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FLT3L-Fc is a cytokine-Fc fusion agonizing receptor-type tyrosine-protein kinase FLT3 (fms-related tyrosine kinase 3; CD135). FLT3 is expressed on dendritic cells (DCs) as well as myeloid and lymphoid progenitors. Nonclinical pharmacokinetics, pharmacodynamics and safety of FLT3L-Fc were investigated in rats and cynomolgus monkeys. FLT3L-Fc induced robust pharmacodynamic responses, evidenced by marked expansion of peripheral blood cDC1s, cDC2s, and pDCs (up to 301-fold in rats and 378-fold in monkeys), peaking at 8-10 days after the first dose. FLT3L-Fc was well tolerated with no adverse findings at doses up to 10 mg/kg administered intravenously twice three weeks apart. In both species, major clinical pathology findings consisted of expansion of white blood cell (WBC) populations including lymphocytes, monocytes, neutrophils, basophils, and large unstained cells, which were pronounced after the first dose. The WBC findings were associated microscopically with histiocytic and mononuclear cell infiltrates in multiple organs. Tissue immunohistochemistry in monkeys showed that the leukocyte infiltrates consisted of hematopoietic progenitor cells and histiocytes with a reactive morphology and were associated with a slight stimulation of regional T and B cell populations. Additional FLT3L-Fc-associated changes included decreases in red blood cell (RBC) mass, increases in RBC distribution width, variable changes in reticulocytes, and transient alterations in platelet counts (rats only). The RBC and WBC findings were associated microscopically with increased hematopoietic cellularity of the bone marrow in both species and increased splenic megakaryocytic extramedullary hematopoiesis in rats. The totality of nonclinical safety data support the clinical development of FLT3L-Fc.
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Proteínas de la Membrana , Neoplasias , Ratas , Animales , Células Dendríticas , Células Madre Hematopoyéticas , InmunoterapiaRESUMEN
Type I interferons are essential for host response to viral infections, while dysregulation of their response can result in autoinflammation or autoimmunity. Among IFNα (alpha) responses, 13 subtypes exist that signal through the same receptor, but have been reported to have different effector functions. However, the lack of available tools for discriminating these closely related subtypes, in particular at the protein level, has restricted the study of their differential roles in disease. We developed a digital ELISA with specificity and high sensitivity for the IFNα2 subtype. Application of this assay, in parallel with our previously described pan-IFNα assay, allowed us to study different IFNα protein responses following cellular stimulation and in diverse patient cohorts. We observed different ratios of IFNα protein responses between viral infection and autoimmune patients. This analysis also revealed a small percentage of autoimmune patients with high IFNα2 protein measurements but low pan-IFNα measurements. Correlation with an ISG score and functional activity showed that in this small sub group of patients, IFNα2 protein measurements did not reflect its biological activity. This unusual phenotype was partly explained by the presence of anti-IFNα auto-antibodies in a subset of autoimmune patients. This study reports ultrasensitive assays for the study of IFNα proteins in patient samples and highlights the insights that can be obtained from the use of multiple phenotypic readouts in translational and clinical studies.
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Antivirales/inmunología , Autoinmunidad/inmunología , Interferón-alfa/inmunología , Virosis/inmunología , Adolescente , Adulto , Anciano , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
Antitumor immune responses depend on the infiltration of solid tumors by effector T cells, a process guided by chemokines. In particular, the chemokine CXCL10 has been shown to play a critical role in mediating recruitment of CXCR3 + cytolytic T and NK cells in tumors, though its use as a therapeutic agent has not been widely explored. One of the limitations is due to the rapid inactivation of CXCL10 by dipeptidyl peptidase 4 (DPP4), a broadly expressed enzyme that is active in plasma and other bodily fluids. In the present study, we describe a novel method to produce synthetic CXCL10 that is resistant to DPP4 N-terminal truncation. Using a Fmoc solid-phase peptide synthesis approach, synthetic murine WT CXCL10 was produced, showing similar biochemical and biological properties to the recombinant protein. This synthesis method supported production of natural (amino acid substitution, insertion or deletion) and non-natural (chemical modifications) variants of CXCL10. In association with a functional screening cascade that assessed DPP4-mediated cleavage, CXCR3 signaling potency and chemotactic activity, we successfully generated 20 murine CXCL10 variants. Among those, two non-natural variants with N-methylated Leu3 (MeLeu3) and a reduced amide bond between Pro2 and Leu3 (rLeu3), respectively, showed resistance to DPP4 truncation but decreased CXCR3 signaling and chemotactic activity. Interestingly, MeLeu3 and rLeu3 CXCL10 behaved as DPP4 inhibitors, preventing the truncation of WT CXCL10. This study highlights the potential of using Fmoc solid-phase chemistry in association with biochemical and biological characterization to rapidly identify CXCL10 variants with desired properties. These novel methods unlock the opportunity to develop DPP4 resistant CXCL10 variants, as well as other chemokine substrates, while maintaining chemotactic properties.
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Quimiocina CXCL10/farmacología , Dipeptidil Peptidasa 4/metabolismo , Inhibidores de la Dipeptidil-Peptidasa IV/farmacología , Quimiocina CXCL10/síntesis química , Quimiocina CXCL10/química , Inhibidores de la Dipeptidil-Peptidasa IV/síntesis química , Inhibidores de la Dipeptidil-Peptidasa IV/química , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Relación Estructura-ActividadRESUMEN
Tumor cell heterogeneity and tumor cell-stromal interactions are being explored as determinants of disease progression and treatment resistance in solid tumor and hematological malignancies. As such, tools simultaneously capable of highly multiplexed profiling of tissues' protein and RNA content, as well as interrogation of rare or single cells, are required to precisely characterize constituent tumor cell populations, infiltrating lymphocytes and stromal elements. Access to spatial relationships will enable more precise characterization of tumors, support patient stratification and may help to identify novel drug targets. Multiple platforms are being developed to address these critical unmet needs. The NanoString digital spatial profiling (DSP) platform enables highly multiplexed, spatial assessment of protein and/or RNA targets in tissues by detecting oligonucleotide barcodes conjugated via a photocleavable linker to primary antibodies or nucleic acid probes. Although this platform enables high-dimensional spatial interrogation of tissue protein and RNA expression, a detailed understanding of its composition, function and chemistry is advisable to guide experimental design and data interpretation. The purpose of this review is to provide an independent, comprehensive description of the DSP technology, including an overview of NanoString's capture and antibody barcode conjugation chemistries, experimental workflow, data output and analysis methods. The DSP technology will be discussed in the context of other highly multiplexed immunohistochemistry methods, including imaging mass cytometry and multiplexed ion beam imaging, to inform potential users of the advantages and limitations of each. Additional issues such as preanalytical variability, sampling and specimen adequacy will be considered with respect to the platforms to inform potential experimental design. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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Biomarcadores de Tumor/metabolismo , Neoplasias/patología , ARN Neoplásico/metabolismo , Análisis de Datos , Progresión de la Enfermedad , Procesamiento Automatizado de Datos , Humanos , Hibridación in SituRESUMEN
Along with CD4+ T lymphocytes, macrophages are a major cellular source of HIV-1 replication and a potential viral reservoir. Following entry and reverse transcription in macrophages, cloaking of the viral cDNA by the HIV-1 capsid limits its cytosolic detection, enabling efficient replication. However, whether incoming HIV-1 particles are sensed by macrophages prior to reverse transcription remains unclear. Here, we show that HIV-1 triggers a broad expression of interferon (IFN)-stimulated genes (ISG) in monocyte-derived macrophages within a few hours after infection. This response does not require viral reverse transcription or the presence of HIV-1 RNA within particles, but viral fusion is essential. This response is elicited by viruses carrying different envelope proteins and thus different receptors to proceed for viral entry. Expression of ISG in response to viral entry requires TBK1 activity and type I IFNs signaling. Remarkably, the ISG response is transient but affects subsequent viral spread. Together, our results shed light on an early step of HIV-1 sensing by macrophages at the level of entry, which confers an early protection through type I IFN signaling and has potential implications in controlling the infection.IMPORTANCE HIV infection is restricted to T lymphocytes and macrophages. HIV-1-infected macrophages are found in many tissues of infected patients, even under antiretroviral therapy, and are considered a viral reservoir. How HIV-1 is detected and what type of responses are elicited upon sensing remain in great part elusive. The kinetics and localization of the production of cytokines such as interferons in response to HIV is of critical importance to understanding how the infection and the immune response are established. Our study provides evidence that macrophages can detect HIV-1 as soon as it enters the cell. Interestingly, this sensing is independent of the presence of viral nucleic acids within the particles but requires their fusion with the macrophages. This triggers a low interferon response, which activates an antiviral program protecting cells against further viral challenge and thus potentially limiting the spread of the infection.
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VIH-1/inmunología , VIH-1/fisiología , Inmunidad Innata , Interferón Tipo I/metabolismo , Macrófagos/inmunología , Macrófagos/virología , Internalización del Virus , Células Cultivadas , Humanos , Proteínas Serina-Treonina Quinasas/metabolismo , Factores de TiempoRESUMEN
The existence of HIV reservoirs in infected individuals under combined antiretroviral therapy (cART) represents a major obstacle toward cure. Viral reservoirs are assessed by quantification of HIV nucleic acids, a method which does not discriminate between infectious and defective viruses, or by viral outgrowth assays, which require large numbers of cells and long-term cultures. Here, we used an ultrasensitive p24 digital assay, which we report to be 1,000-fold more sensitive than classical enzyme-linked immunosorbent assays (ELISAs) in the quantification of HIV-1 Gag p24 production in samples from HIV-infected individuals. Results from ultrasensitive p24 assays were compared to those from conventional viral RNA reverse transcription-quantitative PCR (RT-qPCR)-based assays and from outgrowth assay readout by flow cytometry. Using serial dilutions and flow-based single-cell sorting, we show that viral proteins produced by a single infected cell can be detected by the ultrasensitive p24 assay. This unique sensitivity allowed the early (as soon as day 1 in 43% of cases) and more efficient detection and quantification of p24 in phytohemagglutinin-L (PHA)-stimulated CD4+ T cells from individuals under effective cART. When seven different classes of latency reversal agents (LRA) in resting CD4+ T cells from HIV-infected individuals were tested, the ultrasensitive p24 assay revealed differences in the extent of HIV reactivation. Of note, HIV RNA production was infrequently accompanied by p24 protein production (19%). Among the drugs tested, prostratin showed a superior capacity in inducing viral protein production. In summary, the ultrasensitive p24 assay allows the detection and quantification of p24 produced by single infected CD4+ T cells and provides a unique tool to assess early reactivation of infectious virus from reservoirs in HIV-infected individuals.IMPORTANCE The persistence of HIV reservoirs in infected individuals under effective antiretroviral treatment represents a major obstacle toward cure. Different methods to estimate HIV reservoirs exist, but there is currently no optimal assay to measure HIV reservoirs in HIV eradication interventions. In the present study, we report an ultrasensitive digital ELISA platform for quantification of the HIV-1 protein p24. This method was employed to assess the early reactivation of infectious virus from reservoirs in HIV-1-infected individuals. We found that viral proteins produced by a single infected cell can be detected by an ultrasensitive p24 assay. This unprecedented resolution showed major advantages in comparison to other techniques currently used to assess viral replication in reactivation studies. In addition, such a highly sensitive assay allows discrimination of drug-induced reactivation of productive HIV based on protein expression. The present study heralds new opportunities to evaluate the HIV reservoir and the efficacy of drugs used to target it.
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Proteína p24 del Núcleo del VIH/análisis , VIH-1/efectos de los fármacos , VIH-1/fisiología , Virología/métodos , Activación Viral/efectos de los fármacos , Latencia del Virus/efectos de los fármacos , Linfocitos T CD4-Positivos/virología , Infecciones por VIH/virología , Humanos , Sensibilidad y EspecificidadRESUMEN
Delay in the diagnosis of multiple sclerosis (MS) stems from the lack of specific clinical and analytical markers to assist in the early diagnosis and prediction of progressive course. We propose a decision-tree model that better defines early at onset MS patients and those with the progressive form by analysing a 12-biomarkers panel in serum and CSF samples of patients with MS, other neurological diseases (OND) and healthy contols. Thus, patients at onset of neurological disease were first classified by serum IL-7 levels <141pg/ml (OR=6.51, p<0.001). Combination of IL-7 and CXCL10 indicated risk for a specific MS clinical form, where IL-7<141 and CXCL10<570pg/ml were associated with the highest risk for PP-MS (OR=22, p=0.01). Unexpectedly, both PP-MS and RR-MS patients shared significantly decreased prototypical biomarkers of inflammation and tissue regeneration in CSF than OND suggesting a defective intrinsic immune response playing a role at the beginning of the disease.
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Esclerosis Múltiple Crónica Progresiva/diagnóstico , Esclerosis Múltiple Recurrente-Remitente/diagnóstico , Área Bajo la Curva , Biomarcadores/sangre , Biomarcadores/líquido cefalorraquídeo , Estudios de Casos y Controles , Quimiocina CCL11 , Quimiocina CCL2 , Quimiocina CCL4 , Quimiocina CCL5 , Quimiocina CXCL10/sangre , Quimiocina CXCL10/líquido cefalorraquídeo , Quimiocina CXCL9/sangre , Quimiocina CXCL9/líquido cefalorraquídeo , Árboles de Decisión , Dipeptidil Peptidasa 4/sangre , Dipeptidil Peptidasa 4/líquido cefalorraquídeo , Diagnóstico Precoz , Factor de Crecimiento Epidérmico , Factor 2 de Crecimiento de Fibroblastos/sangre , Factor 2 de Crecimiento de Fibroblastos/líquido cefalorraquídeo , Factor de Crecimiento de Hepatocito , Humanos , Proteína Antagonista del Receptor de Interleucina 1/sangre , Proteína Antagonista del Receptor de Interleucina 1/líquido cefalorraquídeo , Interleucina-7/sangre , Interleucina-7/líquido cefalorraquídeo , Esclerosis Múltiple Crónica Progresiva/sangre , Esclerosis Múltiple Crónica Progresiva/líquido cefalorraquídeo , Esclerosis Múltiple Recurrente-Remitente/sangre , Esclerosis Múltiple Recurrente-Remitente/líquido cefalorraquídeo , Análisis Multivariante , Enfermedades del Sistema Nervioso/sangre , Enfermedades del Sistema Nervioso/líquido cefalorraquídeo , Enfermedades del Sistema Nervioso/diagnóstico , Pronóstico , Medición de RiesgoRESUMEN
Murid γ-herpesvirus-4 (MuHV-4) promotes polyclonal B cell activation and establishes latency in memory B cells via unclear mechanisms. We aimed at exploring whether B cell receptor specificity plays a role in B cell susceptibility to viral latency and how this is related to B cell activation. We first observed that MuHV-4-specific B cells represent a minority of the latent population, and to better understand the influence of the virus on non-MuHV-4 specific B cells we used the SWHEL mouse model, which produce hen egg lysozyme (HEL)-specific B cells. By tracking HEL+ and HEL- B cells, we showed that in vivo latency was restricted to HEL- B cells while the two populations were equally sensitive to the virus in vitro. Moreover, MuHV-4 induced two waves of B cell activation. While the first wave was characterized by a general B cell activation, as shown by HEL+ and HEL- B cells expansion and upregulation of CD69 expression, the second wave was restricted to the HEL- population, which acquired germinal center (GC) and plasma cell phenotypes. Antigenic stimulation of HEL+ B cells led to the development of HEL+ GC B cells where latent infection remained undetectable, indicating that MuHV-4 does not benefit from acute B cell responses to establish latency in non-virus specific B cells but relies on other mechanisms of the humoral response. These data support a model in which the establishment of latency in B cells by γ-herpesviruses is not stochastic in terms of BCR specificity and is tightly linked to the formation of GCs.
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Linfocitos B/inmunología , Infecciones por Herpesviridae/inmunología , Muramidasa/inmunología , Infecciones Tumorales por Virus/inmunología , Latencia del Virus/inmunología , Animales , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Infecciones por Herpesviridae/virología , Inmunidad Celular , Inmunización , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Rhadinovirus/patogenicidad , Infecciones Tumorales por Virus/virologíaRESUMEN
UNLABELLED: Viral hepatitis is the leading cause of liver disease worldwide and can be caused by several agents, including hepatitis A (HAV), B (HBV), and C (HCV) virus. We employed multiplexed protein immune assays to identify biomarker signatures of viral hepatitis in order to define unique and common responses for three different acute viral infections of the liver. We performed multianalyte profiling, measuring the concentrations of 182 serum proteins obtained from acute HAV- (18), HBV- (18), and HCV-infected (28) individuals, recruited as part of a hospital-based surveillance program in Cairo, Egypt. Virus-specific biomarker signatures were identified and validation was performed using a unique patient population. A core signature of 46 plasma proteins was commonly modulated in all three infections, as compared to healthy controls. Principle component analysis (PCA) revealed a host response based upon 34 proteins, which could distinguish HCV patients from HAV- and HBV-infected individuals or healthy controls. When HAV and HBV groups were compared directly, 34 differentially expressed serum proteins allowed the separation of these two patient groups. A validation study was performed on an additional 111 patients, confirming the relevance of our initial findings, and defining the 17 analytes that reproducibly segregated the patient populations. CONCLUSIONS: This combined discovery and biomarker validation approach revealed a previously unrecognized virus-specific induction of host proteins. The identification of hepatitis virus specific signatures provides a foundation for functional studies and the identification of potential correlates of viral clearance.
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Hepatitis A/sangre , Hepatitis A/diagnóstico , Hepatitis B/sangre , Hepatitis B/diagnóstico , Hepatitis C/sangre , Hepatitis C/diagnóstico , Enfermedad Aguda , Adulto , Algoritmos , Biomarcadores/sangre , Estudios de Casos y Controles , Egipto/epidemiología , Monitoreo Epidemiológico , Femenino , Hepatitis A/epidemiología , Hepatitis B/epidemiología , Hepatitis C/epidemiología , Humanos , Hígado/metabolismo , Hígado/virología , Masculino , Persona de Mediana Edad , Análisis MultivarianteRESUMEN
FLT3L-Fc is a half-life extended, effectorless Fc-fusion of the native human FLT3-ligand. In cynomolgus monkeys, treatment with FLT3L-Fc leads to a complex pharmacokinetic/pharmacodynamic (PK/PD) relationship, with observed nonlinear PK and expansion of different immune cell types across different dose levels. A minimal physiologically based PK/PD model with expansion-enhanced target-mediated drug disposition (TMDD) was developed to integrate the molecule's mechanism of action, as well as the complex preclinical and clinical PK/PD data, to support the preclinical-to-clinical translation of FLT3L-Fc. In addition to the preclinical PK data of FLT3L-Fc in cynomolgus monkeys, clinical PK and PD data from other FLT3-agonist molecules (GS-3583 and CDX-301) were used to inform the model and project the expansion profiles of conventional DC1s (cDC1s) and total DCs in peripheral blood. This work constitutes an essential part of our model-informed drug development (MIDD) strategy for clinical development of FLT3L-Fc by projecting PK/PD in healthy volunteers, determining the first-in-human (FIH) dose, and informing the efficacious dose in clinical settings. Model-generated results were incorporated in regulatory filings to support the rationale for the FIH dose selection.
RESUMEN
Plasmacytoid dendritic cells (pDCs) are the professional type I interferon (IFN)-producing cells, and upon activation they traffic to lymph organs, where they bridge innate and adaptive immunity. Using multianalyte profiling (MAP), we have mapped the key chemokines and cytokines produced in response to pDC activation, taking into consideration the role of autocrine IFN, as well as paracrine effects on other innate cells (e.g., monocytes and conventional DCs). Interestingly, we identify four distinct cytokine/chemokine loops initiated by Toll-like receptor engagement. Finally, we applied this analytic approach to the study of pDC activity in chronic hepatitis C patients. Based on the activation state of pDCs in fresh blood, the lack of agonistic activity of infectious virions, the production of a broad array of cytokines/chemokines once stimulated, and the direct effects of pDCs on other PBMCs, we conclude that the pDCs from hepatitis C virus (HCV)-infected individuals are fully functional and are, indeed, a viable drug target. In sum, this study provides insight into the use of MAP technology for characterizing cytokine networks, and highlights how a rare cell type integrates the activation of other inflammatory cells. Furthermore, this work will help evaluate the therapeutic application of pDC agonists in diseases such as chronic HCV infection.
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Citocinas/inmunología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Hepatitis C Crónica/tratamiento farmacológico , Hepatitis C Crónica/inmunología , Comunicación Autocrina/inmunología , Diferenciación Celular/inmunología , Células Cultivadas , Citocinas/biosíntesis , Citocinas/metabolismo , Células Dendríticas/citología , Células Dendríticas/efectos de los fármacos , Hepacivirus/inmunología , Hepacivirus/fisiología , Hepatitis C Crónica/virología , Humanos , Tolerancia Inmunológica , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/patología , Receptores Toll-Like/metabolismo , Replicación ViralRESUMEN
Mycolactone is a polyketide toxin produced by Mycobacterium ulcerans (Mu), the causative agent of the skin disease Buruli ulcer (BU). Surprisingly, infected tissues lack inflammatory infiltrates. Structural similarities between mycolactone and immunosuppressive agents led us to investigate the immunomodulatory properties of mycolactone on dendritic cells (DCs), the key initiators and regulators of immune responses. At noncytotoxic concentrations, phenotypic and functional maturation of both mouse and human DCs was inhibited by mycolactone. Notably, mycolactone blocked the emigration of mouse-skin DCs to draining lymph nodes, as well as their maturation in vivo. In human peripheral blood-derived DCs, mycolactone inhibited the ability to activate allogeneic T cell priming and to produce inflammatory molecules. Interestingly, production of the cytokines interleukin (IL) 12, tumor necrosis factor alpha, and IL-6 was only marginally affected, whereas production of the chemokines macrophage inflammatory protein (MIP) 1alpha, MIP-1beta, regulated on activation, normal T cell expressed and secreted, interferon gamma-inducible protein 10, and monocyte chemoattractant protein 1 was abolished at nanomolar concentrations. Importantly, mycolactone endogenously expressed by Mu mediated similar inhibitory effects on beta-chemokine production by DCs. In accordance with the histopathological features of BUs, our results suggest that bacterial production of mycolactone may limit both the initiation of primary immune responses and the recruitment of inflammatory cells to the infection site. Moreover, they highlight a potential interest in mycolactone as a novel immunosuppressive agent.
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Toxinas Bacterianas/toxicidad , Células Dendríticas/efectos de los fármacos , Inmunosupresores/toxicidad , Animales , Toxinas Bacterianas/inmunología , Movimiento Celular/efectos de los fármacos , Citocinas/metabolismo , Células Dendríticas/fisiología , Femenino , Humanos , Inmunosupresores/inmunología , Activación de Linfocitos/efectos de los fármacos , Macrólidos , Ratones , Ratones Endogámicos C57BL , Mycobacterium ulcerans/químicaRESUMEN
γδ T lymphocytes are commonly viewed as embracing properties of both adaptive and innate immunity. Contributing to this is their responsiveness to pathogen products, either with or without the involvement of the TCR and its coreceptors. This study clarifies this paradoxical behavior by showing that these two modes of responsiveness are the properties of two discrete sets of murine lymphoid γδ T cells. Thus, MyD88 deficiency severely impaired the response to malaria infection of CD27((-)), IL-17A-producing γδ T cells, but not of IFN-γ-producing γδ cells. Instead, the latter compartment was severely contracted by ablating CD27, which synergizes with TCRγδ in the induction of antiapoptotic mediators and cell cycle-promoting genes in CD27((+)), IFN-γ-secreting γδ T cells. Hence, innate versus adaptive receptors differentially control the peripheral pool sizes of discrete proinflammatory γδ T cell subsets during immune responses to infection.
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Inmunidad Adaptativa , Inmunidad Innata , Interferón gamma/biosíntesis , Interleucina-17/biosíntesis , Receptores de Antígenos de Linfocitos T gamma-delta/fisiología , Transducción de Señal/inmunología , Subgrupos de Linfocitos T/inmunología , Inmunidad Adaptativa/genética , Animales , Supervivencia Celular/genética , Supervivencia Celular/inmunología , Células Cultivadas , Técnicas de Cocultivo , Inmunidad Innata/genética , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Recuento de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Plasmodium berghei/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/biosíntesis , Receptores de Antígenos de Linfocitos T gamma-delta/deficiencia , Rhadinovirus/inmunología , Transducción de Señal/genética , Subgrupos de Linfocitos T/parasitología , Subgrupos de Linfocitos T/virología , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/deficiencia , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/genética , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/fisiologíaRESUMEN
Microbial pathogens have evolved mechanisms to overcome immune responses and successfully infect their host. Here, we studied how Listeria monocytogenes evades immune detection by peptidoglycan (PGN) modification. By analyzing L. monocytogenes muropeptides, we detected O-acetylated muramic acid residues. We identified an O-acetyltransferase gene, oatA, in the L. monocytogenes genome sequence. Comparison of PGN from parental and isogenic oatA mutant strains showed that the O-acetyltransferase OatA O-acetylates Listeria PGN. We also found that PGN O-acetylation confers resistance to different types of antimicrobial compounds targeting bacterial cell wall such as lysozyme, ß-lactam antibiotics, and bacteriocins and that O-acetylation is required for Listeria growth in macrophages. Moreover, oatA mutant virulence is drastically affected in mice following intravenous or oral inoculation. In addition, the oatA mutant induced early secretion of proinflammatory cytokines and chemokines in vivo. These results suggest an important role for OatA in limiting innate immune responses and promoting bacterial survival in the infected host.
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Acetiltransferasas/inmunología , Citocinas/metabolismo , Listeria monocytogenes/inmunología , Listeriosis/inmunología , Peptidoglicano/inmunología , Factores de Virulencia/inmunología , Acetilación , Acetiltransferasas/genética , Animales , Línea Celular , Femenino , Humanos , Inmunidad Innata , Dosificación Letal Mediana , Listeria monocytogenes/genética , Listeria monocytogenes/crecimiento & desarrollo , Listeria monocytogenes/patogenicidad , Listeriosis/genética , Hígado/metabolismo , Hígado/microbiología , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Microbiana , Ácidos Murámicos/metabolismo , Peptidoglicano/química , Bazo/microbiología , Células TH1/metabolismo , Células Th2/metabolismo , Factores de Virulencia/genéticaRESUMEN
The incidence of hepatitis C virus (HCV) genotype 4 infection in Egypt provides a unique opportunity to study the innate immune response to symptomatic acute HCV infection. We investigated whether plasmacytoid dendritic cells (pDCs) are activated as a result of HCV infection. We demonstrate that, even during symptomatic acute infection, circulating pDCs maintained a similar precursor frequency and resting phenotype, compared with pDCs in healthy individuals. Moreover, stimulation with a Toll-like receptor 9 agonist resulted in an intact inflammatory response. These data support the growing consensus that pDCs are not directly activated by HCV and therefore are viable targets for immunotherapy throughout HCV infection.
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Células Dendríticas/inmunología , Hepacivirus/inmunología , Hepatitis C/inmunología , Egipto , Genotipo , Hepacivirus/clasificación , Hepacivirus/genética , Humanos , Fenotipo , ARN Viral/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Receptores Toll-LikeRESUMEN
Buruli ulcer disease (BUD) is an emerging human disease caused by infection with Mycobacterium ulcerans, which leads to the development of necrotic skin lesions. The pathogenesis of the ulcer is closely associated with the production of mycolactone, a diffusible cytotoxin with immunomodulatory properties. To identify immunological correlates of BUD, we performed a broad screen of inflammatory mediators in serum samples and stimulated whole-blood supernatants of patients. We found that patients with active ulcers displayed a distinctive profile of immune suppression, marked by the down-modulation of selected chemokines and an impaired capacity to produce Th1, Th2, and Th17 cytokines on stimulation with mitogenic agents. These immunological defects were induced early in the disease and resolved after anti-BUD therapy, establishing their association with the presence of M. ulcerans. Interestingly, some of the defects in cytokine and chemokine response could be mimicked in vitro by incubation of CD4(+) peripheral blood lymphocytes with mycolactone. Our findings support the hypothesis that mycolactone contributes to bacterial persistence in human hosts by limiting the generation of adaptive cellular responses. Moreover, we identified immunological markers of BUD, which may be helpful for confirmatory diagnosis of the disease and, especially, for monitoring the response to antibiotic treatment.
Asunto(s)
Úlcera de Buruli/inmunología , Mycobacterium ulcerans/inmunología , Tuberculosis Cutánea/inmunología , Adolescente , Adulto , Antibióticos Antituberculosos/farmacología , Toxinas Bacterianas/farmacología , Úlcera de Buruli/sangre , Úlcera de Buruli/microbiología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Quimiocinas/sangre , Quimiocinas/inmunología , Niño , Preescolar , Estudios de Cohortes , Citocinas/inmunología , Citocinas/metabolismo , Femenino , Humanos , Activación de Linfocitos/efectos de los fármacos , Macrólidos , Masculino , Persona de Mediana Edad , Estadísticas no Paramétricas , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Tuberculosis Cutánea/sangre , Tuberculosis Cutánea/microbiologíaRESUMEN
UNLABELLED: Both strong antigenic avidity and acquisition of proper effector functions contribute to the efficacy of antiviral T cell responses. To correlate these parameters with the outcome of hepatitis C virus (HCV) infection, we characterized HCV-specific CD8 T cell lines isolated after immunomagnetic sorting of peripheral blood mononuclear cells from human leukocyte antigen A*02 (HLA-A*02) individuals with various HCV serological statuses, using recombinant HLA-A*0201 multimers loaded with three immunodominant HCV genotype 1-derived epitopes. CD8 T cells specific for these three epitopes were derived from most HLA-A*0201 individuals, regardless of their HCV serology or clinical outcome. Donors recovered from genotype 1 HCV infection were enriched for high-avidity T cells with enhanced interferon gamma (IFN-gamma), tumor necrosis factor alpha, and cytotoxic T lymphocyte responses, when compared with seronegative donors and seropositive patients infected with irrelevant HCV genotypes. Patients chronically infected with genotype 1 strain yielded almost exclusively low-avidity T cells, whose hyporesponsiveness was primarily attributable to low T cell receptor (TCR) avidity rather than intrinsic functional defects. CONCLUSION: This study suggests that strong IFN-gamma responses associated with efficient viral clearance primarily result from Ag-driven selection/survival of HCV-specific T cells expressing high-avidity TCR. It also suggests a link between the quality of the initial HCV-specific T cell repertoire and susceptibility to chronic infection.
Asunto(s)
Afinidad de Anticuerpos/inmunología , Linfocitos T CD8-positivos/inmunología , Hepatitis C/inmunología , Inmunidad Celular/fisiología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/patología , Estudios de Casos y Controles , Línea Celular , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/inmunología , Genotipo , Antígenos HLA-A/genética , Antígenos HLA-A/inmunología , Antígeno HLA-A2 , Hepacivirus/inmunología , Hepatitis C/metabolismo , Hepatitis C/patología , Humanos , Interferón gamma/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismo , Linfocitos T Citotóxicos/patología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Type I interferons (IFNs) are essential mediators of antiviral responses. These cytokines have been implicated in the pathogenesis of autoimmunity, most notably systemic lupus erythematosus (SLE), diabetes mellitus, and dermatomyositis, as well as monogenic type I interferonopathies. Despite a fundamental role in health and disease, the direct quantification of type I IFNs has been challenging. Using single-molecule array (Simoa) digital ELISA technology, we recorded attomolar concentrations of IFNα in healthy donors, viral infection, and complex and monogenic interferonopathies. IFNα protein correlated well with functional activity and IFN-stimulated gene expression. High circulating IFNα levels were associated with increased clinical severity in SLE patients, and a study of the cellular source of IFNα protein indicated disease-specific mechanisms. Measurement of IFNα attomolar concentrations by digital ELISA will enhance our understanding of IFN biology and potentially improve the diagnosis and stratification of pathologies associated with IFN dysregulation.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática/métodos , Interferón-alfa/sangre , Humanos , Factores Reguladores del Interferón/sangre , Factores Reguladores del Interferón/líquido cefalorraquídeo , Interferón-alfa/líquido cefalorraquídeo , Lupus Eritematoso Sistémico/sangre , Sensibilidad y Especificidad , Índice de Severidad de la Enfermedad , Linfocitos T/metabolismo , Estomatitis Vesicular/inmunologíaRESUMEN
Experimental cancer models must consider the role of the immune system.