagnosis, which is essential to prevent spreading and progression of this disease.
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Lepra/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Mycobacterium leprae/aislamiento & purificación , ADN Bacteriano/genética , Femenino , Genoma Bacteriano/genética , Humanos , Secuencias Repetitivas Esparcidas/genética , Lepra/sangre , Lepra/microbiología , Mycobacterium leprae/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Sensibilidad y EspecificidadRESUMEN
Juvenile idiopathic arthritis (JIA) is a wide group of diseases, characterized by synovial inflammation and joint tissue damage. Due to the delay in the implementation of biomarkers into clinical practice and the association with severe sequels, there is an imperative need for new JIA diagnosis strategies. Electrochemical biosensors based on screen-printed electrodes and peptides are promising alternatives for molecular diagnosis. In this work, a novel biosensor for detecting juvenile idiopathic arthritis (JIA) was developed based on the immobilization of the PRF+1 mimetic peptide, as recognition biological element, on the surface of screen-printed carbon electrode. This biosensor was able to discriminate the JIA positive and negative serum samples from different individuals using differential pulse voltammetry, presenting limits of detection and quantification in diluted samples of 1:784 (v/v) and 1:235 (v/v), respectively. Evaluation by electrochemical impedance spectroscopy showed RCT 3 times higher for JIA positive sample than for a pool of human serum samples from healthy individuals. Surface analysis of the biosensor by atomic force microscopy, after contact with JIA positive serum, presented great globular clusters irregularly distributed. The long-term stability of the biosensor was evaluated, remaining functional for over 40 days of storage (after storage at 8°C). Therefore, a simple, miniaturized and selective biosensor was developed, being the first one based on mimetic peptide and screen-printed carbon electrode, aiming at the diagnosis of the juvenile idiopathic arthritis in real serum samples.
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Artritis Juvenil/diagnóstico , Técnicas Biosensibles/métodos , Péptidos/química , Artritis Juvenil/sangre , Técnicas Biosensibles/instrumentación , Espectroscopía Dieléctrica , Técnicas Electroquímicas/instrumentación , Técnicas Electroquímicas/métodos , Electrodos , Diseño de Equipo , Humanos , Modelos MolecularesRESUMEN
Temporal lobe epilepsy (TLE) is characterized by spontaneous recurrent seizures, starting from secondary functional disorders due to several insults, including self-sustaining continuous seizures identified as status epilepticus (SE). Although hypoglycemia has been associated with SE, the effect of inhibition of the Na(+)/glucose cotransporters (SGLTs) on hippocampus during SE is still unknown. Here we evaluated the functional role of SGLT in the pattern of limbic seizures and neurodegeneration process after pilocarpine (PILO)-induced SE. Vehicle (VEH, 1µL) or phlorizin, a specific SGLT inhibitor (PZN, 1µL, 50µg/µL), was administered in the hippocampus of rats 30min before PILO (VEH+PILO or PZN+PILO, respectively). The limbic seizures were classified using the Racine's scale, and the amount of wet dog shakes (WDS) was quantified before and during SE. Neurodegeneration process was evaluated by Fluoro-Jade C (FJ-C), and FJ-C-positive neurons (FJ-C+) were counted 24h and 15days after SE. The PZN-treated rats showed higher (p<0.05) number of WDS when compared with VEH+PILO. There was no difference in seizure severity between PZN+PILO and VEH+PILO groups. However, the pattern of limbic seizures significantly changed in PZN+PILO. Indeed, the class 5 seizures repeated themselves more times (p<0.05) than the other classes in the PZN group at 50min after SE induction. The PZN+PILO animals had a higher (p<0.05) number of FJ-C+ cells in the dentate gyrus (DG), hilus, and CA3 and CA1 of hippocampus, when compared with VEH+PILO. The PZN+PILO animals had a decreased number (p<0.05) of FJ-C+ cells in CA1 compared with VEH+PILO 15days after SE induction. Taken together, our data suggest that SGLT inhibition with PZN increased the severity of limbic seizures during SE and increased neurodegeneration in hippocampus 24h after SE, suggesting that SGLT1 and SGLT2 could participate in the modulation of earlier stages of epileptogenic processes.
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Hipocampo/efectos de los fármacos , Degeneración Nerviosa/patología , Neuronas/efectos de los fármacos , Florizina/farmacología , Convulsiones/patología , Proteínas de Transporte de Sodio-Glucosa/antagonistas & inhibidores , Estado Epiléptico/patología , Animales , Hipocampo/metabolismo , Hipocampo/patología , Masculino , Degeneración Nerviosa/inducido químicamente , Degeneración Nerviosa/metabolismo , Neuronas/metabolismo , Neuronas/patología , Pilocarpina , Ratas , Ratas Wistar , Convulsiones/inducido químicamente , Convulsiones/metabolismo , Estado Epiléptico/inducido químicamente , Estado Epiléptico/metabolismoRESUMEN
HIV targets the gut mucosa early in infection, causing immune and epithelial barrier dysfunction and disease progression. However, gut mucosal sensing and innate immune signaling through mucosal pattern recognition receptors (PRRs) during HIV infection and disease progression are not well defined. Using the simian immunodeficiency virus (SIV)-infected rhesus macaque model of AIDS, we found a robust increase in PRRs and inflammatory cytokine gene expression during the acute SIV infection in both peripheral blood and gut mucosa, coinciding with viral replication. PRR expression remained elevated in peripheral blood following the transition to chronic SIV infection. In contrast, massive dampening of PRR expression was detected in the gut mucosa, despite the presence of detectable viral loads. Exceptionally, expression of Toll-like receptor 4 (TLR4) and TLR8 was downmodulated and diverged from expression patterns for most other TLRs in the gut. Decreased mucosal PRR expression was associated with increased abundance of several pathogenic bacterial taxa, including Pasteurellaceae members, Aggregatibacter and Actinobacillus, and Mycoplasmataceae family. Early antiretroviral therapy led to viral suppression but only partial maintenance of gut PRRs and cytokine gene expression. In summary, SIV infection dampens mucosal innate immunity through PRR dysregulation and may promote immune activation, gut microbiota changes, and ineffective viral clearance.
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Disbiosis/inmunología , Microbioma Gastrointestinal/inmunología , Infecciones por VIH/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Virus de la Inmunodeficiencia de los Simios/fisiología , Animales , Enfermedad Crónica , Regulación de la Expresión Génica , Infecciones por VIH/microbiología , Humanos , Evasión Inmune , Inmunidad Mucosa , Macaca mulatta , Receptores de Reconocimiento de Patrones/metabolismo , Transducción de Señal , Síndrome de Inmunodeficiencia Adquirida del Simio/microbiología , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismo , Receptor Toll-Like 8/genética , Receptor Toll-Like 8/metabolismo , Carga Viral , Replicación ViralRESUMEN
BACKGROUND: This study aimed to identify novel biomarkers for thyroid carcinoma diagnosis and prognosis. METHODS: We have constructed a human single-chain variable fragment (scFv) antibody library that was selected against tumour thyroid cells using the BRASIL method (biopanning and rapid analysis of selective interactive ligands) and phage display technology. RESULTS: One highly reactive clone, scFv-C1, with specific binding to papillary thyroid tumour proteins was confirmed by ELISA, which was further tested against a tissue microarray that comprised of 229 thyroid tissues, including: 110 carcinomas (38 papillary thyroid carcinomas (PTCs), 42 follicular carcinomas, 30 follicular variants of PTC), 18 normal thyroid tissues, 49 nodular goitres (NG) and 52 follicular adenomas. The scFv-C1 was able to distinguish carcinomas from benign lesions (P=0.0001) and reacted preferentially against T1 and T2 tumour stages (P=0.0108). We have further identified an OTU domain-containing protein 1, DUBA-7 deubiquitinating enzyme as the scFv-binding antigen using two-dimensional polyacrylamide gel electrophoresis and mass spectrometry. CONCLUSIONS: The strategy of screening and identifying a cell-surface-binding antibody against thyroid tissues was highly effective and resulted in a useful biomarker that recognises malignancy among thyroid nodules and may help identify lower-risk cases that can benefit from less-aggressive management.
Asunto(s)
Adenocarcinoma Folicular/metabolismo , Biomarcadores de Tumor/metabolismo , Carcinoma Papilar/metabolismo , Neoplasias de la Tiroides/metabolismo , Proteasas Ubiquitina-Específicas/metabolismo , Adenocarcinoma Folicular/patología , Biomarcadores de Tumor/inmunología , Carcinoma Papilar/patología , Línea Celular Tumoral , Diagnóstico Diferencial , Ensayo de Inmunoadsorción Enzimática , Humanos , Immunoblotting , Biblioteca de Péptidos , Anticuerpos de Cadena Única/química , Neoplasias de la Tiroides/patología , Proteasas Ubiquitina-Específicas/inmunologíaRESUMEN
Leprosy epidemiological studies have been restricted to Mycobacterium leprae DNA detection in nasal and oral mucosa samples with scarce literature on peripheral blood. We present the largest study applying quantitative real-time PCR (qPCR) for the detection of M. leprae DNA in peripheral blood samples of 200 untreated leprosy patients and 826 household contacts, with results associated with clinical and laboratory parameters. To detect M. leprae DNA a TaqMan qPCR assay targeting the M. leprae ML0024 genomic region was performed. The ML0024 qPCR in blood samples detected the presence of bacillus DNA in 22.0% (44/200) of the leprosy patients: 23.2% (16/69) in paucibacillary (PB), and 21.4% (28/131) in multibacillary (MB) patients. Overall positivity among contacts was 1.2% (10/826), with similar percentages regardless of whether the index case was PB or MB. After a follow-up period of 7 years, 26 contacts have developed leprosy. Comparing the results of healthy contacts with those that become ill, ML0024 qPCR positivity at the time of diagnosis of their index case represented an impressive 14.78-fold greater risk for leprosy onset (95% CI 3.6-60.8; p <0.0001). In brief, contacts with positive PCR in blood at diagnosis of index cases are at higher risk of later leprosy onset and this marker might be combined with other prognostic markers for management of contacts, which requires further studies.
Asunto(s)
ADN Bacteriano/sangre , Lepra Multibacilar/sangre , Lepra Multibacilar/transmisión , Lepra Paucibacilar/sangre , Lepra Paucibacilar/transmisión , Mycobacterium leprae/genética , Proteínas Bacterianas/genética , Portador Sano/sangre , Estudios de Seguimiento , Humanos , Lepra Multibacilar/epidemiología , Lepra Paucibacilar/epidemiología , Mycobacterium leprae/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de RiesgoRESUMEN
Equine infectious anemia caused by equine infectious anemia virus is an important disease due to its high severity and incidence in animals. We used a phage display library to isolate peptides that can be considered potential markers for equine infectious anemia diagnosis. We selected peptides using IgG purified from a pool comprised of 20 sera from animals naturally infected with equine infectious anemia virus. The diagnostic potential of these peptides was investigated by ELISA, Western blot and dot blot with purified IgG and serum samples. Based on the results, we chose a peptide mimetic for glycoprotein gp45 epitopes of equine infectious anemia virus, with potential for use as an antigen in indirect diagnostic assays. Synthesis of this peptide has possible applications for the development of new diagnostic tools for this disease.
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Anemia Infecciosa Equina/sangre , Anemia Infecciosa Equina/diagnóstico , Caballos/sangre , Caballos/virología , Péptidos , Secuencia de Aminoácidos , Animales , Western Blotting , Biología Computacional , Ensayo de Inmunoadsorción Enzimática , Inmunoglobulina G/inmunología , Virus de la Anemia Infecciosa Equina/aislamiento & purificación , Datos de Secuencia Molecular , Biblioteca de Péptidos , Péptidos/químicaRESUMEN
Osteopontin splicing isoforms (OPN-SI) present differential expression patterns and specific tumor roles. Our aims were to characterize OPN-SI expression in prostate cancer (PCa) and benign prostate hyperplasia (BPH) tissues, besides evaluating their potential as biomarkers for PCa diagnosis and prognostic implications. Prostatic tissue specimens were obtained from 40 PCa and 30 benign prostate hyperplasia (BPH) patients. Quantitative real time PCR (qRT-PCR) was used to measure OPN-SI mRNA expression. Immunohistochemical analysis was performed using an anti-OPNc polyclonal antibody. Biostatistical analyses evaluated the association of OPN-SI and total Prostate Specific Antigen (PSA) serum levels with clinical and pathological data. PCa tissue samples presented significantly higher levels of OPNa, OPNb and OPNc transcripts (p<0.01) than in BPH specimens. OPN-SI mRNA expression were positively correlated with Gleason Score (p<0.01). ROC curves and logistic regression analyses demonstrated that OPN-SI and PSA were able to distinguish PCa from BPH patients (p<0.01). The OPNc isoform was the most upregulated variant and the best marker to distinguish patients' groups, presenting sensitivity and specificity of 90% and 100%, respectively. Immunohistochemistry analysis also demonstrated OPNc upregulation in PCa samples as compared to BPH tissues. OPNcprotein was also strongly stained PCa tissues presenting High Gleason Score. Multivariate analysis indicated that OPNc expression levels above the cut-off value presented a chance 4-fold higher for PCa occurrence. We conclude that OPN-SI were overexpressed in PCa tissues, strongly associated with PCa occurrence and with tumor cell differentiation. Our results suggest OPNc splicing isoform as an important biomarker contributing to improve PCa diagnosis and prognosis, besides providing insights into early steps of PCa carcinogenesis.
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Osteopontina/genética , Hiperplasia Prostática/genética , Neoplasias de la Próstata/genética , Empalme del ARN/genética , ARN Mensajero/genética , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos , Biomarcadores de Tumor/sangre , Diferenciación Celular , Diagnóstico Diferencial , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Valor Predictivo de las Pruebas , Pronóstico , Hiperplasia Prostática/diagnóstico , Neoplasias de la Próstata/diagnóstico , ARN Mensajero/metabolismo , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Sensibilidad y EspecificidadRESUMEN
Although curable, leprosy requires better diagnostic and prognostic tools to accompany therapeutic strategies. We evaluated the serum samples of leprosy patients from Venezuela and Brazil for reactivity against the specific recombinant proteins, ML0405 and ML2331, and the LID-1 fusion protein that incorporates both of these antigens. Antigen-specific IgG was highest in lepromatous leprosy patients (LL) and decreased across the disease spectrum, such that only a small subset of true tuberculoid patients (TT) tested positive. The impact of multidrug therapy (MDT) on these antibody responses was also examined. Several years after treatment, the vast majority of Venezuelan patients did not possess circulating anti-LID-1, anti-ML0405, and anti-ML2331 IgG, and the seropositivity of the remaining cases could be attributed to irregular treatment. At discharge, the magnitude and proportion of positive responses of Brazilian patients against the proteins and phenolic glycolipid (PGL)-I were lower for most of the clinical forms. The monthly examination of IgG levels in LL patient sera after MDT initiation indicated that these responses are significantly reduced during treatment. Thus, responses against these antigens positively correlate with bacillary load, clinical forms, and operational classification at diagnosis. Our data indicate that these responses could be employed as an auxiliary tool for the assessment of treatment efficacy and disease relapse.
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Anticuerpos Antibacterianos/sangre , Monitoreo de Drogas/métodos , Inmunoglobulina G/sangre , Lepra/diagnóstico , Antibacterianos/uso terapéutico , Antígenos Bacterianos , Brasil , Humanos , Lepra/tratamiento farmacológico , Estudios Longitudinales , Proteínas Recombinantes , Recurrencia , Factores de Tiempo , Resultado del Tratamiento , VenezuelaRESUMEN
Candidate genes have been associated with milk production in bovines, such as the diacylglycerol O-acyltransferase 1 (DGAT1) and leptin (LEP); however, they have not been simultaneously investigated nor have been evaluated in the Brazilian Girolando breed (Gir×Holstein, backcrossed to Holstein). Our aim was to determine the influence of fat-related genes, DGAT1 and LEP, and their polymorphisms on performance traits of milk production in the Girolando breed. Results indicated that the K allele of the DGAT1 gene showed a significant association with total and average daily milk production with additive effect. The LEP gene showed that the A allele and its homozygote are highly prevalent and almost fixed in this population and may have been favorably selected during backcrossing for the origin of this breed. The important impact of the K allele of the DGAT1 gene on milk production corroborates the initiative of performing marker-assisted selections with this gene in breeding programs of the Girolando breed.
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Bovinos/genética , Bovinos/fisiología , Diacilglicerol O-Acetiltransferasa/metabolismo , Leptina/metabolismo , Polimorfismo Genético , Animales , Industria Lechera , Diacilglicerol O-Acetiltransferasa/genética , Femenino , Regulación de la Expresión Génica , Lactancia/genética , Lactancia/fisiología , Leptina/genética , Reacción en Cadena de la Polimerasa/métodos , Reacción en Cadena de la Polimerasa/veterinaria , Polimorfismo de Longitud del Fragmento de Restricción , Polimorfismo Conformacional Retorcido-SimpleRESUMEN
Neurocysticercosis (NC), caused by Taenia solium metacestode, infects the central nervous system and is a devastating parasitic infection. Diagnosis is based on symptoms, imaging, serology and epidemiology. Current markers present variable sensitivity and specificity, frequent cross-reactions and are not able to discriminate NC clinical forms. The aim of this study was to select mimotopes of T. solium metacestode antigens that may be used in NC immunodiagnosis, specifically to discriminate between active and inactive forms. A random peptide phage display library was screened against IgY from chickens immunized with total saline extract from T. solium metacestodes and validated against 110 serum samples, classified into active NC (18), inactive NC (22), cross-reactive parasitic diseases (40) and healthy controls (30). We have successfully selected seven peptides with significant immunoreactivity to IgG of NC patients, with sensitivity ranging from 95.5% to 100% to detect the inactive form and specificity varied from 85.7% to 94.3%. One phage-displayed peptide (Cc48) can be directly used as biomarker to distinguish inactive from active forms with an accuracy of 95.7%, and this novel mimotope may also be used as an auxiliary tool to neuroimaging tests and treatment follow-up.
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Anticuerpos Antihelmínticos/sangre , Neurocisticercosis/diagnóstico , Neurocisticercosis/inmunología , Parasitología/métodos , Biblioteca de Péptidos , Péptidos , Taenia solium/inmunología , Animales , Pollos , Humanos , Inmunoglobulina G/sangre , Péptidos/aislamiento & purificación , Sensibilidad y Especificidad , Pruebas Serológicas/métodos , Suero/químicaRESUMEN
Leprosy is an important health problem in Brazil despite extensive use of multidrug therapy. The nasal mucosa is the preferential site of entry and exit of Mycobacterium leprae, and although lesions have been found in the oral mucosa, its potential involvement in the transmission of leprosy bacilli has never been investigated. We investigated the presence of the M. leprae DNA in buccal swabs of leprosy patients (334) and household contacts (1288) through polymerase chain reaction (PCR), and correlated this with clinical and laboratorial evaluations. The overall positivity for patients and contacts was 18.26% and 6.83%, respectively. Subclinical infection among contacts was considered when PCR and anti-PGL-1 ELISA presented positive results. This study provides evidence that the oral mucosa may be a secondary site of M. leprae transmission and infection, and contacts with bacillary DNA may be actively involved in transmission. We have also shown that bacilli DNA is more frequently found in the oral mucosa of PB patients. Our findings have great epidemiological relevance and indicate an additional strategy for leprosy control programmes and dental clinics.
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ADN Bacteriano/aislamiento & purificación , Lepra/diagnóstico , Lepra/microbiología , Mucosa Bucal/microbiología , Mycobacterium leprae/aislamiento & purificación , Adulto , Anticuerpos Antibacterianos/sangre , Brasil , Estudios Transversales , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mycobacterium leprae/genética , Mycobacterium leprae/inmunología , Reacción en Cadena de la PolimerasaRESUMEN
Peptides derived from a phage display library may mimic essential features of epitopes (mimotopes), including their immunogenicity. A recombinant peptide library of 12 amino acids displayed on the phage capsid was used to obtain peptides that mimic epitopes of antigens that are reactive to specific polyclonal antibodies anti-neuwiedase (NEU), a toxin from Bothrops neuwiedi snake venom. These polyclonal antibodies are protective against NEU activity and were used as target for the peptide library biopannings, resulting in the selection of 80 peptides. Antibody-binding epitopes were obtained by sequence alignment with the primary and tertiary structures of the NEU protein. Antigenicity and specificity of the mimotopes mixture were confirmed by dot blot, immuno dot blot, plaque reduction and Western blot assays. Their immunogenicity was demonstrated by immunization of BALB/c mice and ELISA tests. The NEU toxin is an important antigen that has many common structural regions to several toxic venom metalloproteinases, in which two epitope regions have been detected. The two mapped epitopes were found in primary sequences of several snake venom toxins, thus demonstrating the potential application of these NEU mimotopes as possible antigen components that are toxicity free.
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Bothrops/fisiología , Venenos de Crotálidos/enzimología , Epítopos/química , Epítopos/inmunología , Metaloendopeptidasas/metabolismo , Venenos de Víboras/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos , Biología Computacional , Venenos de Crotálidos/inmunología , Epítopos/genética , Metaloendopeptidasas/química , Ratones , Modelos Moleculares , Biblioteca de Péptidos , Péptidos , Unión Proteica , Conformación Proteica , Conejos , Venenos de Víboras/químicaRESUMEN
Pregnancy loss can be caused by several factors involved in human reproduction. Although up to 50% of cases remain unexplained, it has been postulated that the major cause of failed pregnancy is an error of embryo implantation. Transmembrane mucin-1 (MUC-1) is a glycoprotein expressed on the endometrial cell surface which acts as a barrier to implantation. The gene that codes for this molecule is composed of a polymorphic tandem repeat of 60 nucleotides. Our objective was to determine if MUC-1 genetic polymorphism is associated with implantation failure in patients with a history of recurrent abortion. The study was conducted on 10 women aged 25 to 35 years with no history of successful pregnancy and with a diagnosis of infertility. The control group consisted of 32 patients aged 25 to 35 years who had delivered at least two full-term live children and who had no history of abortions or fetal losses. MUC-1 amplicons were obtained by PCR and observed on agarose and polyacrylamide gel after electrophoresis. Statistical analysis showed no significant difference in the number of MUC-1 variable number of tandem repeats between these groups (P > 0.05). Our results suggest that there is no effect of the polymorphic MUC-1 sequence on the implantation failure. However, the data do not exclude MUC-1 relevance during embryo implantation. The process is related to several associated factors such as the mechanisms of gene expression in the uterus, specific MUC-1 post-translational modifications and appropriate interactions with other molecules during embryo implantation.
Asunto(s)
Aborto Habitual/genética , Implantación del Embrión/genética , Infertilidad Femenina/genética , Mucina-1/genética , Polimorfismo Genético , Adulto , Estudios de Casos y Controles , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Reacción en Cadena de la Polimerasa , EmbarazoRESUMEN
Pregnancy loss can be caused by several factors involved in human reproduction. Although up to 50 percent of cases remain unexplained, it has been postulated that the major cause of failed pregnancy is an error of embryo implantation. Transmembrane mucin-1 (MUC-1) is a glycoprotein expressed on the endometrial cell surface which acts as a barrier to implantation. The gene that codes for this molecule is composed of a polymorphic tandem repeat of 60 nucleotides. Our objective was to determine if MUC-1 genetic polymorphism is associated with implantation failure in patients with a history of recurrent abortion. The study was conducted on 10 women aged 25 to 35 years with no history of successful pregnancy and with a diagnosis of infertility. The control group consisted of 32 patients aged 25 to 35 years who had delivered at least two full-term live children and who had no history of abortions or fetal losses. MUC-1 amplicons were obtained by PCR and observed on agarose and polyacrylamide gel after electrophoresis. Statistical analysis showed no significant difference in the number of MUC-1 variable number of tandem repeats between these groups (P > 0.05). Our results suggest that there is no effect of the polymorphic MUC-1 sequence on the implantation failure. However, the data do not exclude MUC-1 relevance during embryo implantation. The process is related to several associated factors such as the mechanisms of gene expression in the uterus, specific MUC-1 post-translational modifications and appropriate interactions with other molecules during embryo implantation.
Asunto(s)
Adulto , Femenino , Humanos , Embarazo , Aborto Habitual/genética , /genética , Implantación del Embrión/genética , Infertilidad Femenina/genética , Polimorfismo Genético , Estudios de Casos y Controles , Electroforesis en Gel de Poliacrilamida , Reacción en Cadena de la PolimerasaRESUMEN
The transmembrane mucin glycoprotein (MUC1) has an anti-adhesive role, and functions to maintain a non-receptive uterine state. A polymorphic variation of the MUC1 gene has been associated with female infertility due to suspected failure of embryo implantation, based on the significant greater size of the lower allele observed in infertile women. The aim of this study was to confirm this preliminary observation using long polymerase chain reaction (PCR), which has amplified the 60-bp polymorphic variable number of tandem repeat (VNTR) associated to the binding domain of the MUC1 glycoprotein. DNA samples were obtained from 20 women, 10 fertile and 10 infertile, and the VNTR region was amplified through a long PCR procedure. The VNTR size range from 1.6 to 2.9 kb (22-44 motifs). The average size for the lower allele was 1.69 kb for both groups, and for the upper allele was 2.35 and 2.49 kb (P > 0.05) for fertile and infertile groups respectively. The VNTR polymorphism of the MUC1 gene was not associated with female infertility, although its significance cannot be discarded. It is suggested that other regulatory molecules and signals may interact with the MUC1 gene variations, favouring endometrial receptivity and embryo attachment.