RESUMEN
Staphylococcus aureus is a common human commensal and the leading cause of diverse infections. To identify distinctive parameters associated with infection and colonization, we compared the immune and inflammatory responses of patients with a diagnosis of invasive S. aureus disease to healthy donors. We analyzed the inflammatory responses founding a pattern of distinctive cytokines significantly higher in the patients with invasive disease. The measure of antibody levels revealed a wide antibody responsiveness from all subjects to most of the antigens, with significantly higher response for some antigens in the invasive patients compared to control. Moreover, functional antibodies against toxins distinctively associated with the invasive disease. Finally, we examined the genomic variability of isolates, showing no major differences in genetic distribution compared to a panel of representative strains. Overall, our study shows specific signatures of cytokines and functional antibodies in patients with different primary invasive diseases caused by S. aureus. These data provide insight into human responses towards invasive staphylococcal infections and are important for guiding the identification of novel preventive and therapeutic interventions against S. aureus.
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Infecciones Estafilocócicas/inmunología , Adulto , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/inmunología , Niño , Citocinas/sangre , Humanos , Inmunoglobulina G/sangre , Análisis por Matrices de Proteínas , Infecciones Estafilocócicas/sangre , Infecciones Estafilocócicas/genética , Staphylococcus aureus/inmunología , Factores de Virulencia/inmunologíaRESUMEN
BACKGROUND: Intramuscular immunisation with a vaccine composed of three recombinant Helicobacter pylori antigens-vacuolating cytotoxin A (VacA), cytotoxin-associated antigen (CagA), and neutrophil-activating protein (NAP)-prevented infection in animal models and was well tolerated and highly immunogenic in healthy adults. We aimed to assess the efficacy of the vaccine in prevention of a H pylori infection after challenge with a CagA-positive strain (BCM 300) in healthy volunteers. METHODS: In this randomised phase 1/2, observer-blind, placebo-controlled, single-centre study, healthy non-pregnant adults aged 18-40 years who were confirmed negative for H pylori infection were randomly assigned (3:4) to three intramuscular doses of either placebo or vaccine at 0, 1, and 2 months. Randomisation was via a computer-generated list with study numbers ensuring the correct ratio within a block size of seven. Participants were consecutively assigned in a double-blind manner to existing study numbers of the study protocol. Investigators and participants were blinded to allocation throughout the study. One month after the third immunisation, participants underwent challenge with a CagA-positive H pylori strain, which, for safety reasons, was initially administered in a subset of participants. The primary efficacy outcome was the efficacy of the vaccine as measured by the proportion of participants infected with H pylori 12 weeks after the challenge. At the end of the study, participants infected with H pylori were treated for 14 days with combination therapy consisting of a proton pump inhibitor and two antibiotics twice daily. Safety and immunogenicity were monitored at pre-established visits. This trial is registered with ClinicalTrials.gov, number NCT00736476, and is completed. FINDINGS: 63 patients were randomly assigned, 27 to placebo and 36 to the vaccine. 34 participants (19 in the vaccinated group and 15 in the placebo group) underwent infectious challenge, all but one of whom experienced transient mild-to-moderate epigastric symptoms. 12 weeks after infectious challenge, six (32%) of 19 people in the vaccinated group and six (40%) of 15 people in the placebo group remained positive for H pylori. Eradication was successful in everyone who remained infected at 12 weeks. The geometric mean concentrations of antibodies specific to CagA (202 [95% CI 69-588] vs 4·73 [95% CI 1·41-16]; p=0·001), VacA (1469 [838-2577] vs 73 [39-138]; p=0·001), and NAP (208 [139-313] vs 8·01 [5·05-13]; p=0·001) were significantly higher in the vaccine group than in the placebo group 12 weeks after infectious challenge. INTERPRETATION: Compared with placebo, the vaccine did not confer additional protection against H pylori infection after challenge with a CagA-positive strain, despite increased systemic humoral responses to key H pylori antigens. The finding of spontaneous clearance of H pylori infection in more than half the participants in the placebo group is remarkable and suggests important immune protection in the healthy adult population. FUNDING: Novartis Vaccine and Diagnostics.
Asunto(s)
Vacunas Bacterianas/inmunología , Vacunas Bacterianas/uso terapéutico , Gastritis/prevención & control , Infecciones por Helicobacter/prevención & control , Helicobacter pylori/inmunología , Inmunogenicidad Vacunal , Adulto , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Vacunas Bacterianas/efectos adversos , Quimiocina CXCL1/inmunología , Método Doble Ciego , Femenino , Gastritis/microbiología , Humanos , Inmunidad Celular , Inmunoglobulina G/sangre , Inyecciones Intramusculares , Masculino , Vacunas Sintéticas/efectos adversos , Vacunas Sintéticas/inmunología , Vacunas Sintéticas/uso terapéutico , Adulto JovenRESUMEN
Despite high vaccination coverage worldwide, pertussis has re-emerged in many countries. This randomized, controlled, observer-blind phase I study and extension study in Belgium (March 2012-June 2015) assessed safety and immunogenicity of investigational acellular pertussis vaccines containing genetically detoxified pertussis toxin (PT) (NCT01529645; NCT02382913). 420 healthy adults (average age: 26.8 ± 5.5 years, 60% female) were randomized to 1 of 10 vaccine groups: 3 investigational aP vaccines (containing pertussis antigens PT, filamentous hemagglutinin [FHA] and pertactin [PRN] at different dosages), 6 investigational TdaP (additionally containing tetanus toxoid [TT] and diphtheria toxoid [DT]), and 1 TdaP comparator containing chemically inactivated PT. Antibody responses were evaluated on days 1, 8, 30, 180, 365, and approximately 3 years post-booster vaccination. Cell-mediated immune responses and PT neutralization were evaluated in a subset of participants in pre-selected groups. Local and systemic adverse events (AEs), and unsolicited AEs were collected through day 7 and 30, respectively; serious AEs and AEs leading to study withdrawal were collected through day 365 post-vaccination. Antibody responses against pertussis antigens peaked at day 30 post-vaccination and then declined but remained above baseline level at approximately 3 years post-vaccination. Responses to FHA and PRN were correlated to antigen dose. Antibody responses specific to PT, toxin neutralization activity and persistence induced by investigational formulations were similar or significantly higher than the licensed vaccine, despite lower PT doses. Of 15 serious AEs, none were considered vaccination-related; 1 led to study withdrawal (premature labor, day 364; aP4 group). This study confirmed the potential benefits of genetically detoxified PT antigen. All investigational study formulations were well tolerated.
Asunto(s)
Vacunas contra Difteria, Tétanos y Tos Ferina Acelular/administración & dosificación , Inmunización Secundaria/métodos , Toxina del Pertussis/inmunología , Vacuna contra la Tos Ferina/administración & dosificación , Vacunación/métodos , Tos Ferina/prevención & control , Adulto , Anticuerpos Antibacterianos/análisis , Bélgica , Vacunas contra Difteria, Tétanos y Tos Ferina Acelular/efectos adversos , Vacunas contra Difteria, Tétanos y Tos Ferina Acelular/genética , Vacunas contra Difteria, Tétanos y Tos Ferina Acelular/inmunología , Femenino , Humanos , Inmunidad Celular , Inmunogenicidad Vacunal , Masculino , Toxina del Pertussis/genética , Vacuna contra la Tos Ferina/efectos adversos , Vacuna contra la Tos Ferina/genética , Vacuna contra la Tos Ferina/inmunología , Resultado del Tratamiento , Tos Ferina/sangre , Tos Ferina/inmunología , Adulto JovenRESUMEN
CD4+ T follicular helper cells (T(FH)) have been identified as the T-cell subset specialized in providing help to B cells for optimal activation and production of high affinity antibody. We recently demonstrated that the expansion of peripheral blood influenza-specific CD4(+)IL-21(+)ICOS1(+) T helper (T(H)) cells, three weeks after vaccination, associated with and predicted the rise of protective neutralizing antibodies to avian H5N1. In this study, healthy adults were vaccinated with plain seasonal trivalent inactivated influenza vaccine (TIIV), MF59(®)-adjuvanted TIIV (ATIIV), or saline placebo. Frequencies of circulating CD4(+) T(FH)1 ICOS(+) T(FH) cells and H1N1-specific CD4(+-)IL-21(+)ICOS(+) CXCR5(+) T(FH) and CXCR5(-) T(H) cell subsets were determined at various time points after vaccination and were then correlated with hemagglutination inhibition (HI) titers. All three CD4(+) T cell subsets expanded in response to TIIV and ATIIV, and peaked 7 days after vaccination. To demonstrate that these T(FH) cell subsets correlated with functional antibody titers, we defined an alternative endpoint metric, decorrelated HI (DHI), which removed any correlation between day 28/day 168 and day 0 HI titers, to control for the effect of preexisting immunity to influenza vaccine strains. The numbers of total circulating CD4(+)T(FH)1 ICOS(+) cells and of H1N1-specific CD4(+)IL-21(+)ICOS(+) CXCR5(+), measured at day 7, were significantly associated with day 28, and day 28 and 168 DHI titers, respectively. Altogether, our results show that CD4(+) T(FH) subsets may represent valuable biomarkers of vaccine-induced long-term functional immunity.
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Formación de Anticuerpos/inmunología , Inmunidad , Recuento de Linfocitos , Subgrupos de Linfocitos T/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Vacunación , Adolescente , Adulto , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Biomarcadores , Pruebas de Inhibición de Hemaglutinación , Humanos , Inmunofenotipificación , Proteína Coestimuladora de Linfocitos T Inducibles/metabolismo , Subtipo H1N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Activación de Linfocitos/inmunología , Pronóstico , Vigilancia en Salud Pública , Receptores CXCR5/metabolismo , Subgrupos de Linfocitos T/metabolismo , Linfocitos T Colaboradores-Inductores/metabolismo , Factores de Tiempo , Adulto JovenRESUMEN
Protection against influenza is mediated by neutralizing antibodies, and their induction at high and sustained titers is key for successful vaccination. Optimal B cells activation requires delivery of help from CD4(+) T lymphocytes. In lymph nodes and tonsils, T-follicular helper cells have been identified as the T cells subset specialized in helping B lymphocytes, with interleukin-21 (IL-21) and inducible costimulatory molecule (ICOS1) playing a central role for this function. We followed the expansion of antigen-specific IL-21(+) CD4(+) T cells upon influenza vaccination in adults. We show that, after an overnight in vitro stimulation, influenza-specific IL-21(+) CD4(+) T cells can be measured in human blood, accumulate in the CXCR5(-)ICOS1(+) population, and increase in frequency after vaccination. The expansion of influenza-specific ICOS1(+)IL-21(+) CD4(+) T cells associates with and predicts the rise of functionally active antibodies to avian H5N1. We also show that blood-derived CXCR5(-)ICOS1(+) CD4(+) T cells exert helper function in vitro and support the differentiation of influenza specific B cells in an ICOS1- and IL-21-dependent manner. We propose that the expansion of antigen-specific ICOS1(+)IL-21(+) CD4(+) T cells in blood is an early marker of vaccine immunogenicity and an important immune parameter for the evaluation of novel vaccination strategies.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Vacunas contra la Influenza/inmunología , Gripe Humana/inmunología , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Humanos , Virus de la Influenza A/inmunología , Vacunas contra la Influenza/administración & dosificación , Gripe Humana/sangre , Gripe Humana/prevención & control , Interleucinas , VacunaciónRESUMEN
Protective antibody responses to a single dose of 2009 pandemic vaccines have been observed in the majority of healthy subjects aged more than 3 years. These findings suggest that immune memory lymphocytes primed by previous exposure to seasonal influenza antigens are recruited in the response to A/H1N1 pandemic vaccines and allow rapid seroconversion. However, a clear dissection of the immune memory components favoring a fast response to pandemic vaccination is still lacking. Here we report the results from a clinical study where antibody, CD4+ T cell, plasmablast and memory B cell responses to one dose of an MF59-adjuvanted A/H1N1 pandemic vaccine were analyzed in healthy adults. While confirming the rapid appearance of antibodies neutralizing the A/H1N1 pandemic virus, we show here that the response is dominated by IgG-switched antibodies already in the first week after vaccination. In addition, we found that vaccination induces the rapid expansion of pre-existing CD4+ T cells and IgG-memory B lymphocytes cross-reactive to seasonal and pandemic A/H1N1 antigens. These data shed light on the different components of the immune response to the 2009 H1N1 pandemic influenza vaccination and may have implications in the design of vaccination strategies against future influenza pandemics.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Memoria Inmunológica , Subtipo H1N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Polisorbatos/administración & dosificación , Escualeno/administración & dosificación , Adolescente , Adulto , Linfocitos B/inmunología , Linfocitos T CD4-Positivos/inmunología , Reacciones Cruzadas , Femenino , Humanos , Vacunas contra la Influenza/administración & dosificación , Masculino , Persona de Mediana Edad , Vacunación/métodos , Adulto JovenRESUMEN
The complex pathology of B. pertussis infection is due to multiple virulence factors having disparate effects on different cell types. We focused our investigation on the ability of B. pertussis to modulate host immunity, in particular on the role played by adenylate cyclase toxin (CyaA), an important virulence factor of B. pertussis. As a tool, we used human monocyte derived dendritic cells (MDDC), an ex vivo model useful for the evaluation of the regulatory potential of DC on T cell immune responses. The work compared MDDC functions after encounter with wild-type B. pertussis (BpWT) or a mutant lacking CyaA (BpCyaA-), or the BpCyaA- strain supplemented with either the fully functional CyaA or a derivative, CyaA*, lacking adenylate cyclase activity. As a first step, MDDC maturation, cytokine production, and modulation of T helper cell polarization were evaluated. As a second step, engagement of Toll-like receptors (TLR) 2 and TLR4 by B. pertussis and the signaling events connected to this were analyzed. These approaches allowed us to demonstrate that CyaA expressed by B. pertussis strongly interferes with DC functions, by reducing the expression of phenotypic markers and immunomodulatory cytokines, and blocking IL-12p70 production. B. pertussis-treated MDDC promoted a mixed Th1/Th17 polarization, and the activity of CyaA altered the Th1/Th17 balance, enhancing Th17 and limiting Th1 expansion. We also demonstrated that Th1 effectors are induced by B. pertussis-MDDC in the absence of IL-12p70 through an ERK1/2 dependent mechanism, and that p38 MAPK is essential for MDDC-driven Th17 expansion. The data suggest that CyaA mediates an escape strategy for the bacterium, since it reduces Th1 immunity and increases Th17 responses thought to be responsible, when the response is exacerbated, for enhanced lung inflammation and injury.
Asunto(s)
Toxina de Adenilato Ciclasa/metabolismo , Bordetella pertussis/fisiología , Células Dendríticas/inmunología , Sistema de Señalización de MAP Quinasas , Linfocitos T Colaboradores-Inductores/inmunología , Toxina de Adenilato Ciclasa/fisiología , Citocinas/biosíntesis , Humanos , Fosfatidilinositol 3-Quinasas/metabolismoRESUMEN
Genetically detoxified pertussis toxin (dPT) maintains the protein structure and the immunological properties, but not the enzymatic activity. In search of an adjuvant able to direct polarization of T cells to induce/potentiate protective immune response to a variety of infectious disease, we investigated the role played by dPT on human dendritic cell-driven Th polarization and analyzed the intracellular signaling events. To reach these aims, we used a highly purified dPT preparation devoid of contamination and monocyte-derived dendritic cells, a well-characterized model to study ex vivo the polarization of the immune responses. First, we analyzed dPT-induced monocyte-derived dendritic cell maturation, longevity, and cytokine production and, in a second step, we analyzed TLR4/2 engagement by dPT, the connected signaling events, and their relevance to the skewing of Th cell polarization. These approaches allowed us to clarify some of the mechanisms that are responsible for dPT-driven regulation of T cell polarization. We demonstrated that dPT acts utilizing TLR4/TLR2 engagement, being the signaling induced by the former stronger. dPT, through a crucial role played by MAPK and IL-10, favors the expansion of the Th1/Th17 immunity. Indirect evidences indicated that dPT-induced Th17 expansion is counterregulated by the PI3K pathway. For its properties and being already used in humans as vaccine Ag in pertussis, dPT may represents a valid candidate adjuvant to foster immune protective response in vaccines against infectious diseases where Th1/Th17 are mediating host immunity.
Asunto(s)
Interleucina-10/inmunología , Proteínas Quinasas Activadas por Mitógenos/inmunología , Toxina del Pertussis/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Proliferación Celular , Citocinas/análisis , Células Dendríticas/citología , Humanos , Interleucina-17/inmunología , Toxina del Pertussis/genética , Linfocitos T Colaboradores-Inductores/citología , Células TH1/inmunología , Receptor Toll-Like 2 , Receptor Toll-Like 4RESUMEN
The invasion and the immunomodulatory effect of a Bordetella pertussis natural deficient strain 00141(PRN-) on human dendritic cells (MDDC) and its in vivo infection ability in a mouse model were evaluated in comparison with the reference B. pertussis strain ATCC 97-97 (18323). The mutant was isolated from a case of pertussis which occurred in a 22-month-old infant with typical symptoms of the disease. The results showed that this natural B. pertussis PRN deficient strain presented higher invasion ability of human MDDC compared to the reference strain. This natural mutant similar to the B. pertussis reference strain had immunomodulatory properties, inducing maturation in the DC phenotype which resulted in the acquisition of potent T cell-activating properties and down-regulated IL-12 production, and secretion of IL-10. The ability of PRN- strain to infect the lungs of CD1 mice was comparable to the reference strain and no difference was observed in the kinetics of clearance. Overall, these results show that the enhanced ability of the PRN- strain to invade/infect MDCC suggest that the PRN antigen may play a role in survival of the microorganism in the host.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Bordetella pertussis/patogenicidad , Células Dendríticas/inmunología , Células Dendríticas/microbiología , Factores de Virulencia de Bordetella/metabolismo , Tos Ferina , Animales , Antígenos CD/biosíntesis , Antígeno B7-1/biosíntesis , Bordetella pertussis/aislamiento & purificación , Bordetella pertussis/metabolismo , Técnicas de Cultivo de Célula , Células Dendríticas/metabolismo , Antígenos HLA-DR/biosíntesis , Humanos , Inmunoglobulinas/biosíntesis , Lactante , Interleucinas/biosíntesis , Glicoproteínas de Membrana/biosíntesis , Ratones , Monocitos/citología , Monocitos/inmunología , Virulencia , Tos Ferina/inmunología , Tos Ferina/microbiología , Antígeno CD83RESUMEN
Bordetella pertussis and B. parapertussis are the etiological agents of pertussis, yet the former has a higher incidence and is the cause of a more severe disease, in part due to pertussis toxin. To identify other factors contributing to the different pathogenicity of the two species, we analyzed the capacity of structurally different lipooligosaccharide (LOS) from B. pertussis and LPS from B. parapertussis to influence immune functions regulated by dendritic cells. Either B. pertussis LOS and B. parapertussis LPS triggered TLR4 signaling and induced phenotypic maturation and IL-10, IL-12p40, IL-23, IL-6, and IL-1beta production in human monocyte-derived dendritic cells (MDDC). B. parapertussis LPS was a stronger inducer of all these activities as compared with B. pertussis LOS, with the notable exception of IL-1beta, which was equally produced. Only B. parapertussis LPS was able to induce IL-27 expression. In addition, although MDDC activation induced by B. parapertussis LPS was greatly dependent on soluble CD14, B. pertussis LOS activity was CD14-independent. The analysis of the intracellular pathways showed that B. parapertussis LPS and B. pertussis LOS equally induced IkappaBalpha and p38 MAPK phosphorylation, but B. pertussis LOS triggered ERK1/2 phosphorylation more rapidly and at higher levels than B. parapertussis LPS. Furthermore, B. pertussis LOS was unable to induce MyD88-independent gene induction, which was instead activated by B. parapertussis LPS, witnessed by STAT1 phosphorylation and induction of the IFN-dependent genes, IFN regulatory factor-1 and IFN-inducible protein-10. These differences resulted in a divergent regulation of Th cell responses, B. pertussis LOS MDDC driving a predominant Th17 polarization. Overall, the data observed reflect the different structure of the two LPS and the higher Th17 response induced by B. pertussis LOS may contribute to the severity of pertussis in humans.
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Bordetella parapertussis , Bordetella pertussis , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Lipopolisacáridos/farmacología , Linfocitos T Colaboradores-Inductores/efectos de los fármacos , Linfocitos T Colaboradores-Inductores/inmunología , Bordetella parapertussis/metabolismo , Bordetella pertussis/metabolismo , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/inmunología , Línea Celular , Células Dendríticas/citología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Humanos , Receptores de Lipopolisacáridos/inmunología , Lipopolisacáridos/biosíntesis , Monocitos/inmunología , Factor 88 de Diferenciación Mieloide/metabolismo , Unión Proteica , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Receptor Toll-Like 4/metabolismo , Activación TranscripcionalRESUMEN
Dendritic cells (DCs) are central players in immunity and are used in immune-adoptive vaccine protocols in humans. IFN-gamma, mandatory in Th-1 polarization and endowed with regulatory properties, is currently used to condition monocyte-derived DCs (MDDC) in cancer therapy and in clinical trials to treat chronic infectious diseases. We therefore performed a wide analysis of IFN-gamma signaling consequences on MDDC multiple effector functions. IFN-gamma itself induced IL-27p28 expression and survival but did not promote relevant CCR7-driven migration or activated Th-1 cell recruitment capacity in MDDC. Administered in association with classical maturation stimuli such as CD40 or TLR-4 stimulation, IFN-gamma up-regulated IL-27 and IL-12 production, CCR7-driven migration, and activated Th-1 cell recruitment, whereas it decreased IL-10 production and STAT3 phosphorylation. CD38 signaling, which orchestrates migration, survival, and Th-1 polarizing ability of mature MDDC, was involved in IFN-gamma-mediated effects. Thus, IFN-gamma is a modulator of multiple DC effector functions that can be helpful in MDDC-based vaccination protocols. These data also help understand the dual role exerted by this cytokine as both an inducer and a regulator of inflammation and immune response.
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Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Interferón gamma/farmacología , Células TH1/inmunología , ADP-Ribosil Ciclasa 1/metabolismo , Células Presentadoras de Antígenos/efectos de los fármacos , Células Presentadoras de Antígenos/inmunología , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Humanos , Interleucina-10/metabolismo , Interleucina-12/metabolismo , Monocitos/inmunología , Receptores CCR7 , Receptores Toll-Like/metabolismo , Regulación hacia ArribaRESUMEN
Tests based on the gamma interferon (IFN-gamma) assay (IGA) are used as adjunctive tools for the diagnosis of Mycobacterium tuberculosis infection. Here we compared in-house and commercial whole-blood IGAs to identify a suitable assay for the surveillance of tuberculosis in population studies. The IGAs were selected on the basis of the ease with which they are performed and because they require a small amount of a biological sample and do not require cell purification. Since a "gold standard" for latently M. tuberculosis-infected individuals is not available, the sensitivities and the specificities of the IGAs were determined with samples from patients with clinically diagnosed active tuberculosis and in Mycobacterium bovis BCG-unvaccinated healthy controls. The in-house tests consisted of a bulk assay based on diluted whole blood and a single-cell assay based on IFN-gamma intracellular staining. The commercial assays used were the QuantiFERON-TB-Gold (Q-TB) and the Q-TB in-tube tests. When the purified protein derivative was used as the antigen, in-house whole-blood intracellular staining was found to be highly discriminatory between active tuberculosis patients and BCG-vaccinated healthy controls, whereas the other IGAs did not discriminate between the two categories of patients. When M. tuberculosis-specific antigens were used, a very strong agreement between the results of the Q-TB in-tube assay and the clinical diagnosis was observed, while the Q-TB assay, performed according to the manufacturer's instructions, showed a significantly lower performance. Intriguingly, when the test was performed with RD1 proteins instead of peptides, its sensitivity was significantly increased. The in-house assay with diluted whole blood showed an elevated sensitivity and an elevated specificity, and the results agreed with the clinical diagnosis. Considering that the in-house assay uses 1/20 of the sample compared with the amount of sample used in the commercial IGA, it appears to be particularly promising for use in pediatric studies. Overall, the different assays showed different performance characteristics that need to be considered for surveillance of tuberculosis in population studies.
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Antígenos Bacterianos/inmunología , Inmunoensayo/métodos , Interferón gamma/análisis , Mycobacterium tuberculosis/inmunología , Tuberculosis/diagnóstico , Adulto , Sangre/inmunología , Proliferación Celular , Diagnóstico Diferencial , Humanos , Leucocitos Mononucleares/inmunología , Mycobacterium bovis/inmunología , Sensibilidad y Especificidad , Vacunas contra la Tuberculosis/inmunologíaRESUMEN
Bordetella pertussis has a distinctive cell wall lipooligosaccharide (LOS) that is released from the bacterium during bacterial division and killing. LOS directly participates in host-bacterial interactions, in particular influencing the dendritic cells' (DC) immune regulatory ability. We analyze LOS mediated toll-like receptor (TLR) activation and dissect the role played by LOS on human monocyte-derived (MD)DC functions and polarization of the host T cell response. LOS activates TLR4-dependent signaling and induces mature MDDC able to secrete IL-10. LOS-matured MDDC enhance allogeneic presentation and skew T helper (Th) cell polarization towards a Th2 phenotype. LOS protects MDDC from undergoing apoptosis, prolonging their longevity and their functions. Compared to Escherichia coli lipopolysaccharide (LPS), the classical DC maturation stimulus, LOS was a less efficient inducer of TLR4 signaling, MDDC maturation, IL-10 secretion and allogeneic T cell proliferation and it was not able to induce IL-12p70 production in MDDC. However, the MDDC apoptosis protection exerted by LOS and LPS were comparable. In conclusion, LOS treated MDDC are able to perform antigen presentation in a context that promotes licensing of Th2 effectors. Considering these properties, the use of LOS in the formulation of acellular pertussis vaccines to potentiate protective and adjuvant capacity should be taken into consideration.
Asunto(s)
Bordetella pertussis/inmunología , Células Dendríticas/inmunología , Lipopolisacáridos/inmunología , Células Th2/inmunología , Factores de Virulencia de Bordetella/inmunología , Tos Ferina/inmunología , Apoptosis/inmunología , Proliferación Celular , Células Dendríticas/microbiología , Citometría de Flujo , Humanos , Inmunofenotipificación , Interleucina-10/genética , Interleucina-10/inmunología , Lipopolisacáridos/farmacología , ARN/química , ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/inmunología , Linfocitos T/inmunología , Linfocitos T/microbiología , Células Th2/microbiología , Receptor Toll-Like 4/inmunología , Factores de Virulencia de Bordetella/farmacología , Tos Ferina/microbiologíaRESUMEN
Clostridium difficile, an etiological agent of most cases of antibiotic-associated diarrhea, exerts its pathological action mainly by the activity of toxin A and toxin B. Less known is the role that S-layer proteins (SLPs), predominant surface components of the bacterium, may play in pathogenesis. Here, we evaluate the ability of SLPs to modulate the function of human monocytes and dendritic cells (DC) and to induce inflammatory and regulatory cytokines, influencing the natural and adaptive immune response. To this aim, SLPs were extracted from the clinical isolate C253 and characterized for their effects on immune cells. SLPs induced the release of elevated amounts of interleukin (IL)-1beta and IL-6 pro-inflammatory cytokines by resting monocytes, induced maturation of human monocyte-derived DC (MDDC), and enhanced proliferation of allogeneic T cells. C253-SLP-treated MDDC also secreted large amounts of IL-10 and IL-12p70 and induced a mixed Th1/Th2 orientation of immune response in naïve CD4 T cells. In conclusion, C. difficile SLPs may contribute to the pathogenicity of the bacterium by perturbing the fine balance of inflammatory and regulatory cytokines. These data are of interest also in the light of the possible use of SLPs in a multicomponent vaccine against C. difficile infections for high-risk patients.
Asunto(s)
Proteínas Bacterianas/inmunología , Clostridioides difficile/inmunología , Citocinas/biosíntesis , Células Dendríticas/inmunología , Glicoproteínas de Membrana/inmunología , Monocitos/inmunología , Proteínas Bacterianas/aislamiento & purificación , Proliferación Celular , Activación de Linfocitos , Glicoproteínas de Membrana/aislamiento & purificación , Linfocitos T/inmunologíaRESUMEN
Bordetella pertussis, the causative agent of whooping cough, possesses an array of virulence factors, including adenylate cyclase toxin (ACT), relevant in the establishment of infection. Here we better define the impact of cyclic AMP (cAMP) intoxication due to the action of ACT on dendritic cell (DC)-driven immune response, by infecting monocyte-derived DC (MDDC) with an ACT-deficient B. pertussis mutant (ACT- 18HS19) or its parental strain (WT18323). Both strains induced MDDC maturation and antigen-presenting cell functions; however, only ACT- 18HS19 infected MDDC-induced production of interleukin-12 (IL-12) p70. Gene expression analysis of the IL-12 cytokine family subunits revealed that both strains induced high levels of p40 (protein chain communal to IL-12 p70 and IL-23) as well as p19, a subunit of IL-23. Conversely only ACT- 18HS19 infection induced consistent transcription of IL-12 p35, a subunit of IL-12 p70. Addition of the cAMP analogous D-butyril-cAMP (D-cAMP) abolished IL-12 p70 production and IL-12 p35 expression in ACT- 18HS19-infected MDDC. ACT- 18HS19 infection induced the expression of the transcription factors interferon regulatory factor 1 (IRF-1) and IRF-8 and of beta interferon, involved in IL-12 p35 regulation, and the expression of these genes was inhibited by D-cAMP addition and in WT18323-infected MDDC. The concomitant expression of IL-12 p70 and IL-23 allowed ACT- 18HS19 to trigger a more pronounced T helper 1 polarization compared to WT18323. The present study suggests that ACT-dependent cAMP induction leads to the inhibition of pathways ultimately leading to IL-12 p35 production, thus representing a mechanism for B. pertussis to escape the host immune response.
Asunto(s)
Toxina de Adenilato Ciclasa/fisiología , Bordetella pertussis/patogenicidad , AMP Cíclico/biosíntesis , Células Dendríticas/metabolismo , Interleucina-12/antagonistas & inhibidores , Monocitos/citología , Subunidades de Proteína/antagonistas & inhibidores , Polaridad Celular , Células Cultivadas , Humanos , Factor 1 Regulador del Interferón/genética , Factores Reguladores del Interferón/genética , Interferón beta/genética , Subunidad p35 de la Interleucina-12 , Células TH1/fisiologíaRESUMEN
Infection, in particular by Chlamydia pneumoniae (Cp), has been associated with atherosclerosis and coronary heart disease. Immune reactions to heat shock proteins (HSPs) have been advocated to link infection to atherosclerosis and its acute sequelae based on molecular mimicry with host HSPs. We have here evaluated the role played by recombinant Cp-HSP60 and Cp-HSP10 for their ability to induce maturation of human monocyte-derived dendritic cells (MDDC) and T cell polarization. Cp-HSP60, but not Cp-HSP10, induced a strong MDCC maturation, as assessed by the expression of co-stimulatory molecules and other markers. Secretion of regulatory cytokines and enhancement of antigen presenting ability of mature (m)MDDC toward a clear T helper (Th) 1 pattern were also induced by Cp-HSP60. An analysis of the IL-12 cytokine family demonstrated that Cp-HSP60-matured MDDC were able to express p35 and p40 mRNA subunits to form IL-12, and p19 and p40 subunits to form IL-23. Thus, preferential Th1 polarization of immune response induced by Cp-HSP60-matured MDDC appears to be due to the concomitant expression of IL-12 and IL-23. Our data suggest that Cp-HSP60-matured DC may contribute to T-cell mediated immunopathology of atherosclerosis via a chronic stimulation of Th1 immune responses.
Asunto(s)
Chaperonina 60/inmunología , Chlamydophila pneumoniae/inmunología , Células Dendríticas/inmunología , Interleucina-12/biosíntesis , Interleucinas/biosíntesis , Monocitos/citología , Células TH1/inmunología , Células Cultivadas , Chaperonina 60/metabolismo , Chlamydophila pneumoniae/metabolismo , Células Dendríticas/citología , Células Dendríticas/metabolismo , Regulación de la Expresión Génica , Humanos , Interleucina-12/genética , Interleucina-12/metabolismo , Interleucina-23 , Subunidad p19 de la Interleucina-23 , Interleucinas/genética , Interleucinas/metabolismo , Activación de Macrófagos , Células TH1/metabolismoRESUMEN
Bordetella pertussis, the causative agent of whooping cough, is internalized by several cell types, including epithelial cells, monocytes, and neutrophils. Although its ability to survive intracellularly is still debated, it has been proven that cell-mediated immunity (CMI) plays a pivotal role in protection. In this study we aimed to clarify the interaction of B. pertussis with human monocyte-derived dendritic cells (MDDC), evaluating the ability of the bacterium to enter MDDC, to survive intracellularly, to interfere with the maturation process and functional activities, and to influence the host immune responses. The results obtained showed that B. pertussis had a low capability to be internalized by-and to survive in-MDDC. Upon contact with the bacteria, immature MDDC were induced to undergo phenotypic maturation and acquired antigen-presenting-cell functions. Despite the high levels of interleukin-10 (IL-10) and the barely detectable levels of IL-12 induced by B. pertussis, the bacterium induced maturation of MDDC and T helper 1 (Th1) polarized effector cells. Gene expression analysis of the IL-12 cytokine family clearly demonstrated that B. pertussis induced high levels of the p40 and p19 subunits of IL-23 yet failed to induce the expression of the p35 subunit of IL-12. Overall our findings show that B. pertussis, even if it survives only briefly in MDDC, promotes the synthesis of IL-23, a newly discovered Th1 polarizing cytokine. A Th1-oriented immune response is thus allowed, relevant in the induction of an adequate CMI response, and typical of protection induced by natural infection or vaccination with whole-cell vaccines.