RESUMEN
The establishment of an "interferon (IFN) signature" to subset SLE patients on disease severity has led to therapeutics targeting IFNα. Here, we investigate IFN signaling in SLE using multiplexed protein arrays and single cell cytometry by time of flight (CyTOF). First, the IFN signature for SLE patients (n=81) from the Stanford Lupus Registry is determined using fluidigm qPCR measuring 44 previously determined IFN-inducible transcripts. IFN-high (IFN-H) patients have increased SLE criteria and renal/CNS/immunologic involvement, and increased autoantibody reactivity against spliceosome-associated antigens. CyTOF analysis is performed on non-stimulated and stimulated (IFNα, IFNγ, IL-21) PBMCs from SLE patients (n=25) and HCs (n=9) in a panel identifying changes in phosphorylation of intracellular signaling proteins (pTOF). Another panel is utilized to detect changes in intracellular cytokine (ICTOF) production in non-stimulated and stimulated (PMA/ionomycin) PBMCs from SLE patients (n=31) and HCs (n=17). Bioinformatic analysis by MetaCyto and OMIQ reveal phenotypic changes in immune cell subsets between IFN-H and IFN-low (IFN-L) patients. Most notably, IFN-H patients exhibit increased STAT1/3/5 phosphorylation downstream of cytokine stimulation and increased phosphorylation of non-canonical STAT proteins. These results suggest that IFN signaling in SLE modulates STAT phosphorylation, potentially uncovering possible targets for future therapeutic approaches.
Asunto(s)
Interferón Tipo I/fisiología , Interleucinas/fisiología , Leucocitos Mononucleares/metabolismo , Lupus Eritematoso Sistémico/metabolismo , Factor de Transcripción STAT1/metabolismo , Adulto , Femenino , Citometría de Flujo , Humanos , Interferón Tipo I/análisis , Interleucinas/análisis , Masculino , Persona de Mediana Edad , Fosforilación , Transducción de Señal , Análisis de la Célula IndividualRESUMEN
Systemic lupus erythematosus (SLE) is an autoimmune disorder associated with inflammation and multiple organ involvement. Individually, dendritic cells (DCs) and oxidative stress have been well discussed for their critical involvement in the pathogenesis of disease but the precise impact of oxidative stress on DCs in relation to SLE disease activity is yet to be scrutinized. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/Kelch-like ECH-associated protein 1 (Keap1) pathway is the cellular mechanism to combat increased reactive oxygen species (ROS). The current study was framed in order to understand redox regulation in DCs along with an argument in context to disease activity. Here, 23 SLE patients along with 10 healthy controls were enrolled and disease activity was calculated as the recent change in SLEDAI score. We found the percentage of circulating plasmacytoid DCs (pDCs) was increased with an increase in disease activity. Altered DCs functionality along with disease activity was further supported with the differential concentration of Type I IFNs. The disease activity was positively associated with increased levels of ROS. A relevant reason for increased ROS was further explained with the decreased levels of transcription factor Nrf2. Hence, the present study suggests that SLE specific DCs displayed elevation in ROS and this outcome might be due to impaired free radical clearance by Nrf2. Correlation studies further established an association of disease activity with increased ROS, Type I IFNs levels and decreased activity of oxidative stress regulating enzymes.
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Interferón Tipo I/metabolismo , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Lupus Eritematoso Sistémico/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Estrés Oxidativo/fisiología , Adolescente , Adulto , Células Dendríticas/metabolismo , Células Dendríticas/patología , Femenino , Humanos , Interferón Tipo I/análisis , Lupus Eritematoso Sistémico/patología , Persona de Mediana Edad , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Adulto JovenRESUMEN
Interferon (IFN), the first ever-described cytokine, has a potent activity against viruses. Soon since its discovery, quantification of IFN has been an important issue. Most of the traditional methods to measure IFN biological activity rely on indirect methods that quantify dyes retained by IFN-protected cells against a lytic virus, or by techniques that indirectly quantify viral replication by measuring the expression level of viral-encoded reporter proteins such as the green fluorescent protein (GFP). In both cases, the IFN units are determined by the quantification of an effective dose 50, defined as the IFN dose that prevents 50% cell death of 50% reduction of the maximal amount of GFP intensity. In this study we propose the use of an alternative approach to measure IFN activity by calculating the minimal IFN dose 50 as the amount of IFN able to completely protect 50% of the cells from infection measured by the total absence of virus-dependent GFP signal in a cell culture plate. This sensitive approach could be used to easily quantify the Z value to determine IFN bioassay robustness. We believe that this approximation could be interesting to be considered by the IFN community.
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Bioensayo , Interferón Tipo I/análisis , Animales , Células Cultivadas , Chlorocebus aethiops , Humanos , Proteínas Recombinantes/análisis , Virus Sendai/genética , Virus Sendai/crecimiento & desarrollo , Virus Sendai/aislamiento & purificación , Células VeroRESUMEN
BACKGROUND: Experimental data show that type I interferon has a key role in innate immune response against influenza infection. OBJECTIVE: We compared nasal levels of interferon-α2 and ß among inpatients and outpatients with influenza. STUDY DESIGN: Children younger than 5 years of age with influenza-like illness seeking care at the emergency department within the first 72 h of disease onset were prospectively included. Clinical and demographic data and secretions through nasal wash were obtained. Influenza infection was assessed through reverse-transcription polymerase chain reaction and nasal levels of interferon-α2 and ß were measured by enzyme-linked immunosorbent assay. All patients followed until the end of the disease. RESULTS: One hundred patients were included, of which 24 had confirmed influenza infection, and 5 of them were hospitalized. Subtypes A (H3N2) and B were confirmed in 10 and 14 patients, respectively. Seventy-six patients without influenza, including 48% of outpatients, were recruited as controls. All hospitalized patients were significantly younger regardless of influenza status (age <6 months in 59% vs. 23.2%, p < 0.001). All other data were similar among the groups. Comparing median levels of interferon-α2 among children with influenza, levels were significantly higher in outpatients than in hospitalized patients and were 263.2 pg/mL (25-75 interquartile range: 58.3-634) and detectable in only one patient (90 pg/mL), respectively. The levels of interferon-α2 in controls and those of interferon-ß in all groups were not detected. CONCLUSIONS: Higher levels of interferon-α2 in patients with less severe influenza reinforce experimental evidence about the protective role of interferon-α2 against influenza infection.
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Inmunidad Innata , Gripe Humana/inmunología , Interferón Tipo I/análisis , Nariz/inmunología , Infecciones del Sistema Respiratorio/inmunología , Secreciones Corporales/virología , Preescolar , Estudios de Cohortes , Femenino , Hospitalización , Humanos , Lactante , Subtipo H3N2 del Virus de la Influenza A/genética , Subtipo H3N2 del Virus de la Influenza A/inmunología , Pacientes Internos/estadística & datos numéricos , Interferón Tipo I/inmunología , Interferón alfa-2/análisis , Interferón alfa-2/inmunología , Interferón beta/análisis , Interferón beta/inmunología , Masculino , Nariz/virología , Pacientes Ambulatorios/estadística & datos numéricos , Infecciones del Sistema Respiratorio/virologíaRESUMEN
PURPOSE: To evaluate the different degrees of residual structure in the unfolded state of interferon-τ using chemical denaturation as a function of temperature by both urea and guanidinium hydrochloride. METHODS: Asymmetrical flow field-flow fractionation (AF4) using both UV and multi-angle laser light scattering (MALLS). Flow Microscopy. All subvisible particle imaging measurements were made using a FlowCAM flow imaging system. RESULTS: The two different denaturants provided different estimates of the conformational stability of the protein when extrapolated back to zero denaturant concentration. This suggests that urea and guanidinium hydrochloride (GnHCl) produce different degrees of residual structure in the unfolded state of interferon-τ. The differences were most pronounced at low temperature, suggesting that the residual structure in the denatured state is progressively lost when samples are heated above 25°C. The extent of expansion in the unfolded states was estimated from the m-values and was also measured using AF4. In contrast, the overall size of interferon-τ was determined by AF4 to decrease in the presence of histidine, which is known to bind to the native state, thereby providing conformational stabilization. Addition of histidine as the buffer resulted in formation of fewer subvisible particles over time at 50°C. Finally, the thermal aggregation was monitored using AF4 and the rate constants were found to be comparable to those determined previously by SEC and DLS. The thermal aggregation appears to be consistent with a nucleation-dependent mechanism with a critical nucleus size of 4 ± 1. CONCLUSION: Chemical denaturation of interferon-τ by urea or GnHCl produces differing amounts of residual structure in the denatured state, leading to differing estimates of conformational stability. AF4 was used to determine changes in size, both upon ligand binding as well as upon denaturation with GnHCl. Histidine appears to be the preferred buffer for interferon-τ, as shown by slower formation of soluble aggregates and reduced levels of subvisible particles when heated at 50°C.
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Interferón Tipo I/química , Proteínas Gestacionales/química , Agregado de Proteínas , Desnaturalización Proteica , Desplegamiento Proteico , Agua/química , Interferón Tipo I/análisis , Interferón Tipo I/metabolismo , Soluciones Farmacéuticas/química , Soluciones Farmacéuticas/metabolismo , Espectroscopía de Fotoelectrones/métodos , Proteínas Gestacionales/análisis , Proteínas Gestacionales/metabolismo , Agregado de Proteínas/fisiología , Agua/metabolismoRESUMEN
OBJECTIVE(S): Type I interferon (IFN-I) responses confer both protective and pathogenic effects in persistent virus infections. IFN-I diversity, stage of infection and tissue compartment may account for this dichotomy. The gut is a major site of early HIV-1 replication and microbial translocation, but the nature of the IFN-I response in this compartment remains unclear. DESIGN: Samples were obtained from two IRB-approved cross-sectional studies. The first study included individuals with chronic, untreated HIV-1 infection (nâ=â24) and age/sex-balanced uninfected controls (nâ=â14). The second study included antiretroviral-treated, HIV-1-infected individuals (nâ=â15) and uninfected controls (nâ=â15). METHODS: The expression of 12 IFNα subtypes, IFNß and antiviral IFN-stimulated genes (ISGs) were quantified in peripheral blood mononuclear cells (PBMCs) and colon biopsies using real-time PCR and next-generation sequencing. In untreated HIV-1-infected individuals, associations between IFN-I responses and gut HIV-1 RNA levels as well as previously established measures of colonic and systemic immunological indices were determined. RESULTS: IFNα1, IFNα2, IFNα4, IFNα5 and IFNα8 were upregulated in PBMCs during untreated chronic HIV-1 infection, but IFNß was undetectable. By contrast, IFNß was upregulated and all IFNα subtypes were downregulated in gut tissue. Gut ISG levels positively correlated with gut HIV-1 RNA and immune activation, microbial translocation and inflammation markers. Gut IFN-I responses were not significantly different between HIV-1-infected individuals on antiretroviral treatment and uninfected controls. CONCLUSION: The IFN-I response is compartmentalized during chronic untreated HIV-1 infection, with IFNß being more predominant in the gut. Gut IFN-I responses are associated with immunopathogenesis, and viral replication is likely a major driver of this response.
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Colon/patología , Infecciones por VIH/patología , Factores Inmunológicos/análisis , Interferón Tipo I/análisis , Mucosa Intestinal/patología , Adulto , Biopsia , Estudios Transversales , Femenino , Perfilación de la Expresión Génica , Humanos , Leucocitos Mononucleares/patología , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
Type I interferon (IFN) pathways are significant in SLE pathogenesis. Less is known about the utility of measuring markers of IFN activity in patients, or whether patient subsets with different profiles exist. We explored the longitudinal associations of IFN-induced chemokines with disease activity in a cohort of SLE patients. We calculated a validated composite score (IFN-CK) of three type I IFN-inducible chemokines (CCL2/CXCL10/CCL19) measured in 109 SLE patients (median 7 occasions over 3.2 years). Longitudinal associations of IFN-CK score with disease activity (SLEDAI-2K) and other variables were assessed using general estimating equation (GEE) methods. IFN-CK was detectable in all patients. SLEDAI-2K was significantly associated with IFN-CK, damage score and prednisolone dose. SLEDAI-2K remained significantly associated with IFN-CK over time after adjustment of covariates. Patients with high time-adjusted mean IFN-CK had lower complement and higher time-adjusted disease activity. Concordance between IFN-CK and SLEDAI-2K varied widely among patients, with some individuals having none, others weak, and a subset very high concordance. In summary in our cohort of SLE patients, serum IFN-CK varied over time with disease activity, but with wide variation in concordance. Differing relationships between IFN pathway activation and disease activity may be valuable in assigning patients to emerging IFN-pathway targeting treatments.
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Quimiocina CCL19/análisis , Quimiocina CCL2/análisis , Quimiocina CXCL10/análisis , Interferón Tipo I/análisis , Lupus Eritematoso Sistémico/patología , Adulto , Australia , Femenino , Humanos , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Índice de Severidad de la EnfermedadRESUMEN
The innate immune system directly senses microbial viability via the detection of a special class of viability-associated pathogen-associated molecular patterns (vita-PAMPs), such as prokaryotic messenger RNA. In the case of Gram-negative bacteria, detection of bacterial viability by phagocytes leads to a unique activation of inflammasome and type I interferon pathways, resulting in a robust pro-inflammatory innate response and a vigorous adaptive immune response. This protocol describes the methods required to study activation of both inflammasome and type I interferon pathways after stimulation of mouse bone marrow-derived macrophages with live or killed Gram-negative and Gram-positive bacteria. It covers the generation and handling of bone marrow-derived macrophages, the culture and killing of bacteria, the preparation of bacterial messenger RNA, and the stimulation of macrophages with live or killed bacteria. Lastly, this protocol describes the techniques employed to measure the hallmarks of inflammasome (secretion of interleukin-1ß) and type I interferon (activation of TBK1, IRF3 and secretion of type I interferon) pathways.
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Bacterias/inmunología , Inflamasomas/análisis , Interferón Tipo I/análisis , Macrófagos/inmunología , Viabilidad Microbiana/inmunología , Animales , Bacterias/crecimiento & desarrollo , Inmunidad Innata , Inflamasomas/inmunología , Interferón Tipo I/inmunología , Ratones , Transducción de SeñalRESUMEN
Superoxide dismutase 2 (SOD2) is essential in radical scavenging, which balances the intracellular level of reactive oxygen species (ROS). The dysfunction of SOD2 is associated with increasing incidence of various human diseases, including cancer, neuron diseases, and myocardial defects. However, the connections between SOD2-mediated oxidative homeostasis and innate immune response remain unclear. In this study, we report that SOD2 is a crucial regulator of antiviral signaling. Depletion of SOD2 impairs RNA virus-induced type I interferon (IFN) and proinflammatory cytokine production, resulting in enhanced viral replication. Type I IFN production is highly sensitive to cellular level of ROS. SOD2 deficiency-mediated ROS accumulation potently inhibits RIG-I-like receptor (RLR)-induced innate immune responses through the regulation of nuclear factor-kappa B (NF-κB) and interferon regulatory factor-3 activation. These findings uncover a novel role for SOD2 in regulating RLR-mediated antiviral innate immune signaling.
Asunto(s)
Inmunidad Innata/inmunología , Especies Reactivas de Oxígeno/inmunología , Virus Sendai/inmunología , Superóxido Dismutasa/inmunología , Vesiculovirus/inmunología , Animales , Chlorocebus aethiops , Células HEK293 , Humanos , Factor 3 Regulador del Interferón/metabolismo , Interferón Tipo I/análisis , Interferón Tipo I/inmunología , Interleucina-6/análisis , Interleucina-6/inmunología , FN-kappa B/metabolismo , ARN Viral/genética , Especies Reactivas de Oxígeno/análisis , Superóxido Dismutasa/genética , Células VeroRESUMEN
Interferon alpha and its surrogates, including IP-10 and SIGLEC1, paralleled changes of disease activity in systemic lupus erythematosus (SLE). However, the whole blood interferon signature (WBIFNS)-the current standard for type I IFN assessment in SLE-does not correlate with SLE disease activity in individual patients over time. The underlying causes for this apparent contradiction have not been convincingly demonstrated. Using a multicenter dataset of gene expression data from leukocyte subsets in SLE, we identify distinctive subset-specific contributions to the WBIFNS. In a subsequent analysis, the effects of type I interferon on cellular blood composition in patients with SLE and hepatitis B were also studied over time. We found that type I interferon mediates significant alterations in whole blood composition, including a neutropenia and relative lymphocytosis. Given different effects of type 1 interferon on different leukocyte subsets, these shifts confound measurement of a type 1 interferon signature in whole blood. To minimize and overcome these limitations of the WBIFNS, we suggest to measure IFN-induced transcripts or proteins in a specific leukocyte subset to improve clinical impact of interferon biomarkers. KEY MESSAGES: Myeloid cells contribute more to the WBIFNS in SLE than their lymphocytic counterpart. Very similar leukocyte subsets reveal distinctive IFN signatures. IFN alpha mixes up composition of blood and leads to a preferential neutropenia, yielding relative lymphocytosis.
Asunto(s)
Hepatitis B/genética , Hepatitis C/genética , Interferón Tipo I/genética , Leucocitos/patología , Lupus Eritematoso Sistémico/genética , Transcriptoma , Adulto , Biomarcadores/análisis , Biomarcadores/sangre , Femenino , Hepatitis B/sangre , Hepatitis B/patología , Hepatitis C/sangre , Hepatitis C/patología , Humanos , Interferón Tipo I/análisis , Interferón Tipo I/sangre , Leucocitos/metabolismo , Lupus Eritematoso Sistémico/sangre , Lupus Eritematoso Sistémico/patología , Linfocitosis/sangre , Linfocitosis/genética , Linfocitosis/patología , Masculino , Persona de Mediana EdadRESUMEN
Plasmacytoid dendritic cells (pDCs) play a major role in antiviral immunity via the production of type I interferons (IFNs). There is some evidence that pDCs interact with bacteria but it is not yet clear whether they are protective or contribute to bacterial pathogenicity. We wished to investigate whether Coxiella burnetii, the agent of Q fever, interacts with pDCs. The stimulation of pDCs with C. burnetii increased the expression of activation and migratory markers (CD86 and CCR7) as determined by flow cytometry and modulated gene expression program as revealed by a microarray approach. Indeed, genes encoding for pro-inflammatory cytokines, chemokines, and type I INF were up-regulated. The up-regulation of type I IFN was correlated with an increase in IFN-α release by C. burnetii-stimulated pDCs. We also investigated pDCs in patients with Q fever endocarditis. Using flow cytometry and a specific gating strategy, we found that the number of circulating pDCs was significantly lower in patients with Q fever endocarditis as compared to healthy donors. In addition, the remaining circulating pDCs expressed activation and migratory markers. As a whole, our study identified non-previously reported activation of pDCs by C. burnetii and their modulation during Q fever.
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Coxiella burnetii/inmunología , Citocinas/análisis , Células Dendríticas/inmunología , Células Dendríticas/microbiología , Interferón Tipo I/análisis , Fiebre Q/inmunología , Citometría de Flujo , Perfilación de la Expresión Génica , Humanos , Análisis por MicromatricesAsunto(s)
Inmunoterapia , Mycobacterium tuberculosis/inmunología , Tuberculosis Pulmonar/terapia , Animales , Dinoprostona/metabolismo , Modelos Animales de Enfermedad , Eicosanoides/análisis , Eicosanoides/metabolismo , Humanos , Interferón Tipo I/análisis , Interferón Tipo I/biosíntesis , Interleucina-1/análisis , Interleucina-1/antagonistas & inhibidores , Interleucina-1/inmunología , Ratones , Mycobacterium tuberculosis/crecimiento & desarrollo , Tuberculosis Pulmonar/inmunologíaAsunto(s)
Células Dendríticas/inmunología , Ectima Contagioso/inmunología , Molusco Contagioso/inmunología , Adolescente , Adulto , Niño , Ectima Contagioso/patología , Femenino , Humanos , Interferón Tipo I/análisis , Lectinas Tipo C/análisis , Masculino , Glicoproteínas de Membrana/análisis , Persona de Mediana Edad , Proteínas de Resistencia a Mixovirus/análisis , Virus del Orf , Receptores Inmunológicos/análisis , Adulto JovenRESUMEN
This study was performed to elucidate the changes in IFNT messenger RNA (mRNA) levels in in vivo-fertilized and parthenogenetic bovine embryos and their interferon-τ (IFNT) secretion amounts during the elongation phase. We assessed the induction capability of maternal recognition of pregnancy by parthenogenetic embryos and attempted cotransfer of in vivo-fertilized and parthenogenetic embryos. The expression level of IFNT mRNA in in vivo-fertilized embryos peaked on Day 18 after estrus, and the highest amount of uterine IFNT was observed on Day 20. Transfer of 10 parthenogenetic embryos produced a detectable amount of uterine IFNT. Transfer of one or three parthenogenetic embryos inhibited luteolysis. An increase in ISG15 mRNA levels in peripheral granulocytes was induced by the transfer of three parthenogenetic embryos. Cotransfer of three parthenogenetic embryos significantly improved the pregnancy rate on Day 40 in code 3 in vivo-fertilized embryos compared with single transfer without parthenogenetic embryos (65% vs. 35%). However, the pregnancy rate on Day 90 (35%) in cotransfer of code 3 in vivo-fertilized embryos did not differ from that upon single transfer (29%), because the cotransfer group had a higher incidence of pregnancy loss than with single transfer (47% vs. 17%) after Day 40. Cotransfer did not affect the pregnancy rate of code 2 in vivo-fertilized embryos. The incidence of pregnancy loss was higher in cotransfer of code 2 in vivo-fertilized embryos than in single transfer (30% vs. 7%). In conclusion, parthenogenetic embryos in the elongation phase secreted IFNT, enabling induction of maternal recognition of pregnancy. The present study revealed that enhancement of the maternal recognition of pregnancy using parthenogenetic embryos promoted the viability of poor-quality embryos until Day 40 of gestation. However, the incidence of pregnancy loss increased after Day 40 in the cotransfer of parthenogenetic embryos. A technique for promoting the full-term survival of poor-quality embryos is needed.
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Bovinos/fisiología , Transferencia de Embrión/veterinaria , Partenogénesis , Aborto Veterinario/epidemiología , Animales , Blastocisto/metabolismo , Enfermedades de los Bovinos/epidemiología , Transferencia de Embrión/métodos , Femenino , Fertilización In Vitro/veterinaria , Edad Gestacional , Interferón Tipo I/análisis , Interferón Tipo I/genética , Interferón Tipo I/fisiología , Embarazo , Mantenimiento del Embarazo , Proteínas Gestacionales/análisis , Proteínas Gestacionales/genética , Proteínas Gestacionales/fisiología , ARN Mensajero/análisis , Útero/químicaRESUMEN
Primary Sjögren's syndrome (pSS) is a systemic autoimmune disease characterized by lymphocytic infiltrates in salivary and lacrimal glands. Clinical manifestations range from ocular and oral dryness to vasculitis and severe fatigue. pSS is a disease with heterogeneous symptoms and a variable response to the available treatment. Recently, a key role for Interferon (IFN) type I has been implicated in the pathogenesis of pSS. As type I IFN consists of 17 different subtypes, it cannot be easily assessed using a conventional ELISA. Therefore the expression of type I IFN inducible genes--the so-called type I IFN signature--is assessed in salivary gland tissue and blood from patients as a readout for type I IFN activity. In this review we discuss the potential of type I IFN as a novel biomarker for disease activity, subclassification of patients, prediction of therapy response and most importantly as a target for therapeutic intervention.
Asunto(s)
Biomarcadores/análisis , Interferón Tipo I/biosíntesis , Síndrome de Sjögren/inmunología , Transcriptoma , Humanos , Interferón Tipo I/análisis , Interferón Tipo I/inmunología , Glándulas Salivales/inmunología , Glándulas Salivales/metabolismo , Síndrome de Sjögren/genéticaRESUMEN
OBJECTIVES: The aim of this study was to describe the pattern of expression of Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4) in skin biopsies of patients with American tegumentary leishmaniasis (ATL) caused by Leishmania braziliensis. METHODS: This prospective study evaluated 12 patients with ATL caused by Leishmania braziliensis confirmed by polymerase chain reaction. Immunohistochemistry was performed to determine the expression of TLR2 and TLR4. The number of NK cells, dendritic cells and macrophages in the tissue were calculated. The cytokine expression was determined using the anti-TNF-α, anti-IFN-Γ, anti-IL-1 and anti-IL-6. Double immunostaining reactions were used to determine the cell expressing TLR2 and TLR4. RESULTS: The numbers of cells expressing TLR2 and TLR4 were 145.48 ± 82.46 cell/mm² and 3.26 ± 4.11 cell/mm² respectively (p < 0.05). There was no correlation of TLR2 and TLR4 with the amount of cytokines and the number of NK cells, dendritic cells or macrophages. The double immunostaining revealed that TLR2 was expressed by macrophages. CONCLUSION: In human cutaneous leishmaniasis caused by Leishmania braziliensis, TLR2 is the most common TLR expressed during active disease, mainly by macrophages although without correlation with the amount of cytokines and number of cells.
OBJETIVOS: O objetivo deste estudo foi descrever o padrão de expressão dos receptores toll-like 2 e 4 (TLR2 e TLR4) em biópsias de pele de pacientes com leishmaniose tegumentar americana (LTA). MÉTODOS: Este estudo prospectivo avaliou 12 pacientes com LTA causada por Leishmania braziliensis confirmada por reação em cadeia da polimerase. Imunohistoquímica foi realizada para determinar a expressão de TLR2 e TLR4. O número de células NK, células dendríticas e macrófagos foi calculado no tecido. A expressão de citocinas foi determinada usando anti-TNF-α, anti-IFN-Γ, anti-IL-1 e anti-IL-6. Dupla marcação foi usada para determinar a célula responsável pela expressão de TLR2 e TLR4. RESULTADOS: O número de células expressando TLR2 e TLR4 foi 145.48±82.46 cell/mm² e 3.26 ± 4.11 cell/mm² respectivamente (p < 0.05). Não houve correlação entre a quantidade de expressão de TLR2 e TLR4 com a quantidade de citocinas e o número de células NK, macrófagos e células dendríticas. A dupla marcação revelou que o TLR2 é expresso por macrófagos. CONCLUSÃO: Na LTA causada por Leishmania braziliensis, TLR2 é o TLR mais comum na doença ativa, principalmente por macrófagos sem correlação com a quantidade de citocinas e outras células.
Asunto(s)
Adolescente , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adulto Joven , Citocinas/análisis , Leishmaniasis Cutánea/metabolismo , /metabolismo , /metabolismo , Recuento de Células , Células Dendríticas/metabolismo , Inmunohistoquímica , Interferón Tipo I/análisis , Interferón Tipo I/inmunología , Interleucina-1/análisis , Interleucina-1/inmunología , /análisis , /inmunología , Células Asesinas Naturales/metabolismo , Leishmaniasis Cutánea/inmunología , Macrófagos/metabolismo , Reacción en Cadena de la Polimerasa , Estudios Prospectivos , /inmunología , /inmunología , Factor de Necrosis Tumoral alfa/análisis , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
OBJECTIVES: The aim of this study was to describe the pattern of expression of Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4) in skin biopsies of patients with American tegumentary leishmaniasis (ATL) caused by Leishmania braziliensis. METHODS: This prospective study evaluated 12 patients with ATL caused by Leishmania braziliensis confirmed by polymerase chain reaction. Immunohistochemistry was performed to determine the expression of TLR2 and TLR4. The number of NK cells, dendritic cells and macrophages in the tissue were calculated. The cytokine expression was determined using the anti-TNF-α, anti-IFN-Γ, anti-IL-1 and anti-IL-6. Double immunostaining reactions were used to determine the cell expressing TLR2 and TLR4. RESULTS: The numbers of cells expressing TLR2 and TLR4 were 145.48 ± 82.46 cell/mm² and 3.26 ± 4.11 cell/mm² respectively (p < 0.05). There was no correlation of TLR2 and TLR4 with the amount of cytokines and the number of NK cells, dendritic cells or macrophages. The double immunostaining revealed that TLR2 was expressed by macrophages. CONCLUSION: In human cutaneous leishmaniasis caused by Leishmania braziliensis, TLR2 is the most common TLR expressed during active disease, mainly by macrophages although without correlation with the amount of cytokines and number of cells.
Asunto(s)
Citocinas/análisis , Leishmaniasis Cutánea/metabolismo , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 4/metabolismo , Adolescente , Adulto , Recuento de Células , Células Dendríticas/metabolismo , Femenino , Humanos , Inmunohistoquímica , Interferón Tipo I/análisis , Interferón Tipo I/inmunología , Interleucina-1/análisis , Interleucina-1/inmunología , Interleucina-6/análisis , Interleucina-6/inmunología , Células Asesinas Naturales/metabolismo , Leishmaniasis Cutánea/inmunología , Macrófagos/metabolismo , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Estudios Prospectivos , Receptor Toll-Like 2/inmunología , Receptor Toll-Like 4/inmunología , Factor de Necrosis Tumoral alfa/análisis , Factor de Necrosis Tumoral alfa/inmunología , Adulto JovenRESUMEN
Type I interferons (IFN) comprise a family of cytokines that signal through a common cellular receptor to induce a plethora of genes with antiviral and other activities. Recombinant IFNs are used for the treatment of hepatitis C virus infection, multiple sclerosis, and certain malignancies. The capability of type I IFN to suppress virus replication and resultant cytopathic effects is frequently used to measure their bioactivity. However, these assays are time-consuming and require appropriate biosafety containment. In this study, an improved IFN assay is presented which is based on a recombinant vesicular stomatitis virus (VSV) replicon encoding two reporter proteins, firefly luciferase and green fluorescent protein. The vector lacks the essential envelope glycoprotein (G) gene of VSV and is propagated on a G protein-expressing transgenic cell line. Several mammalian and avian cells turned out to be susceptible to infection with the complemented replicon particles. Infected cells readily expressed the reporter proteins at high levels five hours post infection. When human fibroblasts were treated with serial dilutions of human IFN-ß prior to infection, reporter expression was accordingly suppressed. This method was more sensitive and faster than a classical IFN bioassay based on VSV cytopathic effects. In addition, the antiviral activity of human IFN-λ (interleukin-29), a type III IFN, was determined on Calu-3 cells. Both IFN-ß and IFN-λ were acid-stable, but only IFN-ß was resistant to alkaline treatment. The antiviral activities of canine, porcine, and avian type I IFN were analysed with cell lines derived from the corresponding species. This safe bioassay will be useful for the rapid and sensitive quantification of multi-species type I IFN and potentially other antiviral cytokines.
Asunto(s)
Bioensayo/métodos , Interferón Tipo I/análisis , Interferón Tipo I/farmacología , Replicón/genética , Vesiculovirus/efectos de los fármacos , Vesiculovirus/genética , Animales , Antivirales/análisis , Antivirales/química , Antivirales/farmacología , Línea Celular Tumoral , Perros , Genes Reporteros/genética , Proteínas Fluorescentes Verdes/genética , Humanos , Concentración de Iones de Hidrógeno , Interferón Tipo I/química , Luciferasas de Luciérnaga/genética , Ratones , Estabilidad Proteica , Seguridad , Especificidad de la Especie , Temperatura , Factores de TiempoRESUMEN
The need for a simple and high-throughput method for identifying the tertiary structure of protein pharmaceuticals has increased. In this study, a simple method for mapping the protein fold is proposed for use as a complementary quality test. This method is based on cross-linking a protein using a [bis(sulfosuccinimidyl)suberate (BS(3))], followed by peptide mapping by LC-MS. Consensus interferon (CIFN) was used as the model protein. The tryptic map obtained via liquid chromatography tandem mass spectroscopy (LC-MS/MS) and the mass mapping obtained via matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy were used to identify cross-linked peptides. While LC-MS/MS analyses found that BS(3) formed cross-links in the loop region of the protein, which was regarded as the biologically active site, sodium dodecyl-sulfate polyacrylamide gel electrophoresis demonstrated that cross-linking occurred within a protein molecule, but not between protein molecules. The occurrence of cross-links at the active site depends greatly on the conformation of the protein, which is determined by the denaturing conditions. Quantitative evaluation of the tertiary structure of CIFN was thus possible by monitoring the amounts of cross-linked peptides generated. Assuming that background information is available at the development stage, this method may be applicable to process development as a complementary test for quality control.