RESUMEN
Recently, a salivary gland transcriptome study demonstrated that the transcripts of a putative cystatin gene (SeqID AAEL013287; Aacystatins) from Aedes aegypti were increased in DENV2-infected mosquitoes and that silencing of the Aacystatin gene resulted in an increase in DENV titres. In this work, Aacystatin was biochemically characterized; the purified recombinant inhibitor was able to inhibit typical cysteine proteases with a Ki in the nM range. Pulldown assays using Aag2 cell extracts identified a cathepsin L-like peptidase (AaCatL) as a possible target of Aacystatin. Purified recombinant AaCatL had an optimal pH of 5.0 and displayed a preference for Leu, Val and Phe residues at P2, which is common for other cathepsin L-like peptidases. Transcription analysis of Aacystatin and AaCatL in the salivary glands and midgut of DENV2-infected mosquitoes revealed a negative correlation between DENV2 titres and levels of the inhibitor and peptidase, suggesting their involvement in DENV2-mosquito interactions. Considering that apoptosis may play an important role during viral infections, the possible involvement of Aacystatin in staurosporine-induced apoptosis in Aag2 cells was investigated; the results showed higher expression of the inhibitor in treated cells; moreover, pre incubation with rAacystatin was able to increase Aag2 cell viability.
Asunto(s)
Aedes , Catepsina L , Cistatinas , Virus del Dengue/metabolismo , Proteínas de Insectos , Aedes/enzimología , Aedes/genética , Aedes/virología , Animales , Catepsina L/química , Catepsina L/genética , Catepsina L/metabolismo , Línea Celular , Cistatinas/química , Cistatinas/genética , Cistatinas/metabolismo , Proteínas de Insectos/química , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismoRESUMEN
INTRODUCTION: Receptor tyrosine kinases have been implicated in various vascular remodeling processes and cardiovascular disease. However, their role in the regulation of vascular tone is poorly understood. Herein, we evaluate the contribution of c-Kit signaling to vasoactive responses. METHODS: The vascular reactivity of mesenteric arteries was assessed under isobaric conditions in c-Kit deficient (KitW/W-v) and littermate control mice (Kit+/+) using pressure myography. Protein levels of soluble guanylyl cyclase beta 1 (sGCß1) were quantified by Western blot. Mean arterial pressure was measured after high salt (8% NaCl) diet treatment using the tail-cuff method. RESULTS: Smooth muscle cells (SMCs) from c-Kit deficient mice showed a 5-fold downregulation of sGCß1 compared to controls. Endothelium-dependent relaxation of mesenteric arteries demonstrated a predominance of prostanoid vs. nitric oxide (NO) signaling in both animal groups. The dependence on prostanoid-induced dilation was higher in c-Kit mutant mice than in controls, as indicated by a significant impairment in vasorelaxation with indomethacin with respect to the latter. Endothelium-independent relaxation showed significant dysfunction of NO signaling in c-Kit deficient SMCs compared to controls. Mesenteric artery dilation was rescued by addition of a cGMP analog, but not with a NO donor, indicating a deficiency in cGMP production in c-Kit deficient SMCs. Finally, c-Kit deficient mice developed higher blood pressure on an 8% NaCl diet compared to their control littermates. CONCLUSION: c-Kit deficiency inhibits NO signaling in SMCs. The existence of this c-Kit/sGC signaling axis may be relevant for vascular reactivity and remodeling.
Asunto(s)
Miocitos del Músculo Liso/metabolismo , Óxido Nítrico/metabolismo , Proteínas Proto-Oncogénicas c-kit/deficiencia , Transducción de Señal , Animales , Arterias/efectos de los fármacos , Arterias/fisiología , Presión Sanguínea/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiología , Ratones , Prostaglandinas/farmacología , Proteínas Proto-Oncogénicas c-kit/metabolismo , Transducción de Señal/efectos de los fármacos , Cloruro de Sodio Dietético , Vasodilatación/efectos de los fármacosRESUMEN
Introduction: Helicobacter pylori strains expressing cytotoxic CagA protein are more commonly associated with peptic ulceration, atrophic gastritis and gastric adenocarcinoma than those lacking CagA. Determination of anti-CagA antibodies, therefore, acquires a relevant clinical significance in the serological detection of H. pylori infection and disease risk prediction. However, the CagA-serology has been questioned due to the differences found in their performance evaluations in different populations. Objective: To obtain a recombinant CagA fragment useful for serodiagnosis of H. pylori infection Methods: A fragment of the cagA gene was cloned into a prokaryotic T7 RNA polymerase expression vector. A recombinant C-terminal His 6 -tagged CagA was expressed, subsequently solubilized with urea and purified by immobilized metal affinity chromatography. The performance of the recombinant protein was evaluated using 180 human serum samples with an in-house Western blot assay compared to the Helicoblot 2.1 reference test. Results: The expressed His 6 -tagged CagA showed an immunoreactive 80kDa band as was revealed by SDS-PAGE and Western blot analysis using two different specific anti-CagA polyclonal antibodies. The recombinant protein was successfully purified obtaining a 93% of purity. The performance analysis of the purified recombinant antigen showed good immunoreactivity and exhibited values of sensitivity, specificity and accuracy of 88.1%, 100% and 92.7%, respectively. Conclusion: The CagA fragment of the study may constitute a useful tool for serological diagnosis of CagA-positive H. pylori infection.
Introducción. Las cepas de Helicobacter pylori que expresan la citotoxina CagA, se asocian más frecuentemente con úlcera péptica, gastritis atrófica y adenocarcinoma gástrico que las que carecen de esta citotoxina. Por lo anterior, el determinar la presencia de anticuerpos anti-CagA adquiere gran importancia clínica en la detección serológica de la infección por H. pylori y la predicción del riesgo de enfermedades. Sin embargo, los métodos serológicos que emplean CagA han sido cuestionados debido a las diferencias encontradas en las evaluaciones de su desempeño en diversas poblaciones. Objetivo. Obtener un fragmento recombinante de la proteína CagA para el serodiagnóstico de la infección por H. pylori . Materiales y métodos. Un fragmento del gen cagA fue clonado en un vector de expresión procariota que contenía el promotor de la T7 ARN polimerasa. El fragmento de la proteína CagA con seis histidinas en la región C-terminal, se expresó, se solubilizó con urea y se purificó por cromatografía de afinidad con iones metálicos inmovilizados. El desempeño de la proteína recombinante se evaluó empleando un método in house de Western Blot y 180 sueros humanos. Los resultados se compararon con la prueba de referencia Helicoblot 2.1. Resultados. La proteína CagA expresada mostró una banda inmunorreactiva de 80 kDa en el Western Blot al emplear dos anticuerpos policlonales anti-CagA específicos. La proteína recombinante fue purificada hasta un 93 % de pureza y el análisis de desempeño del antígeno recombinante purificado mostró buena inmunorreacción y exhibió valores de sensibilidad, especificidad y exactitud de 88,1 %, 100 % y 92,7 %, respectivamente. Conclusiones. El fragmento de la proteína CagA del estudio puede constituir una herramienta útil para el diagnóstico serológico de la infección por cepas de H. pylori positivas para CagA.
Asunto(s)
Adolescente , Adulto , Humanos , Persona de Mediana Edad , Adulto Joven , Antígenos Bacterianos/sangre , Proteínas Bacterianas/sangre , Infecciones por Helicobacter/sangre , Infecciones por Helicobacter/diagnóstico , Helicobacter pylori , Antígenos Bacterianos/biosíntesis , Antígenos Bacterianos/genética , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Clonación Molecular , Expresión Génica , Helicobacter pylori/genética , Helicobacter pylori/metabolismo , Proteínas Recombinantes , Pruebas SerológicasRESUMEN
Pattern-recognition receptors have been highly conserved in evolution. They recognize danger signals including both pathogen- and damage-associated molecular patterns, also known as alarmins. Several signaling pathways leading to an inflammatory reaction as part of an effective defensive response, are thus triggered. RAGE, a receptor initially considered for advanced glycation end-products, is also known to be activated by several danger signals, thus functioning as a pattern-recognition receptor. As a new member of this family, attempts to unraveling its functioning show that RAGE activation not only results in innate immune response but also contributes to promote and shape the acquired immune reaction. As reported for other members of the family, RAGE presents many polymorphic variants and additional studies are needed to elucidate its significance in immune response and disease susceptibility. Here we describe recent advances unraveling RAGE functions, as well as its significance and challenges in immunobiology.
Asunto(s)
Enfermedades Cardiovasculares/inmunología , Nefropatías Diabéticas/inmunología , Productos Finales de Glicación Avanzada/metabolismo , Polimorfismo Genético , Receptores Inmunológicos/genética , Inmunidad Adaptativa , Enfermedades Cardiovasculares/genética , Enfermedades Cardiovasculares/patología , Nefropatías Diabéticas/genética , Nefropatías Diabéticas/patología , Regulación de la Expresión Génica , Humanos , Inmunidad Innata , Inflamación , Ligandos , Especificidad de Órganos , Receptor para Productos Finales de Glicación Avanzada , Receptores Inmunológicos/inmunología , Transducción de SeñalRESUMEN
INTRODUCTION: Helicobacter pylori strains expressing cytotoxic CagA protein are more commonly associated with peptic ulceration, atrophic gastritis and gastric adenocarcinoma than those lacking CagA. Determination of anti-CagA antibodies, therefore, acquires a relevant clinical significance in the serological detection of H. pylori infection and disease risk prediction. However, the CagA-serology has been questioned due to the differences found in their performance evaluations in different populations. OBJECTIVE: To obtain a recombinant CagA fragment useful for serodiagnosis of H. pylori infection METHODS: A fragment of the cagA gene was cloned into a prokaryotic T7 RNA polymerase expression vector. A recombinant C-terminal His 6 -tagged CagA was expressed, subsequently solubilized with urea and purified by immobilized metal affinity chromatography. The performance of the recombinant protein was evaluated using 180 human serum samples with an in-house Western blot assay compared to the Helicoblot 2.1 reference test. RESULTS: The expressed His 6 -tagged CagA showed an immunoreactive 80kDa band as was revealed by SDS-PAGE and Western blot analysis using two different specific anti-CagA polyclonal antibodies. The recombinant protein was successfully purified obtaining a 93% of purity. The performance analysis of the purified recombinant antigen showed good immunoreactivity and exhibited values of sensitivity, specificity and accuracy of 88.1%, 100% and 92.7%, respectively. CONCLUSION: The CagA fragment of the study may constitute a useful tool for serological diagnosis of CagA-positive H. pylori infection.
Asunto(s)
Antígenos Bacterianos/sangre , Proteínas Bacterianas/sangre , Infecciones por Helicobacter/sangre , Infecciones por Helicobacter/diagnóstico , Helicobacter pylori , Adolescente , Adulto , Antígenos Bacterianos/biosíntesis , Antígenos Bacterianos/genética , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Clonación Molecular , Expresión Génica , Helicobacter pylori/genética , Helicobacter pylori/metabolismo , Humanos , Persona de Mediana Edad , Proteínas Recombinantes , Pruebas Serológicas , Adulto JovenRESUMEN
INTRODUCTION: It is known that polymorphisms in C-terminal region of CagA influence gastric disease development on Helicobacter pylori infection. Additionally, the geographic distribution of these polymorphisms has been associated with the appearance of more severe gastroduodenal pathologies. Objective. To determine the CagA phosphorylation motifs pattern (EPIYA pattern) in Cuban H. pylori isolates, and to study its association with patient´s pathologies. MATERIALS AND METHODS: DNAs from 95 H. pylori cagA-positive strains were used to amplify the 3´ variable region of cagA gene by PCR using two different strategies. Additionally, new primers were designed to identify either Western or Eastern CagAEPIYA motiftype by PCR. To confirm the PCR results, PCR products from 14 representative isolates were purified and sequenced. RESULTS: The distribution of the EPIYA motif found was: 2 AB (2.1 %), 1 AC (1.1 %), 1 BC (1.1 %), 70 ABC (73.6 %), 19 ABCC (20 %), and 2 ABCCC (2.1 %). Sequencing analysis confirmed the PCR classification in the 14 studied strains and showed three strains with unusual nucleotide sequences, not reported before. Distribution of the EPIYA-ABC pattern was equivalent in all pathologies (78.9 % in gastric ulcer, 72.5 % in duodenal ulcer and 72.2 % in non-ulcer dyspepsia). CONCLUSION: The PCR results using the new primers confirmed that all studied strains carried the Western CagA type. No specific EPIYA motif was associated with peptic ulcer. This is the first report that shows EPIYA motif distribution in H. pylori isolates from the Caribbean region.
Asunto(s)
Antígenos Bacterianos/química , Proteínas Bacterianas/química , Gastritis/microbiología , Infecciones por Helicobacter/microbiología , Helicobacter pylori/genética , Úlcera Péptica/microbiología , Procesamiento Proteico-Postraduccional , Regiones no Traducidas 3' , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Antígenos Bacterianos/genética , Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cuba/epidemiología , Cartilla de ADN , ADN Bacteriano/genética , Dispepsia/microbiología , Femenino , Infecciones por Helicobacter/epidemiología , Helicobacter pylori/aislamiento & purificación , Helicobacter pylori/patogenicidad , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Fosforilación , Reacción en Cadena de la Polimerasa , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Virulencia , Adulto JovenRESUMEN
AIM: To investigate the prevalence of vacuolating cytotoxin (vacA), cytotoxin associated gene A (cagA) and blood adhesion binding antigen (babA2) genotypes of Helicobacter pylori (H pylori) isolates from Cuban dyspeptic patients. METHODS: DNA was extracted from H pylori-positive cultures taken from 130 dyspeptic patients. Genotyping was performed by PCR, using specific primers for vacA (s1, s2, m1, m2), cagA and babA2 genes. Endoscopic observations and histological examinations were used to determine patient pathologies. RESULTS: vacA alleles s1, s2, m1 and m2 were detected in 96 (73.8%), 34 (26.2%), 75 (57.7%) and 52 isolates (40%), respectively, while the cagA gene was detected in 95 isolates (73.2%). One hundred and seven isolates (82.3%) were babA2-positive. A significant correlation was observed between vacAs1m1 and cagA and between vacAs1m1 and babA2 genotypes (P < 0.001 and P < 0.05, respectively) and between babA2 genotype and cagA status (P < 0.05); but, no correlation was observed between vacAs1 and babA2 genotypes. Eighty five (65.4%) and 73 (56.2%) strains were type 1 (vacAs1-cagA-positive) and "triple-positive" (vacAs1-cagA-babA2-positive), respectively, and their presence was significantly associated with duodenal ulcer (P < 0.01 and P < 0.001, respectively). CONCLUSION: The distribution of the main virulence factors in the Cuban strains in this study resembled that of the Western-type strains, and the more virulent H pylori isolates were significantly associated with duodenal ulcer, ulcer disease being the worst pathology observed in the group studied.
Asunto(s)
Genes Bacterianos , Infecciones por Helicobacter/microbiología , Helicobacter pylori/genética , Adhesinas Bacterianas/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Secuencia de Bases , Cuba , ADN Bacteriano/genética , Dispepsia/microbiología , Femenino , Genotipo , Helicobacter pylori/aislamiento & purificación , Helicobacter pylori/patogenicidad , Humanos , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
Vibrio cholerae 638 is a living candidate cholera vaccine strain attenuated by deletion of the CTXPhi prophage from C7258 (O1, El Tor Ogawa) and by insertion of the Clostridium thermocellum endoglucanase A gene into the hemagglutinin/protease coding sequence. This vaccine candidate was previously found to be well tolerated and immunogenic in volunteers. This article reports a randomized, double-blind, placebo-controlled trial conducted to test short-term protection conferred by 638 against subsequent V. cholerae infection and disease in volunteers in Cuba. A total of 45 subjects were enrolled and assigned to receive vaccine or placebo. The vaccine contained 10(9) CFU of freshly harvested 638 buffered with 1.3% NaHCO(3), while the placebo was buffer alone. After vaccine but not after placebo intake, 96% of volunteers had at least a fourfold increase in vibriocidal antibody titers, and 50% showed a doubling of at least the lipopolysaccharide-specific immunoglobulin A titers in serum. At 1 month after vaccination, five volunteers from the vaccine group and five from the placebo group underwent an exploratory challenge study with 10(9) CFU of DeltaCTXPhi attenuated mutant strain V. cholerae 81. Only two volunteers from the vaccine group shed strain 81 in their feces, but none of them experienced diarrhea; in the placebo group, all volunteers excreted the challenge strain, and three had reactogenic diarrhea. An additional 12 vaccinees and 9 placebo recipients underwent challenge with 7 x 10(5) CFU of virulent strain V. cholerae 3008 freshly harvested from a brain heart infusion agar plate and buffered with 1.3% NaHCO(3). Three volunteers (25%) from the vaccine group and all from the placebo group shed the challenge agent in their feces. None of the 12 vaccinees but 7 volunteers from the placebo group had diarrhea, and 2 of the latter exhibited severe cholera (>5,000 g of diarrheal stool). These results indicate that at 1 month after ingestion of a single oral dose (10(9) CFU) of strain 638, volunteers remained protected against cholera infection and disease provoked by the wild-type challenge agent V. cholerae 3008. We recommend that additional vaccine lots of 638 be prepared under good manufacturing practices for further evaluation.
Asunto(s)
Vacunas contra el Cólera/administración & dosificación , Vacunas contra el Cólera/inmunología , Cólera/prevención & control , Vibrio cholerae/inmunología , Administración Oral , Adolescente , Adulto , Anticuerpos Antibacterianos/sangre , Bacteriófagos/genética , Celulasa/genética , Vacunas contra el Cólera/genética , Clostridium thermocellum , Método Doble Ciego , Heces/microbiología , Eliminación de Gen , Hemaglutininas/genética , Humanos , Masculino , Péptido Hidrolasas/genética , Vacunación , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunología , Vibrio cholerae/genética , Vibrio cholerae/patogenicidad , Vibrio cholerae/virologíaRESUMEN
The main virulence factor of Vibrio cholerae, the cholera toxin, is encoded by the ctxAB operon, which is contained in the genome of the lysogenic filamentous phage CTX phi. This phage transmits ctxAB genes between V. cholerae bacterial populations that express toxin-coregulated pilus (TCP), the CTX phi receptor. In investigating new forms of ctxAB transmission, we found that V. cholerae filamentous phage VGJ phi, which uses the mannose-sensitive hemagglutinin (MSHA) pilus as a receptor, transmits CTX phi or its satellite phage RS1 by an efficient and highly specific TCP-independent mechanism. This is a novel type of specialized transduction consisting in the site-specific cointegration of VGJ phi and CTX phi (or RS1) replicative forms to produce a single hybrid molecule, which generates a single-stranded DNA hybrid genome that is packaged into hybrid viral particles designated HybP phi (for the VGJ phi/CTX phi hybrid) and HybRS phi (for the VGJ phi/RS1 hybrid). The hybrid phages replicate by using the VGJ phi replicating functions and use the VGJ phi capsid, retaining the ability to infect via MSHA. The hybrid phages infect most tested strains more efficiently than CTX phi, even under in vitro optimal conditions for TCP expression. Infection and lysogenization with HybP phi revert the V. cholerae live attenuated vaccine strain 1333 to virulence. Our results reinforce that TCP is not indispensable for the acquisition of CTX phi. Thus, we discuss an alternative to the current accepted evolutionary model for the emergence of new toxigenic strains of V. cholerae and the importance of our findings for the development of an environmentally safer live attenuated cholera vaccine.
Asunto(s)
Bacteriófagos/genética , Toxina del Cólera/genética , Transducción Genética , Vibrio cholerae/genética , Vibrio cholerae/virología , Vacunas contra el Cólera , Regulación Viral de la Expresión Génica , Transferencia de Gen Horizontal , Lisogenia , Plásmidos/genética , Vibrio cholerae/patogenicidad , VirulenciaRESUMEN
We describe a novel filamentous phage, designated VGJ phi, isolated from strain SG25-1 of Vibrio cholerae O139, which infects all O1 (classical and El Tor) and O139 strains tested. The sequence of the 7,542 nucleotides of the phage genome reveals that VGJ phi has a distinctive region of 775 nucleotides and a conserved region with an overall genomic organization similar to that of previously characterized filamentous phages, such as CTX phi of V. cholerae and Ff phages of Escherichia coli. The conserved region carries 10 open reading frames (ORFs) coding for products homologous to previously reported peptides of other filamentous phages, and the distinctive region carries one ORF whose product is not homologous to any known peptide. VGJ phi, like other filamentous phages, uses a type IV pilus to infect V. cholerae; in this case, the pilus is the mannose-sensitive hemagglutinin. VGJ phi-infected V. cholerae overexpresses the product of one ORF of the phage (ORF112), which is similar to single-stranded DNA binding proteins of other filamentous phages. Once inside a cell, VGJ phi is able to integrate its genome into the same chromosomal attB site as CTX phi, entering into a lysogenic state. Additionally, we found an attP structure in VGJ phi, which is also conserved in several lysogenic filamentous phages from different bacterial hosts. Finally, since different filamentous phages seem to integrate into the bacterial dif locus by a general mechanism, we propose a model in which repeated integration events with different phages might have contributed to the evolution of the CTX chromosomal region in V. cholerae El Tor.
Asunto(s)
Bacteriófagos/genética , Inovirus/clasificación , Inovirus/genética , Vibrio cholerae/virología , Integración Viral , Sitios de Ligazón Microbiológica , Proteínas Bacterianas/química , Bacteriófagos/fisiología , Secuencia de Bases , Cromosomas Bacterianos , Genoma Viral , Inovirus/fisiología , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Proteínas Virales/químicaRESUMEN
Se presentó un enfermo con poliarteritis nodosa cuyo diagnóstico se sustentó en aspectos clínicos, histológicos y arteriográficos. Se observó, evolutivamente, una inusual y repetida complicación: rotura renal espontánea con hematoma retroperitoneal, inicialmente en el riñón derecho y 8 d después, en el izquierdo. Quedó anéfrico y se sostuvo durante 3 meses con tratamiento hemodialítico hasta su fallecimiento. Se trató de una verdadera rareza dentro de las complicaciones de la poliarteritis nodosa y la presentación bilateral constituyó un hecho excepcional. Se hallaron muy escasos reportes en la literatura médica mundial
Asunto(s)
Nefrectomía , Poliarteritis Nudosa , Rotura Espontánea/complicacionesRESUMEN
The hemagglutinin/protease (HA/P) seems to be an attractive locus for the insertion of heterologous tags in live cholera vaccine strains. A deltaCTXphi spontaneous mutant derived from a pathogenic strain of O139 Vibrio cholerae was sequentially manipulated to obtain hapA Colon, two colons celA derivatives which were later improved in their environmental safety by means of a thyA mutation. All the strains here obtained showed similar phenotypes in traits known to be remarkable for live cholera vaccines irrespective of their motility phenotypes, although the hapA mutants had a 10-fold decrease in their colonisation capacity compared with their parental strains in the infant mouse cholera model. However, the subsequent thyA mutation did not affect their colonisation properties in the same model. These preliminary results pave the way for further clinical assays to confirm the possibilities of these vaccine prototypes as safe and effective tools for the prevention of O139 cholera.