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1.
PLoS Negl Trop Dis ; 14(3): e0008093, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32176691

RESUMO

Human leishmaniasis is a public health problem worldwide for which the development of a vaccine remains a challenge. T cell-mediated immune responses are crucial for protection. Peptide vaccines based on the identification of immunodominant T cell epitopes able to induce T cell specific immune responses constitute a promising strategy. Here, we report the identification of human leukocyte antigen class-I (HLA-I) and -II (HLA-II)-restricted multi-epitope peptides from Leishmania proteins that we have previously described as vaccine candidates. Promastigote Surface Antigen (PSA), LmlRAB (L. major large RAB GTPase) and Histone (H2B) were screened, in silico, for T cell epitopes. 6 HLA-I and 5 HLA-II-restricted multi-epitope peptides, able to bind to the most frequent HLA molecules, were designed and used as pools to stimulate PBMCs from individuals with healed cutaneous leishmaniasis. IFN-γ, IL-10, TNF-α and granzyme B (GrB) production was evaluated by ELISA/CBA. The frequency of IFN-γ-producing T cells was quantified by ELISpot. T cells secreting cytokines and memory T cells were analyzed by flow cytometry. 16 of 25 peptide pools containing HLA-I, HLA-II or HLA-I and -II peptides were able to induce specific and significant IFN-γ levels. No IL-10 was detected. 6 peptide pools were selected among those inducing the highest IFN-γ levels for further characterization. 3/6 pools were able to induce a significant increase of the percentages of CD4+IFN-γ+, CD8+IFN-γ+ and CD4+GrB+ T cells. The same pools also induced a significant increase of the percentages of bifunctional IFN-γ+/TNF-α+CD4+ and/or central memory T cells. We identified highly promiscuous HLA-I and -II restricted epitope combinations from H2B, PSA and LmlRAB proteins that stimulate both CD4+ and CD8+ T cell responses in recovered individuals. These multi-epitope peptides could be used as potential components of a polytope vaccine for human leishmaniasis.


Assuntos
Antígenos de Protozoários/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Leishmania/imunologia , Leishmaniose Cutânea/imunologia , Proteínas Recombinantes de Fusão/imunologia , Adulto , Antígenos de Protozoários/genética , Ensaio de Imunoadsorção Enzimática , Epitopos de Linfócito T/genética , Feminino , Citometria de Fluxo , Granzimas/análise , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Interferon gama/análise , Interleucina-10/análise , Masculino , Pessoa de Meia-Idade , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fator de Necrose Tumoral alfa/análise , Voluntários , Adulto Jovem
2.
Biosci Biotechnol Biochem ; 84(1): 208-215, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31532348

RESUMO

Dihomo-γ-linolenic acid (DGLA, C20: 3n-6) is known to have an anti-inflammatory activity, but its range of effects was not well studied because of its limited natural sources. We addressed these issues by constructing an yeast Saccharomyces cerevisiae strain having a complete metabolic pathway for DGLA synthesis by introducing two desaturase and one elongase genes to convert endogenous oleic acid to DGLA. Taking advantage of well-known safety of S. cerevisiae, we previously investigated the efficacy of heat-killed whole DGLA-producing yeast cells on irritant contact dermatitis, and showed that oral intake of this yeast significantly suppressed inflammatory reactions, whereas no such suppression was observed by the intake of 25 times the amount of purified DGLA. Since this method is considered to be a simple and efficient way to suppress inflammation, we examined its effectiveness against allergic contact dermatitis (ACD) in this study and showed that this method was also effective against ACD.


Assuntos
Ácido 8,11,14-Eicosatrienoico/farmacologia , Terapia Baseada em Transplante de Células e Tecidos/métodos , Dermatite Alérgica de Contato/terapia , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Ácido 8,11,14-Eicosatrienoico/administração & dosagem , Ácido 8,11,14-Eicosatrienoico/metabolismo , Acetona/química , Administração Oral , Animais , Quimiocina CCL2/análise , Quimiocinas/análise , Dermatite Alérgica de Contato/etiologia , Dinitrofluorbenzeno/efeitos adversos , Dinitrofluorbenzeno/imunologia , Orelha Externa/patologia , Feminino , Imunização , Inflamação/terapia , Interferon gama/análise , Camundongos , Ácido Oleico/metabolismo , Azeite de Oliva/química
3.
BMC Biotechnol ; 19(1): 102, 2019 12 23.
Artigo em Inglês | MEDLINE | ID: mdl-31870349

RESUMO

BACKGROUND: Interferon-gamma (IFN-γ) is an important mediator of type I immune response and has antiviral, immunoregulatory and anti-tumor properties, plays a wide range of roles in inflammation and autoimmune diseases. The aim of this study was to obtain monoclonal antibody (mAb) against caprine IFN-γ by immunizing of BALB/c mice with the purified rIFN-γ. RESULTS: Recombinant caprine IFN-γ was expressed in Escherichia coli strain BL21 (DE3) and monoclonal antibodies against caprine IFN-γ were produced by immunizing of BALB/c mice with rIFN-γ. One hybridoma secreting mAb was screened by enzyme-linked immunosorbent assay (ELISA) which was designated as 2C. MAb secreted by this cell line were analyzed through ELISA, western blot and application of the mAb was evaluated by immunofluorescence analysis using goat lip tissues infected with Orf virus. ELISA analysis revealed that mAb 2C can specifically recognize rIFN-γ protein and culture supernatant of goat peripheral blood mononuclear cells (PBMCs) stimulated by concanavalin A (Con A) but cannot recognize the fusion tag protein of pET-32a. Western blot analysis showed that mAb 2C can specifically react with the purified 34.9 kDa rIFN-γ protein but does not react with the fusion tag protein of pET-32a. Immunofluorescence results demonstrated that mAb 2C can detect IFN-γ secreted in histopathological sites of goats infected with Orf virus. CONCLUSIONS: A caprine IFN-γ-specific mAb was successfully developed in this study. Further analyses showed that the mAb can be used to detect IFN-γ expression level during contagious ecthyma in goats.


Assuntos
Anticorpos Monoclonais/análise , Interferon gama/análise , Interferon gama/imunologia , Animais , Anticorpos Monoclonais/metabolismo , Western Blotting , Ectima Contagioso/sangue , Ectima Contagioso/imunologia , Ectima Contagioso/virologia , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Doenças das Cabras/sangue , Doenças das Cabras/imunologia , Doenças das Cabras/virologia , Cabras , Hibridomas/metabolismo , Interferon gama/sangue , Interferon gama/genética , Leucócitos Mononucleares/imunologia , Camundongos Endogâmicos BALB C , Vírus do Orf/fisiologia
4.
Vet Parasitol ; 276: 108990, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31775103

RESUMO

Neospora caninum infection is an important cause of neuromuscular disease in dogs and abortion in cattle, leading to significant economic losses in beef and dairy industries. The protective immunity against apicomplexan parasites, specifically Toxoplasma gondii and N. caninum, is typically achieved by inducing an IL-12-driven Th1 immune response. IL-12 stimulates IFN-γ production, which activates Inducible Nitric Oxide Synthase (iNOS) and promotes consequent Nitric Oxide (NO) synthesis, classically described as one of the main effector mechanisms for parasite elimination. Here, we aimed to evaluate the role played by iNOS during N. caninum infection. Our results show that N. caninum infection in C57BL/6 wild type (WT) mice induce NO production in vivo and in vitro. In agreement, iNOS deficient mice, as well as WT mice treated with iNOS inhibitor aminoguanidine, succumbed during acute infection with a dose lethal to 50 % of the WT mice, and presented significant increase in parasite load when submitted to sub-lethal infection protocols. Interestingly, the lack of control of parasite proliferation observed in iNOS-/- mice was associated with notable CNS inflammation and increased production of the main systemic proinflammatory cytokines (IL-12, IFN-γ, IL-6, TNF and IL-17A). Taken together, our findings show that iNOS plays an important role in restricting N. caninum replication, while also modulates the inflammatory process induced by the infection.


Assuntos
Coccidiose/enzimologia , Neospora/imunologia , Óxido Nítrico Sintase Tipo II/fisiologia , Animais , Coccidiose/parasitologia , Coccidiose/patologia , Interferon gama/análise , Subunidade p40 da Interleucina-12/análise , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/deficiência
5.
Nucleic Acids Res ; 47(16): 8362-8374, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31392985

RESUMO

Many nucleic acid aptamers that bind to target molecules have been reported as antibody alternatives. However, while the affinities of aptamers vary widely, little is known about the relationship between the affinities and their applicabilities for practical use. Here, we developed molecular affinity rulers: a series of DNA aptamers with different affinities that bind to the same area of target molecules, to measure the aptamer and its device applicabilities. For the ruler preparation, we used high-affinity DNA aptamers containing a hydrophobic unnatural base (Ds) as the fifth base. By replacing Ds bases with A bases in Ds-DNA aptamers targeting VEGF165 and interferon-γ, we prepared two sets of DNA aptamers with dissociation constants (KD) ranging from 10-12 to 10-8 M. Using these molecular affinity rulers, we evaluated the sensitivity of DNA aptamers in ELISA (enzyme-linked immunosorbent assay), which showed the clear relationship between aptamer affinities and their detection sensitivities. In sandwich-type ELISA using combinations of aptamers and antibodies, aptamers with KD values lower than ∼10-9 M were required for sufficient sensitivities (limit of detection (LOD) < 10 pM) and signal intensities, but optimizations improved the lower-affinity aptamers' applicabilities. These aptamer affinity rulers could be useful for evaluating and improving aptamer applicabilities.


Assuntos
Adenina/química , Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais , Ensaio de Imunoadsorção Enzimática/métodos , Interferon gama/análise , Fator A de Crescimento do Endotélio Vascular/análise , Animais , Anticorpos Monoclonais/química , Aptâmeros de Nucleotídeos/síntese química , Pareamento de Bases , Sequência de Bases , Biotina/química , Ensaio de Imunoadsorção Enzimática/normas , Humanos , Interações Hidrofóbicas e Hidrofílicas , Interferon gama/química , Cinética , Limite de Detecção , Conformação de Ácido Nucleico , Ligação Proteica , Padrões de Referência , Técnica de Seleção de Aptâmeros , Estreptavidina/química , Fator A de Crescimento do Endotélio Vascular/química
6.
PLoS Negl Trop Dis ; 13(8): e0007113, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31425525

RESUMO

Buruli Ulcer (BU) is a cutaneous disease caused by Mycobacterium ulcerans. The pathogenesis of this disease is closely related to the secretion of the toxin mycolactone that induces extensive destruction of the skin and soft tissues. Currently, there are no effective measures to prevent the disease and, despite availability of antibiotherapy and surgical treatments, these therapeutic options are often associated with severe side effects. Therefore, it is important to develop alternative strategies for the treatment of BU. Endolysins (lysins) are phage encoded enzymes that degrade peptidoglycan of bacterial cell walls. Over the past years, lysins have been emerging as alternative antimicrobial agents against bacterial infections. However, mycobacteria have an unusual outer membrane composed of mycolylarabinogalactan-peptidoglycan. To overcome this complex barrier, some mycobacteriophages encode a lipolytic enzyme, Lysin B (LysB). In this study, we demonstrate for the first time that recombinant LysB displays lytic activity against M. ulcerans isolates. Moreover, using a mouse model of M. ulcerans footpad infection, we show that subcutaneous treatment with LysB prevented further bacterial proliferation, associated with IFN-γ and TNF production in the draining lymph node. These findings highlight the potential use of lysins as a novel therapeutic approach against this neglected tropical disease.


Assuntos
Úlcera de Buruli/tratamento farmacológico , Endopeptidases/administração & dosagem , Micobacteriófagos/enzimologia , Mycobacterium ulcerans/efeitos dos fármacos , Animais , Bacteriólise , Úlcera de Buruli/patologia , Modelos Animais de Doenças , Endopeptidases/farmacologia , Feminino , Interferon gama/análise , Linfonodos/imunologia , Camundongos Endogâmicos BALB C , Mycobacterium ulcerans/virologia , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/farmacologia , Resultado do Tratamento , Fator de Necrose Tumoral alfa/análise
7.
Acta Trop ; 199: 105148, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31425673

RESUMO

Toxoplasma gondii, a ubiquitous and obligate intracellular pathogen, belonging to the phylum Apicomplexa, is capable of infecting a broad range of warm-blooded hosts including birds and mammals that is nearly worldwide. Preventive measures for toxoplasmosis are currently lacking and as such, development of novel vaccines is of urgent need. The plant-like calcium-dependent protein kinases (CDPKs) expressed by T. gondii, play important roles in cell invasion, gliding motility, egress and some other developmental processes, in which T. gondii CDPK3 (TgCDPK3) has been implicated as an important virulence factor. In this study, the immune protective function of recombinant TgCDPK3 (rTgCDPK3) against experimental toxoplasmosis in BALB/c were evaluated. We divided the mice into different dose groups of vaccines and all immunizations with purified rTgCDPK3 protein were injected by intramuscular at weeks 0, 2, and 4 in BALB/c mice. The rTgCDPK3 vaccine provided protection was correlated with the development of humoral and cellular immune responses demonstrated through the antigen-specific spleen cell proliferation, release of Th1 cytokines IFN-γ, and the production of the high titers of IgG antibody with a predominance of IgG2a over IgG1. Vaccination with rTgCDPK3 conferred partial protection against acute toxoplasmosis, as demonstrated by prolonged survival rate after lethal challenge. Additionally, the amount of brain tissues cysts in vaccinated mice led to 46.5% reduction compared with non-vaccinated ones. These data demonstrated that rTgCDPK3 inoculation prevents or attenuates the harmful influence of T. gondii infection, and it is a potential vaccine candidate against toxoplasmosis.


Assuntos
Proteínas Quinases/imunologia , Vacinas Protozoárias/imunologia , Toxoplasma/imunologia , Toxoplasmose Animal/prevenção & controle , Animais , Feminino , Interferon gama/análise , Camundongos , Camundongos Endogâmicos BALB C , Vacinação , Vacinas Sintéticas/imunologia
8.
Nanoscale ; 11(35): 16362-16367, 2019 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-31435631

RESUMO

The monitoring and detection of molecular biomarkers play crucial roles in disease diagnosis and treatment. In this work, we proposed a target-responsive netlike hybridization chain reaction (nHCR) DNA nanostructure construction method, which can offer an exceptional signal enhancement, for highly sensitive fluorescence detection of cytokine, interferon-gamma (IFN-γ). The presence of the target cytokine can lead to the conformational change of the aptamer recognition hairpin probes and the liberation of the nHCR initiator strands, which further trigger the nHCR process between two dye-labeled and double hairpin-structured probes to form netlike DNA nanostructures. The formation of the DNA nanostructures brings the dyes into close proximity, resulting in significantly amplified fluorescence resonance energy transfer signals for sensitive and enzyme-free detection of IFN-γ. The present method has a detection limit of 1.2 pM and a dynamic linear range of 5 to 1000 pM for IFN-γ detection. Besides, with the high specificity of the aptamer probe and the significant signal amplification of the nHCR, such an IFN-γ detection strategy shows excellent selectivity and high sensitivity, which can be potentially applied to detect IFN-γ in human serums. With such a demonstration of the detection of IFN-γ, this proposed method can be extended for detecting different types of biomolecules.


Assuntos
DNA/química , Transferência Ressonante de Energia de Fluorescência , Interferon gama/análise , Nanoestruturas/química , Humanos , Limite de Detecção , Hibridização de Ácido Nucleico
9.
Biosens Bioelectron ; 142: 111532, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31377576

RESUMO

In this paper, a novel label-free electrochemical impedance aptasensor for highly sensitive detection of IFN-γ based on target-induced exonuclease inhibition was constructed. For this purpose, we designed a DNA hairpin modified on the gold electrode whose loop was the aptamer of the IFN-γ, and the stem was 5'-thiol-modified. In the absence of IFN-γ, Exonuclease III (Exo III) and Exonuclease I (Exo I) digested the double-stranded and single-stranded strands of the hairpin DNA, respectively, causing smaller impedance value on the surface of the electrode. In the presence of IFN-γ, the function of Exo III was greatly inhibited by the binding of the aptamer with the target, and it stopped after cutting three bases of the hairpin DNA. Forming a major target-bound aptamer digestion product, it could not be digested by Exo I, so there was larger impedance on the electrode surface. The calibration curve for IFN-γ was linear in the range of 1 pM-50 nM with the detection limit (LOD) of 0.7 pM. The proposed aptasensor proved good selectivity and reproducibility, and low cost. In addition, the biosensor was able to detect IFN-γ in serum samples successfully, which is expected to provide an efficient method for TB diagnosis at early stages.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , Interferon gama/sangue , Técnicas Biossensoriais/instrumentação , Impedância Elétrica , Técnicas Eletroquímicas/instrumentação , Técnicas Eletroquímicas/métodos , Eletrodos , Desenho de Equipamento , Exodesoxirribonucleases/química , Humanos , Interferon gama/análise , Limite de Detecção
10.
Clin Chim Acta ; 497: 48-53, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31310745

RESUMO

BACKGROUND: In clinical practice, pleural and peritoneal effusions are usual diagnosis. We evaluated the performance of a hybrid panel of biomarkers in the diagnosis of the main diseases affecting pleura and/or peritoneum. METHODS: Samples of pleural/ peritoneal fluid from 120 patients were evaluated for: CEA (carcinoembryonic antigen), VEGF-A (vascular endothelial growth factor A), PD-L1/B7-H1 (programmed death-ligand 1), NGAL (neutrophil gelatinase-associated lipocalin), TREM-1 (triggering receptor expressed in myeloid cells type-1) and IFNγ (gamma-interferon) by Luminex®; CALP (Calprotectin) by ELISA, and ADA (adenosine deaminase) by enzymatic deamination. RESULTS: For malignant effusion (ME) diagnosis, CEA and NGAL presented superior performance than VEGF-A, PD-L1 and CALP. A CEA-NGAL association showed good sensitivity (86.6%) and accuracy (79.2%). For non-tuberculous infectious effusion (NTBIE), NGAL presented the best performance with sensitivity (75.0%), specificity (62.0%) and accuracy (65.0%) higher than TREM-1 and CALP; however, when associated, although with good sensitivity, there was important decrease in specificity. For tuberculous pleural effusion (TPE), IFNy-ADA presented excellent sensitivity (100%), specificity (87.6%), NPV (100%) and accuracies (~90%). CONCLUSIONS: CEA, NGAL, ADA and IFNy were useful in discriminating ME and TPE. However, for NTBIE diagnosis, the hybrid panel did not demonstrate advantages over the classic parameters.


Assuntos
Adenosina Desaminase/análise , Interferon gama/análise , Derrame Pleural Maligno/diagnóstico , Adenosina Desaminase/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/análise , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Interferon gama/metabolismo , Masculino , Pessoa de Meia-Idade , Paracentese , Adulto Jovem
11.
Medicina (Kaunas) ; 55(6)2019 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-31181785

RESUMO

Background and Objectives: Interferon-gamma (IFN-γ)/interleukin-4 (IL-4) ratio may indicate a change in the immune response with a potential pathological effect presented in oral lichen planus (OLP) patients. Herein, this meta-analysis evaluated the role of serum and salivary interferon-gamma/interleukin-4 ratio in the severity and development of OLP. Materials and Methods: The Scopus, Cochrane Library, PubMed, and Web of Science databases were systematically searched to retrieve the relevant studies published up from the database inception to March 2019. The crude mean difference (MD) and 95% confidence interval (CI) were calculated by RevMan 5.3 software using a random-effects model. A sensitivity analysis was performed on the results using the CMA 2.0 software. A total of 98 studies were retrieved from the databases, of which at last seven studies were included in this meta-analysis. Results: The findings showed that the pooled MDs of serum and salivary IFN-γ/IL-4 ratio were -0.22 (95% CI: -1.16, 0.72; p = 0.64) and 0.17 (95% CI: -1.50, 1.84; p = 0.84) in OLP patients compared to controls, respectively. In addition, the pooled MDs of serum and salivary IFN-γ/IL-4 ratio were -0.15 (95% CI: -0.53, 0.23; p = 0.43) and -0.39 (95% CI: -0.63, -0.15; p = 0.001) in patients with erythematous/ulcerative subtype compared to patients with reticular subtype, respectively. Conclusions: In conclusion, the results of meta-analysis demonstrated that serum and salivary IFN-γ/IL-4 ratio cannot play a major role in OLP development and severity.


Assuntos
Interferon gama/análise , Interleucina-4/análise , Líquen Plano Bucal/diagnóstico , Fragmentos de Peptídeos/análise , Distribuição de Qui-Quadrado , Humanos , Interferon gama/sangue , Interleucina-4/sangue , Líquen Plano Bucal/sangue , Fragmentos de Peptídeos/sangue , Saliva/metabolismo
12.
ACS Appl Mater Interfaces ; 11(27): 23901-23908, 2019 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-31187614

RESUMO

Barcode nanowires (BNWs) composed of multiple layered segments of different materials are attractive to bioengineering field due to their characteristics that allow the adjustment of physicochemical properties and conjugation with two or more types of biomolecules to facilitate multiple tasks. Here, we report a metallic Fe (iron)-Au (gold) BNW-based platform for capturing CD8 T cells and the interferon-γ (γ) they secrete, both of which play key roles in controlling infectious diseases such as tuberculosis, at the single-cell level. We also describe an efficient approach for conjugating distinct antibodies, which recognize different epitopes to appropriate materials. The platform achieved detection even with 4.45-35.6 µg mL-1 of BNWs. The T cell capture efficiency was close to 100% and the detection limit for interferon-γ was 460 pg mL-1. This work presents a potential guideline for the design of single-cell immunoassay platforms for eliminating diagnostic errors by unambiguously identifying disease-relevant immune mediators.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Separação Celular , Ouro/química , Interferon gama/imunologia , Ferro/química , Nanofios/química , Linfócitos T CD8-Positivos/citologia , Humanos , Imunoensaio , Interferon gama/análise , Sensibilidade e Especificidade
13.
PLoS One ; 14(5): e0216056, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31067281

RESUMO

The airway epithelial barrier is critical for preventing pathogen invasion and translocation of inhaled particles into the lung. Epithelial cells also serve an important sentinel role after infection and release various pro-inflammatory mediators that recruit and activate immune cells. Airway epithelial barrier disruption has been implicated in a growing number of respiratory diseases including viral infections. It is thought that when a pathogen breaks the barrier and gains access to the host tissue, pro-inflammatory mediators increase, which further disrupts the barrier and initiates a vicious cycle of leak. However, it is difficult to study airway barrier integrity in vivo, and little is known about relationship between epithelial barrier function and airway inflammation. Current assays of pulmonary barrier integrity quantify the leak of macromolecules from the vasculature into the airspaces (or "inside/out" leak). However, it is also important to measure the ease with which inhaled particles, allergens, or pathogens can enter the subepithelial tissues (or "outside/in" leak). We challenged mice with inhaled double stranded RNA (dsRNA) and explored the relationship between inside/out and outside/in barrier function and airway inflammation. Using wild-type and gene-targeted mice, we studied the roles of the dsRNA sensors Toll Like Receptor 3 (TLR3) and Melanoma Differentiation-Associated protein 5 (MDA5). Here we report that after acute challenge with inhaled dsRNA, airway barrier dysfunction occurs in a TLR3-dependent manner, whereas leukocyte accumulation is largely MDA5-dependent. We conclude that airway barrier dysfunction and inflammation are regulated by different mechanisms at early time points after exposure to inhaled dsRNA.


Assuntos
Inflamação/induzido quimicamente , Helicase IFIH1 Induzida por Interferon/fisiologia , RNA de Cadeia Dupla/farmacologia , Mucosa Respiratória/efeitos dos fármacos , Receptor 3 Toll-Like/fisiologia , Administração por Inalação , Animais , Líquido da Lavagem Broncoalveolar/química , Quimiocina CCL3/análise , Feminino , Inflamação/metabolismo , Inflamação/fisiopatologia , Interferon gama/análise , Interleucina-6/análise , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA de Cadeia Dupla/metabolismo , Mucosa Respiratória/metabolismo , Mucosa Respiratória/fisiologia
14.
Arch Dermatol Res ; 311(7): 519-527, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31089878

RESUMO

Recent studies have noticed significant role of interleukin (IL)-17, 22, 23, Foxp3, interferon-gamma (IFN-γ) and Wnt5a in oral and cutaneous lichen planus (LP). This study was undertaken to assess whether similar expression exists in lichen planus pigmentosus (LPP). We recruited 30 patients of treatment-naïve 'LPP' (in absence of cutaneous/mucosal LP elsewhere, group 1), 10 patients having active treatment-naïve cutaneous 'LP' (group 2), 10 patients having 'post-LP' hyperpigmentation (in absence of active LP and off treatment for at least past 3 months, group 3), and 10 controls. Quantitative real-time polymerase chain reaction (qRT-PCR, peripheral blood mononuclear cells [PBMCs] and skin) and immunohistochemistry (IHC, skin) was performed. mRNA expression (in PBMCs) of IL-17A, IL-22, IL-23A, IFN-γ and Foxp3 was significantly decreased in group 1 and 3 as compared to group 2 (p < 0.05). Wnt5a expression was maximal in controls; and while there was no difference between group 1 and 2; whereas expression in group 3 was significantly lesser than group 1 and 2 (p < 0.05). qRT-PCR (skin) and IHC (skin) revealed similar results; and mRNA expression and mean fluorescence intensity of IL-17A, IL-22, IL-23A/R was significantly increased in group 2 and 3 compared to group 1 (p < 0.05). Mean fluorescence intensity and mRNA expression of IFN-γ, Foxp3 and Wnt5a were significantly increased in group 2 compared to group 1 (p < 0.05); whereas the difference between group 1 and 3 was not significant. Mean fluorescence intensity and mRNA expression of IL-17A, 1L-22 and IFN-γ showed no difference between group 2 and 3; whereas that of IL-23A/R, foxp3 and wnt5a were significantly higher in group 2 than group 3 (p < 0.05). Overall, maximal expression of IL-17A, IL-22, IL-23A, IFN-γ and Foxp3 (mRNA PBMCs) was observed in LP. Minimal expression of IL-17A, IL-22, IL-23A/R, IFN-γ and Foxp3 (mRNA skin and IHC skin) was seen in LPP patients. In contrast to LP, LPP lacks the expression of IFN-γ, Foxp3 and the cytokines representing Th17 pathway, and thus seems to have a distinct pathogenesis.


Assuntos
Líquen Plano/diagnóstico , Transtornos da Pigmentação/diagnóstico , Pele/patologia , Adolescente , Adulto , Biomarcadores/análise , Biomarcadores/metabolismo , Diagnóstico Diferencial , Feminino , Seguimentos , Fatores de Transcrição Forkhead/análise , Fatores de Transcrição Forkhead/metabolismo , Humanos , Interferon gama/análise , Interferon gama/metabolismo , Interleucina-17/análise , Interleucina-17/metabolismo , Subunidade p19 da Interleucina-23/análise , Subunidade p19 da Interleucina-23/metabolismo , Interleucinas/análise , Interleucinas/metabolismo , Líquen Plano/patologia , Masculino , Pessoa de Meia-Idade , Transtornos da Pigmentação/patologia , Estudos Prospectivos , Pigmentação da Pele , Adulto Jovem
15.
BMC Vet Res ; 15(1): 149, 2019 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-31096976

RESUMO

BACKGROUND: The objective of this study was to determine the sensitivity (Se) and specificity (Sp) of bovine tuberculosis (bTB) screening tests including a single intradermal tuberculin (SIT) test, interferon gamma (IFN-γ) assay, and a commercial ELISA test (M. bovis Ab) in dairy cattle, under field conditions, using a Bayesian approach. RESULTS: The study population consisted of 128 dairy cows from 25 bTB-infected herds in Chiang Mai and Chiang Rai provinces, Thailand. A single-population Bayesian model was implemented assuming conditional dependence between the SIT test and IFN-γ assays. The 95% posterior probability interval (PPI) of the SIT test (severe interpretation) Se ranged from 75.3 to 95.2% (median = 87.6%), while the Sp was slightly lower (median = 83.6%, PPI = 74.2-92.8%). The IFN-γ assay Se was moderate and the 95% PPI ranged from 38.6 to 74.4% (median = 55.7%) with higher Sp (median = 93.5.4%, PPI = 87.0-98.1%). The M. bovis Ab ELISA Se was low, with 95% PPI ranging between 30.0 and 71.2% (median = 47.4%); however, the Sp was high (median = 90.9%, PPI = 84.5-95.5%). CONCLUSION: The SIT test sensitivity was similar to that demonstrated in other regions and can, therefore, be used effectively as part of control programs in this area. The IFN-γ and M. bovis Ab ELISA assays can be applied as supplementary techniques. The test performance of these tests when used as single tests without confirmation, however, are expected to continue to challenge disease eradication efforts.


Assuntos
Ensaio de Imunoadsorção Enzimática/veterinária , Interferon gama/análise , Teste Tuberculínico/veterinária , Tuberculose Bovina/diagnóstico , Animais , Teorema de Bayes , Bovinos , Indústria de Laticínios , Testes Diagnósticos de Rotina/veterinária , Feminino , Mycobacterium bovis , Sensibilidade e Especificidade , Tailândia
16.
Mikrochim Acta ; 186(6): 356, 2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-31098714

RESUMO

The authors describe a versatile aptasensing scheme based on the use of polypyrrole nanoparticles (PPyNPs) and DNA-silver nanoclusters (DNA-AgNCs) for multiple target detection. The DNA-AgNCs consist of two functional domains, viz. (a) a nucleation domain for attaching the metal core of the nanoclusters, and (b) a recognition domain which consists of a single-stranded aptamer. In the absence of analytes, the single-strand recognition domain will be absorbed onto the surface of the PPyNPs through π stacking and hydrophobic interactions. As a result, the red fluorescence of the DNA-AgNCs (with excitation/emission peaks at 535/625 nm) is quenched by the PPyNPs. On introducing the analytes, the DNA-AgNCs will bind them. This leads to the desorption of DNA-AgNCs and the recovery of the red fluorescence. Based on the above strategy, a versatile, sensitive and selective aptasensor was established for detection of adenosine, thrombin and interferon-gamma. The method was applied to the detection of the above targets in (spiked) serum samples and gave satisfactory results, with detection limit of 0.58 nM for IFN-γ, 0.39 nM for adenosine, and 2.2 nM for thrombin. The use of PPyNPs results in uniquely low non-specific absorption and in improved analytical results in case of real-sample analysis when compared to previously reported methods. Graphical abstract Schematic illustration of DNA-silver nanoclusters and polypyrrole nanoparticles in an aptasensor for detection of multiple targets.


Assuntos
Adenosina/análise , DNA/química , Interferon gama/análise , Polímeros/química , Pirróis/química , Prata/química , Trombina/análise , Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais , Fluorometria , Nanoestruturas/química
17.
Avian Pathol ; 48(5): 423-428, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31081347

RESUMO

Salmonella enterica serovar Gallinarum causes a disease in chickens known as fowl typhoid. Interferon-gamma (IFN-γ) has been shown to be crucial in eliminating salmonellosis infection because of its strong association with T-cell responses. This study was undertaken to compare the expression of IFN-γ in chickens generated by different vaccine formulations. Eighty one-day-old Lohmann layer chicks were divided into four groups of 20 birds each for the experiment. This comprised an unvaccinated negative control group (NEG), a group vaccinated with the live 9R vaccine by the injection route (SC), a group vaccinated with alginate-coated chitosan microparticles encapsulating live plasmid-cured S. Gallinarum strain 9 (PC) by the oral route, and a group vaccinated with a weak attenuated live S. Gallinarum strain 9 encapsulated in alginate-coated chitosan microparticles (VM) given orally. Vaccinations were done at 10 and 14 weeks of age followed by challenge at 16 weeks of age. IgG was measured using ELISA. qRT-PCR was used to compare the mRNA fold expression of IFN-γ in the PC, VM and SC groups using the unvaccinated/unchallenged group as the control. There were significant differences in the IgG levels between each vaccinated group and the unvaccinated group (P < 0.05) after booster vaccination and post-challenge. There was 100% protection of the birds in SC and VM groups, 80% protection in PC group and 0% protection in the NEG group. Using 2-ΔΔCT calculation, IFN-γ was more highly expressed in the PC group than in the SC group or VM group. In conclusion, the IFN-γ was more highly expressed in the PC group (though not significantly higher) compared to the SC and VM groups and this could be attributed to the alginate-coated chitosan microparticles which acted as an adjuvant.


Assuntos
Galinhas/imunologia , Interferon gama/análise , Doenças das Aves Domésticas/prevenção & controle , Salmonelose Animal/prevenção & controle , Vacinas contra Salmonella/administração & dosagem , Salmonella enterica/imunologia , Administração Oral , Alginatos/química , Animais , Quitosana/química , Feminino , Imunidade Celular , Imunidade Humoral , Imunização Secundária/veterinária , Plasmídeos/genética , Doenças das Aves Domésticas/microbiologia , Salmonelose Animal/microbiologia , Vacinas contra Salmonella/imunologia , Vacinas Tíficas-Paratíficas/administração & dosagem , Regulação para Cima
18.
Int J Mol Sci ; 20(7)2019 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-30925759

RESUMO

Hepatic intrasinusoidal (HI) natural killer (NK) cells from liver perfusate have unique features that are similar to those of liver-resident NK cells. Previously, we have reported that HI CD56bright NK cells effectively degranulate against SNU398 hepatocellular carcinoma (HCC) cells. Thus, the aim of this study was to further investigate the phenotype and function of HI NK cells. We found that HI CD56bright NK cells degranulated much less to Huh7 cells. HI CD56bright NK cells expressed NKG2D, NKp46, TNF-related apoptosis-inducing ligand (TRAIL), and FAS ligand (FASL) at higher levels than CD56dim cells. SNU398 cells expressed more NKG2D ligands and FAS and less PD-L1 than Huh7 cells. Blockade of NKG2D, TRAIL, and FASL significantly reduced the cytotoxicity of HI NK cells against SNU398 cells, but blockade of PD-L1 did not lead to any significant change. However, HI NK cells produced IFN-γ well in response to Huh7 cells. In conclusion, the cytotoxicity of HI CD56bright NK cells was attributed to the expression of NKG2D, TRAIL, and FASL. The results suggest the possible use of HI NK cells for cancer immunotherapy and prescreening of HCC cells to help identify the most effective NK cell therapy recipients.


Assuntos
Antígeno CD56/imunologia , Carcinoma Hepatocelular/imunologia , Células Matadoras Naturais/imunologia , Neoplasias Hepáticas/imunologia , Adulto , Antígeno CD56/análise , Linhagem Celular Tumoral , Proteína Ligante Fas/análise , Proteína Ligante Fas/imunologia , Feminino , Proteínas Ligadas por GPI/análise , Proteínas Ligadas por GPI/imunologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/análise , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Interferon gama/análise , Interferon gama/imunologia , Masculino , Ligante Indutor de Apoptose Relacionado a TNF/análise , Ligante Indutor de Apoptose Relacionado a TNF/imunologia
19.
Yonsei Med J ; 60(4): 375-380, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30900424

RESUMO

PURPOSE: This study aimed to evaluate ichroma™ IGRA-TB, a novel point-of-care platform for assaying IFN-γ release, and to compare it with QuantiFERON-TB Gold In-Tube (QFT-GIT) for identifying Mycobacterium tuberculosis (M. tb) infection. MATERIALS AND METHODS: We recruited 60 healthy subjects, and blood samples were obtained in QFT-GIT blood collection tubes. The blood collection tubes were incubated at 37°C, and culture supernatant was harvested after 18-24 hours. IFN-γ responses were assessed by the ichroma™ IGRA-TB cartridge and the QFT-GIT IFN-γ enzyme-linked immunosorbent assay. Three active TB patients were recruited as a positive control for M. tb infection. RESULTS: The area under the receiver operating characteristic curve of the ichroma™ IGRA-TB test for differentiating between infected and non-infected individuals was 0.9706 (p<0.001). Inconsistent positivity between the two tests was found in three participants who showed weak positive IFN-γ responses (<1.0 IU/mL) with QFT-GIT. However, the two tests had excellent agreement (95.2%, κ=0.91, p<0.001), and a very strong positive correlation was observed between the IFN-γ values of both tests (r=0.91, p<0.001). CONCLUSION: The diagnostic accuracy demonstrated in this study indicates that the ichroma™ IGRA-TB test could be used as a rapid diagnostic method for detecting latent TB infection. It may be particularly beneficial in resource-limited places that require cost-effective laboratory diagnostics.


Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Testes de Liberação de Interferon-gama/métodos , Interferon gama/sangue , Tuberculose Latente/diagnóstico , Mycobacterium tuberculosis/isolamento & purificação , Sistemas Automatizados de Assistência Junto ao Leito , Teste Tuberculínico/métodos , Adulto , Área Sob a Curva , Estudos de Casos e Controles , Estudos de Viabilidade , Feminino , Humanos , Interferon gama/análise , Tuberculose Latente/etnologia , Tuberculose Latente/imunologia , Masculino , Mycobacterium tuberculosis/imunologia , Curva ROC , Kit de Reagentes para Diagnóstico , República da Coreia/epidemiologia , Adulto Jovem
20.
Klin Lab Diagn ; 64(1): 46-48, 2019.
Artigo em Russo | MEDLINE | ID: mdl-30912884

RESUMO

The identification and analysis of microbiological and immunological disorders in urogenital chlamydiosis of mixed etiology plays an important role in understanding the pathogenesis of the development of the chronic course of the disease with severe complications (infertility, miscarriage). Along with classical microbiological studies (isolating and studying the properties of the microbiota in mixed infections), the role of antigens of all participants of the infectious process (bacteria, viruses, fungi) in the occurrence of adequate immunological reactions (the level of interferon, γ-interferon) as indicators of the immune response.


Assuntos
Infecções por Chlamydia/diagnóstico , Doenças Urogenitais Femininas/diagnóstico , Feminino , Humanos , Imunidade Ativa , Interferon gama/análise , Sistema Urogenital/fisiopatologia
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