RESUMEN
NK-lysin is a potent antimicrobial peptide (AMP) with antimicrobial activity against bacteria, fungi, viruses, and parasites. NK-lysin is a type of granulysin, a member of the saposin-like proteins family first isolated from a pig's small intestine. In previous work, for the first time, we identified four variants of nk-lysin from Atlantic salmon (Salmo salar) using EST sequences. In the present study, we reported and characterized two additional transcripts of NK-lysin from S. salar. Besides, we evaluated the tissue distribution of three NK-lysins from S. salar and assessed the antimicrobial, hemolytic, and immunomodulatory activities and signaling pathways of three NK-lysin-derived peptides. The synthetic peptides displayed antimicrobial activity against Piscirickettsia salmonis (LF-89) and Flavobacterium psychrophilum. These peptides induced the expression of immune genes related to innate and adaptive immune responses in vitro and in vivo. The immunomodulatory activity of the peptides involves the mitogen-activated protein kinases-mediated signaling pathway, including p38, extracellular signal-regulated kinase 1/2, and/or c-Jun N-terminal kinases. Besides, the peptides modulated the immune response induced by pathogen-associated molecular patterns (PAMPs). Our findings show that NK-lysin could be a highly effective immunostimulant or vaccine adjuvant for use in fish aquaculture.
Asunto(s)
Péptidos Antimicrobianos , Proteínas de Peces , Proteolípidos , Salmo salar , Animales , Péptidos Antimicrobianos/metabolismo , Péptidos Antimicrobianos/farmacología , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/microbiología , Proteínas de Peces/metabolismo , Proteínas de Peces/farmacología , Inmunidad Innata , Proteolípidos/metabolismo , Proteolípidos/farmacología , Salmo salar/inmunología , Transducción de SeñalRESUMEN
Lipids, glycolipids and lipopeptides derived from Mycobacterium tuberculosis (Mtb) are presented to T cells by monomorphic molecules known as CD1. This is the case of the Mtb-specific sulfoglycolipid Ac2SGL, which is presented by CD1b molecules and is recognized by T cells found in tuberculosis (TB) patients and in individuals with latent infections. Our group, using filamentous phage display technology, obtained two specific ligands against the CD1b-Ac2SGL complex: (i) a single chain T cell receptor (scTCR) from a human T cell clone recognizing the CD1b-AcSGL complex; and (ii) a light chain domain antibody (dAbκ11). Both ligands showed lower reactivity to a synthetic analog of Ac2SGL (SGL12), having a shorter acyl chain as compared to the natural antigen. Here we put forward the hypothesis that the CD1b endogenous spacer lipid (EnSpacer) plays an important role in the recognition of the CD1b-Ac2SGL complex by specific T cells. To support this hypothesis we combined: (a) molecular binding assays for both the scTCR and the dAbκ11 antibody domain against a small panel of synthetic Ac2SGL analogs having different acyl chains, (b) molecular modeling of the CD1b-Ac2SGL/EnSpacer complex, and (c) modeling of the interactions of this complex with the scTCR. Our results contribute to understand the mechanisms of lipid presentation by CD1b molecules and their interactions with T-cell receptors and other specific ligands, which may help to develop specific tools targeting Mtb infected cells for therapeutic and diagnostic applications.
Asunto(s)
Antígenos Bacterianos/inmunología , Antígenos CD1/inmunología , Modelos Moleculares , Mycobacterium tuberculosis/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Antígenos CD1/genética , Humanos , Proteínas Recombinantes/inmunologíaRESUMEN
OBJECTIVES: The prevalence of fibromyalgia (FM) differs depending on the population studied. The main objective of the EPISER2016 study was to estimate the prevalence of FM in adults in Spain. The secondary objective was to evaluate the association with sociodemographic and anthropometric characteristics and smoking. METHODS: This is a population-based cross-sectional multicentre study. The random selection was based on multistage stratified cluster sampling. The final sample comprised 4916 persons aged ≥20 years. Participants were contacted by telephone for completion of a screening survey. Investigating rheumatologists evaluated positive results (review of medical records and/or telephone interview, with medical visit if needed) to confirm the diagnosis. Prevalence and 95% confidence interval were calculated, taking into account the sample design. Weighing was applied based on age, sex, and geographic origin. Predictive models were constructed to analyse which sociodemographic, anthropometric and lifestyle variables in the call centre questionnaire were associated with the presence of FM. RESULTS: 602 subjects (12.25%) had a positive screening result for FM, of which 24 were missing (3.99%). A total of 141 cases of FM were recorded. The estimated prevalence was 2.45% (95% CI, 2.06-2.90). Female sex was the variable most associated with FM, with an odds ratio (OR) of 10.156 (95% CI, 5.068-20.352). Peak prevalence was at 60-69 years (p=0.009, OR=6.962). FM was 68% more frequent in obese individuals (OR, 1.689; 95% CI, 1.036-2.755). CONCLUSIONS: The prevalence of FM in adults in Spain barely changed between 2000 and 2016 and it is similar to that observed in Europe as a whole.
Asunto(s)
Fibromialgia/epidemiología , Adulto , Estudios Transversales , Femenino , Humanos , Obesidad/complicaciones , Prevalencia , Factores Sexuales , España/epidemiología , Adulto JovenRESUMEN
Vaccination has reduced morbidity and mortality of many diseases that previously caused devastating epidemics and deaths globally. Vaccines as a biological product may contain microorganisms or their derivatives. This aspect together with the fact that they are administered to healthy individuals (mainly children) means that approximately 70% of vaccines development time is dedicated to quality control. Monoclonal antibodies (MAbs) have become essential analytical tools for application in ELISAs, Western and Dot blotting, immunoprecipitation, and flow cytometric assays that ensure the quality control of vaccines. The aim of this work is to present a review of the methods used to obtain a platform of MAbs against Neisseria meningitidis polysaccharide antigens to use as an analytical tool for quality control of anti-meningococcal polysaccharide (Ps) vaccines. The MAbs obtained are used in five sandwich ELISAs developed for Ps quantification. The assays showed good reproducibility and repeatability, with quantitation and detection limits below 1 ng/mL. Dot Blot, as the Identity test of the Ps vaccine, was carried out to positively identify licensed and experimental vaccines. All assays described are suitable for the screening of multiple vaccine samples and could be useful for monitoring lot-to-lot consistency and stability.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Anticuerpos Monoclonales/inmunología , Infecciones Meningocócicas/inmunología , Vacunas Meningococicas/normas , Neisseria meningitidis/inmunología , Polisacáridos Bacterianos/inmunología , Control de Calidad , Humanos , Infecciones Meningocócicas/microbiología , Infecciones Meningocócicas/prevención & control , Vacunas Meningococicas/administración & dosificación , Polisacáridos Bacterianos/clasificaciónRESUMEN
AIMS: To describe the methodology of the EPISER 2016 (study of the prevalence of rheumatic diseases in adult population in Spain), as well its strengths and limitations. The aim of this study is to estimate the prevalence of rheumatoid arthritis (RA), psoriatic arthritis (PsA), ankylosing spondylitis (AS), systemic lupus erythematosus (SLE), Sjögren's syndrome (SS), osteoarthritis (knee, hip, hands, and cervical and lumbar spine), fibromyalgia, gout and clinical osteoporotic fracture. MATERIAL AND METHOD: Population-based, multicenter, cross-sectional study, with the participation of 45 municipalities in the 17 Spanish autonomous communities. The reference population will consist of adults aged 20 years and over residing in Spain. A computer-assisted telephone interview (CATI) system will be used for data collection. Diagnostic suspicions and diagnoses received by the participants will be studied by rheumatologists in the referral hospitals in the selected municipalities. STATISTICAL ANALYSIS: the prevalence of the rheumatic diseases will be calculated using estimators and their 95% confidence intervals. Weights will be calculated in each of the sampling stages in accordance with the probability of selection. The distribution of the population in Spain will be obtained from the Spanish Statistics Institute. CONCLUSIONS: Sociodemographic and lifestyle changes over the last 16 years justify EPISER 2016. This study will provide current data about the prevalences of RA, AS, PsA, SLE, SS, osteoarthritis, fibromyalgia, gout and clinical osteoporotic fracture. The results will allow comparisons with studies from other countries and EPISER 2000.
Asunto(s)
Proyectos de Investigación , Enfermedades Reumáticas/epidemiología , Adulto , Anciano , Anciano de 80 o más Años , Estudios Transversales , Femenino , Humanos , Masculino , Persona de Mediana Edad , Prevalencia , España/epidemiologíaRESUMEN
OBJECTIVE: The objective of this study was to determine the prevalence of clarithromycin-resistant Helicobacter pylori in asymptomatic children in a rural community of Cajamarca (northern Peru). RESULTS: Helicobacter pylori was detected in 17.2% (49/285) of the samples. Unboiled water consumption the most frequent associated factor in patients with positive PCR for H. pylori infection (93.9%). Clarithromycin resistant mutations were found in 79.6% (39/49) of the positive samples for H. pylori. The most frequent mutation was A2142G (46.9%), followed by the double-mutation A2142G-A2143G (28.6%).
Asunto(s)
Claritromicina/farmacología , Farmacorresistencia Bacteriana/genética , Infecciones por Helicobacter/diagnóstico , Helicobacter pylori/genética , Población Rural/estadística & datos numéricos , Adolescente , Antibacterianos/farmacología , Niño , Estudios Transversales , Femenino , Infecciones por Helicobacter/epidemiología , Infecciones por Helicobacter/microbiología , Helicobacter pylori/fisiología , Humanos , Masculino , Mutación , Perú/epidemiología , Prevalencia , ARN Ribosómico 23S/genéticaRESUMEN
Tuberculosis (TB) remains an important cause of mortality and morbidity. The TB vaccine, BCG, is not fully protective against the adult form of the disease and is unable to prevent its transmission although it is still useful against severe childhood TB. Hence, the search for new vaccines is of great interest. In a previous study, we have shown that proteoliposomes obtained from Mycobacterium smegmatis (PLMs) induced cross reactive humoral and cellular response against Mycobacterium tuberculosis (Mtb) antigens. With the objective to evaluate the protective capability of PLMs, a murine model of progressive pulmonary TB was used. Animals immunized with PLMs with and without alum (PLMs/PLMsAL respectively) showed protection compared to non-immunized animals. Mice immunized with PLMsAL induced similar protection as that of BCG. Animals immunized with BCG, PLMs and PLMsAL showed a significant decrease in tissue damage (percentage of pneumonic area/lung) compared to non-immunized animals, with a more prominent effect in BCG vaccinated mice. The protective effect of the administration of PLMs in mice supports its future evaluation as experimental vaccine candidate against Mtb.
Asunto(s)
Mycobacterium smegmatis/inmunología , Proteolípidos/inmunología , Vacunas contra la Tuberculosis , Tuberculosis Pulmonar/prevención & control , Adyuvantes Inmunológicos , Compuestos de Alumbre , Animales , Vacuna BCG , Carga Bacteriana , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Masculino , Ratones Endogámicos BALB C , Mycobacterium tuberculosis/crecimiento & desarrollo , Mycobacterium tuberculosis/aislamiento & purificación , Neumonía Bacteriana/microbiología , Neumonía Bacteriana/patología , Neumonía Bacteriana/prevención & control , Tuberculosis Pulmonar/inmunología , Tuberculosis Pulmonar/microbiología , Tuberculosis Pulmonar/patologíaRESUMEN
OBJECTIVE/BACKGROUND: The development of new tools capable of targeting Mycobacterium tuberculosis (Mtb)-infected cells have potential applications in diagnosis, treatment, and prevention of tuberculosis. In Mtb-infected cells, CD1b molecules present Mtb lipids to the immune system (Mtb lipid-CD1b complexes). Because of the lack of CD1b polymorphism, specific Mtb lipid-CD1b complexes could be considered as universal Mtb infection markers. 2-Stearoyl-3-hydroxyphthioceranoyl-2'-sulfate-α-α'-d-trehalose (Ac2SGL) is specific for Mtb, and is not present in other mycobacterial species. The CD1b-Ac2SGL complexes are expressed on the surface of human cells infected with Mtb. The aim of this study was to generate ligands capable of binding these CD1b-Ac2SGL complexes. METHODS: A synthetic human scFv phage antibody library was used to select phage-displayed antibody fragments that recognized CD1b-Ac2SGL using CD1b-transfected THP-1 cells loaded with Ac2SGL. RESULTS: One clone, D11-a single, light-variable domain (kappa) antibody (dAbκ11)-showed high relative binding to the Ac2SGL-CD1b complex. CONCLUSION: A ligand recognizing the Ac2SGL-CD1b complex was obtained, which is a potential candidate to be further tested for diagnostic and therapeutic applications.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Antígenos CD1/inmunología , Glucolípidos/inmunología , Mycobacterium tuberculosis/inmunología , Anticuerpos de Cadena Única/genética , Tuberculosis/inmunología , Anticuerpos Antibacterianos/genética , Antígenos CD1/genética , Bacteriófagos/genética , Bacteriófagos/metabolismo , Expresión Génica , Humanos , Mycobacterium tuberculosis/genética , Anticuerpos de Cadena Única/inmunología , Tuberculosis/microbiologíaRESUMEN
Tuberculosis (TB) is one of the most important causes of mortality and morbidity due to infectious diseases. BCG, the vaccine in use, is not fully protective against TB. In a previous study, we have shown that proteoliposomes (outer membrane extracts), obtained from BCG (PLBCG) were able to induce humoral immune responses against Mycobacterium tuberculosis (Mtb) antigens. With the objective to evaluate the protective capability of PLBCG alone or as a booster with BCG, a murine model of progressive pulmonary TB was used. Animals immunized with PLBCG adjuvanted with alum (PLBCG-Al) showed similar protection to that conferred by BCG. The group immunized with PLBCG-Al as a booster to BCG gave superior protection than BCG as evidenced by a reduction of bacterial load in lungs 2 months after infection with Mtb. Animals immunized with BCG, PLBCG-Al and this formulation as a booster of BCG, showed a significant decrease of tissue damage (percentage of pneumonic area/lung) compared with non-immunized animals. These results demonstrate that immunization with PLBCG-Al alone or as a booster to BCG induce appropriate protection against challenge with Mtb in mice and support the future evaluation of PLBCG as a promising vaccine candidate against Mtb.
Asunto(s)
Mycobacterium bovis/inmunología , Proteolípidos/inmunología , Vacunas contra la Tuberculosis/inmunología , Tuberculosis/prevención & control , Adyuvantes Inmunológicos/administración & dosificación , Compuestos de Alumbre/administración & dosificación , Animales , Carga Bacteriana , Modelos Animales de Enfermedad , Pulmón/microbiología , Masculino , Ratones Endogámicos BALB C , Mycobacterium bovis/química , Mycobacterium tuberculosis/aislamiento & purificación , Proteolípidos/administración & dosificación , Proteolípidos/aislamiento & purificación , Tuberculosis/inmunología , Vacunas contra la Tuberculosis/administración & dosificación , Vacunas contra la Tuberculosis/aislamiento & purificaciónRESUMEN
Los tumores neuroendocrinos de cuello uterino son extremadamente raros. Las mujeres con diagnóstico de carcinoma neuroendocrino de células pequeñas del cuello uterino tienen mayor frecuencia de metástasis en los ganglios linfáticos, invasión linfovascular, recurrencia y peor pronóstico en comparación con aquellos con otros tipos de neoplasias cervicales. Se presenta el caso de una mujer de 58 años, con un tiempo de enfermedad de seis años antes del ingreso, caracterizado por sangrado vaginal irregular posmenopáusica, además de sintomatología relacionada a anemia crónica. En el examen ginecológico, se evidenció tumoración de 4 cm que ocupaba tercio superior de vagina y protruía por el cérvix. Fue diagnosticado como mioma abortivo y enviada a estudio anatomopatológico. El resultado fue carcinoma neuroendocrino de células pequeñas grado III en el 90% y carcinoma epidermoide en el 10%. La paciente fue sometida a histerectomía radical más salpingo-ooferectomía bilateral y linfadenectomía pélvica bilateral y para-aortica. El estudio anatomopatológico de la pieza quirúrgica encontró endometrio y miometrio comprometido por neoplasia maligna. Parametrios, anexos y ganglios linfáticos se encontraron libres de neoplasia. A la microscopía el resultado fue carcinoma neuroendocrino grado III (carcinoma de células pequeñas, infiltrante), con extensa embolia linfovascular. El estudio de inmunohistoquímica arrojó sinaptofisina positivo en las áreas con diferenciación neuroendocrina.
Neuroendocrine tumors of the cervix are extremely rare. Women diagnosed with small cell neuroendocrine carcinoma of the cervix have a higher frequency of metastases in the lymph nodes, lymphovascular invasion, recurrence and worse prognosis compared to those with other types of cervical neoplasia. We report the case of a 58-year-old female, with a history of six years of postmenopausal irregular vaginal bleeding, in addition to symptoms related to chronic anemia. Gynecological examination showed a tumor of 4 cm that occupied the upper third of the vagina and protruded through the cervix initially diagnosed as an abortifacient myoma, and sent to histopathology study. 90% of the tumor was small cell neuroendocrine carcinoma grade III, and the remaining 10% was squamous cell carcinoma. The patient underwent into a radical hysterectomy plus bilateral salpingo-oophorectomy, and bilateral pelvic and para-aortic lymphadenectomy. Histopathologic examination of the surgical specimen found endometrium and myometrium compromised by malignancy. Parametrium, annexes and lymph nodes were free of neoplasia. At microscopy, the result was a grade III neuroendocrine carcinoma (small cell carcinoma, infiltrating), with extensive lymphovascular emboli. The immune-histochemical study showed synaptophysin positive in areas with neuroendocrine differentiation.
Asunto(s)
Humanos , Femenino , Persona de Mediana Edad , Neoplasias del Cuello Uterino/diagnóstico , Carcinoma Neuroendocrino/diagnóstico , Neoplasias del Cuello Uterino/cirugía , Neoplasias del Cuello Uterino/patología , Sinaptofisina , Carcinoma de Células Pequeñas , Carcinoma Neuroendocrino/cirugía , Carcinoma Neuroendocrino/patología , Histerectomía/métodosRESUMEN
A novel murine hybridoma monoclonal antibody (MAb) was produced against the capsular polysaccharide (CP) of Neisseria meningitidis serogroup X (MenX) in order to develop a sandwich enzyme linked immunosorbent assay (ELISA) for the quantitation of the meningococcal polysaccharide. The MAb only reacted with the CP from MenX and did not react with CPs from N. meningitidis serogroups A, C, Y and W (MenA, MenC, MenY, MenW). The affinity constant (Ka) of the MAb measured by non-competitive ELISA was 7.25 × 10(7) M(-1). The application of this MAb in a sandwich ELISA was demonstrated by its ability to properly quantitate three lots of an experimental meningococcal CP-based vaccine. The MAb obtained in this work could be a valuable reagent for the detection and quantitation of future meningococcal vaccines containing MenX CP.
Asunto(s)
Anticuerpos Monoclonales/química , Infecciones Meningocócicas/prevención & control , Vacunas Meningococicas/química , Neisseria meningitidis/metabolismo , Polisacáridos/química , Animales , Calibración , Ensayo de Inmunoadsorción Enzimática , Límite de Detección , Infecciones Meningocócicas/microbiología , Ratones , Ratones Endogámicos BALB C , Serogrupo , Especificidad de la EspecieRESUMEN
The most important targets for vaccine development are the proteins that are highly expressed by the microorganisms during infection in-vivo. A number of Mycobacterium tuberculosis (Mtb) proteins are also reported to be expressed in-vivo at different phases of infection. In the present study, we analyzed multiple published databases of gene expression profiles of Mtb in-vivo at different phases of infection in animals and humans and selected 38 proteins that are highly expressed in the active, latent and reactivation phases. We predicted T- and B-cell epitopes from the selected proteins using HLAPred for T-cell epitope prediction and BCEPred combined with ABCPred for B-cell epitope prediction. For each selected proteins, regions containing both T- and B-cell epitopes were identified which might be considered as important candidates for vaccine design against tuberculosis.
Asunto(s)
Proteínas Bacterianas/genética , Biología Computacional , Epítopos de Linfocito B/genética , Epítopos de Linfocito T/genética , Mycobacterium tuberculosis/genética , Tuberculosis/genética , Animales , Proteínas Bacterianas/inmunología , Bases de Datos Genéticas , Diseño de Fármacos , Regulación Bacteriana de la Expresión Génica , Humanos , Mycobacterium tuberculosis/inmunología , Mycobacterium tuberculosis/patogenicidad , Redes Neurales de la Computación , Tuberculosis/inmunología , Tuberculosis/microbiología , Tuberculosis/prevención & control , Vacunas contra la Tuberculosis/inmunologíaRESUMEN
Neisseria meningitidis is a Gram negative bacterium that has been classified in 13 serogroups according to the biochemical composition of the capsular polysaccharide (CP). However, invasive infections are most frequently caused by six of these serogroups: A, B, C, W, X and Y (MenA, MenB, MenC, MenW, MenX, MenY). Individual CP quantitation in multivalent meningococcal CP-based vaccines is required for quality control testing of these products. In this regard, four sandwich enzyme-linked immunosorbent assays (ELISAs) were developed for the quantitation of CP. The quantitation and detection limits of the four ELISAs were below 1ng/mL. The assays showed good reproducibility and repeatability as calculated for each point of the standard curve (CV<15%). In addition, five multivalent meningococcal CP-based vaccines were evaluated and the proposed ELISAs showed that these vaccines were found into the accepted range (±30%) of CP content. These assays are suitable for screening multiple plain or conjugated meningococcal CP-based vaccines and could be useful for monitoring lot-to-lot consistency and stability analysis.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática , Infecciones Meningocócicas/inmunología , Vacunas Meningococicas/inmunología , Neisseria meningitidis/inmunología , Polisacáridos Bacterianos , Anticuerpos Antibacterianos/metabolismo , Cápsulas Bacterianas/inmunología , Pruebas Diagnósticas de Rutina , Estudios de Factibilidad , Humanos , Inmunoglobulina G/metabolismo , Infecciones Meningocócicas/prevención & control , Vacunas Meningococicas/clasificación , Control de Calidad , Reproducibilidad de los ResultadosRESUMEN
Microbial control strategies are needed in the food industry to prevent foodborne illnesses and outbreaks and prolong product shelf life. The aim of this study was to investigate and compare the efficacy of the commercial natural antimicrobials white mustard essential oil (WMEO), citrus flavonoid and acid blend (CFAB), olive extract (OE), Nisaplin (a compound containing nisin), and lauric arginate (LAE) alone and in combinations against foodborne pathogens and spoilage microorganisms. MICs of individual and combined antimicrobials against Escherichia coli, Salmonella Enteritidis, Enterobacter aerogenes, Bacillus cereus, Listeria monocytogenes, and Staphylococcus aureus were determined at pH 6.0 and 25 °C. WMEO was most effective against B. cereus and S. aureus, with MICs of 250 and 500 mg/liter, respectively. CFAB inhibited all tested microorganisms, requiring only 12 to 35 mg/liter for gram-positive bacteria. For OE, 2,000 mg/liter was needed to achieve microbial inhibition. Nisaplin at 400 to 1,200 mg/liter inhibited only gram-positive bacteria. LAE was effective at low concentrations and required only 20 to 50 mg/liter to inhibit all tested microorganisms. When WMEO was combined with other antimicrobials, the effects were usually additive except for WMEO plus Nisaplin and WMEO+OE, which had synergistic activity against L. monocytogenes and Salmonella Enteritidis, respectively. An antagonistic effect was observed for WMEO+CFAB against E. aerogenes. For WMEO+LAE+CFAB, additive antimicrobial effects were noted against all strains tested except S. aureus, where a synergistic effect occurred. These findings suggest that these commercial natural antimicrobials have potential to enhance food safety by inhibiting foodborne pathogens and extending product shelf life.
Asunto(s)
Antibacterianos/farmacología , Enfermedades Transmitidas por los Alimentos/prevención & control , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Medios de Cultivo/química , Combinación de Medicamentos , Sinergismo Farmacológico , Microbiología de Alimentos , Bacterias Gramnegativas/crecimiento & desarrollo , Bacterias Grampositivas/crecimiento & desarrollo , Nisina/farmacología , Aceites Volátiles/farmacologíaRESUMEN
Murine hybridoma monoclonal antibodies (MAbs) were produced against the capsular polysaccharide (CPs) of serogroups A, C, W135 and Y meningococci (MenA, MenC, MenW, MenY) in order to develop immunological reagents for the identification of meningococcal polysaccharides. Each serogroup-specific MAb reacted with the CPs from its homologous serogroup only and did not react with CPs from the other three serogroups. The affinity constant (Ka) of the four MAbs measured by non-competitive ELISA was 6.62 × 10(9), 2.76 × 10(9), 1.48 × 10(9) and 3.8 × 10(9) M(-1) for MenA, MenC, MenW and MenY MAbs respectively. The application of these MAbs for identity tests was demonstrated by their abilities to correctly identify the CPs from serogroups A, C, W135 and Y in meningococcal CPs-based vaccines through ELISA. The MAbs obtained in this work are a very valuable set of tools for study meningococcal polysaccharides vaccines.
Asunto(s)
Anticuerpos Antibacterianos , Anticuerpos Monoclonales de Origen Murino , Cápsulas Bacterianas , Neisseria meningitidis Serogrupo A , Neisseria meningitidis Serogrupo C , Neisseria meningitidis Serogrupo W-135 , Neisseria meningitidis Serogrupo Y , Polisacáridos Bacterianos , Animales , Anticuerpos Antibacterianos/química , Anticuerpos Antibacterianos/inmunología , Anticuerpos Monoclonales de Origen Murino/química , Anticuerpos Monoclonales de Origen Murino/inmunología , Especificidad de Anticuerpos , Cápsulas Bacterianas/química , Cápsulas Bacterianas/metabolismo , Ensayo de Inmunoadsorción Enzimática/métodos , Ratones , Neisseria meningitidis Serogrupo A/química , Neisseria meningitidis Serogrupo A/inmunología , Neisseria meningitidis Serogrupo C/química , Neisseria meningitidis Serogrupo C/inmunología , Neisseria meningitidis Serogrupo W-135/química , Neisseria meningitidis Serogrupo W-135/inmunología , Neisseria meningitidis Serogrupo Y/química , Neisseria meningitidis Serogrupo Y/inmunología , Polisacáridos Bacterianos/química , Polisacáridos Bacterianos/inmunologíaRESUMEN
Mycobacterium smegmatis (Ms) is a nonpathogenic mycobacteria of rapid growth, which shares many characteristics with Mycobacterium tuberculosis (MTB), the major causative agent of tuberculosis. MTB has several cell wall glycolipids in common with Ms, which play an important role in the pathogenesis of tuberculosis and the induction of a protective immune response against MTB infection in some animal models. In this study, the humoral immune response and cross reactivity against MTB, of liposomes containing a mixture of cell wall glycolipids of Ms and commercial lipids was evaluated, in order to study its possible use as a component of a vaccine candidate against tuberculosis. Liposomes containing total lipids extracted from Ms, distearoyl phosphatidyl choline and cholesterol were prepared by the dehydration-rehydration technique. Balb/c mice were immunized with the liposomes obtained and the antibody response and cross reactivity against MTB were tested by ELISA. Total lipids extract from Ms showed the presence of several polar glycolipids in common with MTB, such as phosphatidylinositol mannosides. Liposomes that contained glycolipids of Ms were capable of inducing a specific IgG antibody response that allowed the recognition of surface antigens of MTB. The results of this study demonstrated the presence of immunogenic glycolipids in Ms, which could be included to enhance the protective effects of subunit vaccine formulations against tuberculosis.
Asunto(s)
Glucolípidos/inmunología , Liposomas/inmunología , Mycobacterium smegmatis/inmunología , Mycobacterium tuberculosis/inmunología , Vacunas contra la Tuberculosis/inmunología , Tuberculosis/inmunología , Animales , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Colesterol , Reacciones Cruzadas , Glucolípidos/administración & dosificación , Inmunidad Humoral , Liposomas/administración & dosificación , Ratones , Ratones Endogámicos BALB C , Fosfatidilcolinas , Fosfatidilinositoles/inmunología , Tuberculosis/prevención & control , VacunaciónRESUMEN
The only currently available vaccine against tuberculosis (TB) is Mycobacterium bovis Bacille Calmette-Guerin (BCG), which has inconsistent efficacy to protect against the disease in adults. M. tuberculosis (MTB) cell wall components have been implicated in the pathogenicity of TB and therefore have been a prime target for the identification and characterization of cell wall proteins with potential application in vaccine development. In this regard, proteoliposomes (PLs) derived from mycobacteria containing lipids and cell wall proteins could be potential vaccine candidates against TB. In the present study PLs derived from BCG were prepared. These homogeneous population of spherical microparticles was then immunized into Balb/c mice. Sera of immunized animals showed high IgG response and strong cross-reactivity against different MTB antigens.These results showed that BCG PLs could be potential vaccine candidates against TB.
Asunto(s)
Anticuerpos Antibacterianos/sangre , Vacuna BCG/inmunología , Mycobacterium bovis/inmunología , Mycobacterium tuberculosis/inmunología , Proteolípidos/inmunología , Tuberculosis/inmunología , Animales , Anticuerpos Antibacterianos/inmunología , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Pared Celular/inmunología , Reacciones Cruzadas , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Masculino , Lípidos de la Membrana/inmunología , Ratones , Ratones Endogámicos BALB C , Tuberculosis/prevención & controlRESUMEN
BACKGROUND: A monoclonal antibody (mAb) of the IgA isotype, designated TBA61, is specific for the Acr protein of Mycobacterium tuberculosis (MTB). TBA61 has been used in studies exploring protection against tuberculosis (TB), and its efficacy has been proven using different challenge models. To purify the mouse IgA isotype, a combination of methods, such as globulin precipitation, ion exchange, and gel filtration, is usually required to achieve a satisfactory degree of purity. METHODS: To minimise the number of chromatographic steps, we proposed to employ immunoaffinity chromatography using the Acr protein of MTB as a specific ligand for this mAb. For this purpose, the HspX gene was cloned and expressed in Escherichia coli, and recombinant Acr (rAcr) was coupled to a cyanogen bromide-activated Sepharose 4B matrix, which was used to purify TBA61 mAb from ascites produced in mice in a single step. RESULTS: The recovery from the purification procedure was 1.46 mg per mL of ascites. Analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot showed a high purity. The purified mAb retained its reactivity against the Acr protein based on enzyme-linked immunosorbent assay (ELISA) and western blot. CONCLUSION: The purification method used is rapid, simple, and specific and can be easily scaled up.
RESUMEN
Proteoliposomes (PL) obtained from Mycobacterium smegmatis (Ms) were evaluated for their capacity to elicit cross-reactive responses against Mycobacterium tuberculosis (Mtb) antigens in BALB/c mice. Animals immunized with PL adjuvanted with alum (PL-AL) or Freund's Incomplete Adjuvant (PL-IFA) showed significant IgG responses against the PL as well as total Ms lipids. Both groups of animals also showed significant IgG responses against BCG, but only animals immunized with PL-AL produced significant IgG responses against soluble cell wall proteins (SCWP) or whole cell lysate (WCL) of Mtb. Significant DTH responses against WCL were observed in both groups of animals after 24 h, but only PL-AL-immunized mice showed significant DTH responses after 48 h and 72 h. PL-Ms are capable of eliciting cross-reactive humoral and cellular responses against Mtb antigens and thus may be a potential vaccine strategy against tuberculosis.