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1.
Methods Mol Biol ; 2270: 61-76, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33479893

RESUMO

IL-10 is the best known and most studied anti-inflammatory cytokine and, in the last 20 years, it has acquired even greater fame as it has been associated with the regulatory phenotype of B cells. Indeed, although great efforts have been made to find a unique marker, to date IL-10 remains the main way to follow both murine and human regulatory B cells, hence the need of precise and reproducible methods to identify and purify IL-10-producing B cells for both functional and molecular downstream assays. In this chapter, we present our protocols to isolate these cells from the murine spleen and peritoneum and from human peripheral blood. Since the production of IL-10 by B cells is not only a weapon to counteract the adverse effect of pro-inflammatory cytokines but also a response to cellular activation, we focused on those B cells that are prone to IL-10 production and detectable following a short-term stimulation with phorbol-12-myristate-13-acetate, ionomycin, and lipopolysaccharide (murine system) or CpG (human system).


Assuntos
Subpopulações de Linfócitos B/citologia , Linfócitos B Reguladores/citologia , Separação Celular/métodos , Animais , Subpopulações de Linfócitos B/imunologia , Citocinas/imunologia , Expressão Gênica/genética , Expressão Gênica/imunologia , Humanos , Interleucina-10/metabolismo , Ionomicina/farmacologia , Lipopolissacarídeos/farmacologia , Ativação Linfocitária/imunologia , Camundongos , Ésteres de Forbol/farmacologia , Baço/citologia , Acetato de Tetradecanoilforbol/farmacologia
3.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 36(8): 704-711, 2020 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-32958127

RESUMO

Objective To elucidate the mechanisms by which elongation factor Tu GTP binding domain containing 2 (Eftud2) enhances the immune function of murine macrophages by bioinformatics analysis. Methods The bone marrow-derived macrophages (BMDMs) of Eftud2 myeloid cell-specific knockout (MKO) mice (n=10) and wild-type (WT) littermates (n=10) were collected and stimulated by lipopolysaccharide (LPS) (100 ng/mL) for 2 hours. Bioinformatics analysis was conducted to examine the differences in gene expression and mRNA transcription levels. The the differences in gene expression and alternative splicing of mRNA transcription in BMDMs were analyzed by DEGseq and rMATS, respectively. The signaling pathways affected were clarified by Kyoto Encyclopedia of Genes and Genomes (KEGG) classification and enrichment methods. Results Compared with WT counterparts, the expression levels of IL-6, IL-1ß, TNF-α, and the genes related to immune response in MKO BMDMs were down-regulated following LPS stimulation. KEGG pathway analysis showed that the differently expressed genes in BMDMs and alternative splicing mainly affected the signal transduction and immune system-related metabolic pathways, and had a strong correlation with PI3K-AKT signaling pathway. The difference in alternative splicing also existed in ubiquitination and endocytosis. Compared with WT counterparts, there were 232 differences in alternative splicing in MKO BMDMs, among which 125 were skipping exons, accounting for the largest proportion. In addition, the analysis of alternative splicing differences also confirmed the previous experimental results, that is, Eftud2 could participate in the activation of inflammatory signaling pathways by enhancing the alternative splicing of key molecules such as MyD88 in TLR4-NF-κB signaling pathway, thereby augmenting the function of macrophages. Conclusion Eftud2 can promote the release of inflammatory cytokines in BMDMs by regulating gene expression and alternative splicing, and consequently enhance the immune function of macrophages.


Assuntos
Biologia Computacional , Expressão Gênica , Macrófagos , Animais , Citocinas/genética , Citocinas/imunologia , Expressão Gênica/genética , Expressão Gênica/imunologia , Lipopolissacarídeos/farmacologia , Macrófagos/imunologia , Camundongos , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo
4.
J Exp Med ; 217(12)2020 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-32820330

RESUMO

Type I interferons (IFN-I) are a major antiviral defense and are critical for the activation of the adaptive immune system. However, early viral clearance by IFN-I could limit antigen availability, which could in turn impinge upon the priming of the adaptive immune system. In this study, we hypothesized that transient IFN-I blockade could increase antigen presentation after acute viral infection. To test this hypothesis, we infected mice with viruses coadministered with a single dose of IFN-I receptor-blocking antibody to induce a short-term blockade of the IFN-I pathway. This resulted in a transient "spike" in antigen levels, followed by rapid antigen clearance. Interestingly, short-term IFN-I blockade after coronavirus, flavivirus, rhabdovirus, or arenavirus infection induced a long-lasting enhancement of immunological memory that conferred improved protection upon subsequent reinfections. Short-term IFN-I blockade also improved the efficacy of viral vaccines. These findings demonstrate a novel mechanism by which IFN-I regulate immunological memory and provide insights for rational vaccine design.


Assuntos
Imunogenicidade da Vacina/imunologia , Interferon Tipo I/antagonistas & inibidores , Interferon-alfa/imunologia , Receptor de Interferon alfa e beta/imunologia , Vacinas Virais/imunologia , Infecção por Zika virus/imunologia , Zika virus/imunologia , Animais , Anticorpos Bloqueadores/imunologia , Anticorpos Bloqueadores/farmacologia , Anticorpos Antivirais/imunologia , Apresentação do Antígeno/imunologia , Linfócitos T CD8-Positivos/metabolismo , Células Dendríticas/imunologia , Modelos Animais de Doenças , Expressão Gênica/imunologia , Células HEK293 , Humanos , Memória Imunológica , Interferon-alfa/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor de Interferon alfa e beta/genética , Transfecção , Infecção por Zika virus/virologia
5.
Nat Commun ; 11(1): 3871, 2020 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-32747712

RESUMO

Relapses in multiple sclerosis can result in irreversible nervous system tissue injury. If these events could be detected early, targeted immunotherapy could potentially slow disease progression. We describe the use of engineered biomaterial-based immunological niches amenable to biopsy to provide insights into the phenotype of innate immune cells that control disease activity in a mouse model of multiple sclerosis. Differential gene expression in cells from these niches allow monitoring of disease dynamics and gauging the effectiveness of treatment. A proactive treatment regimen, given in response to signal within the niche but before symptoms appeared, substantially reduced disease. This technology offers a new approach to monitor organ-specific autoimmunity, and represents a platform to analyze immune dysfunction within otherwise inaccessible target tissues.


Assuntos
Encefalomielite Autoimune Experimental/terapia , Imunoterapia/métodos , Monitorização Fisiológica/métodos , Esclerose Múltipla/terapia , Animais , Modelos Animais de Doenças , Progressão da Doença , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/imunologia , Feminino , Expressão Gênica/genética , Expressão Gênica/imunologia , Perfilação da Expressão Gênica/métodos , Humanos , Camundongos Endogâmicos , Esclerose Múltipla/genética , Esclerose Múltipla/imunologia , Recidiva , Resultado do Tratamento
6.
Cells ; 9(9)2020 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-32847031

RESUMO

Following influenza infection, rs2248374-G ERAP2 expressing cells may transcribe an alternative spliced isoform: ERAP2/Iso3. This variant, unlike ERAP2-wt, is unable to trim peptides to be loaded on MHC class I molecules, but it can still dimerize with both ERAP2-wt and ERAP1-wt, thus contributing to profiling an alternative cellular immune-peptidome. In order to verify if the expression of ERAP2/Iso3 may be induced by other pathogens, PBMCs and MDMs isolated from 20 healthy subjects were stimulated with flu, LPS, CMV, HIV-AT-2, SARS-CoV-2 antigens to analyze its mRNA and protein expression. In parallel, Calu3 cell lines and PBMCs were in vitro infected with growing doses of SARS-CoV-2 (0.5, 5, 1000 MOI) and HIV-1BAL (0.1, 1, and 10 ng p24 HIV-1Bal/1 × 106 PBMCs) viruses, respectively. Results showed that: (1) ERAP2/Iso3 mRNA expression can be prompted by many pathogens and it is coupled with the modulation of several determinants (cytokines, interferon-stimulated genes, activation/inhibition markers, antigen-presentation elements) orchestrating the anti-microbial immune response (Quantigene); (2) ERAP2/Iso3 mRNA is translated into a protein (western blot); (3) ERAP2/Iso3 mRNA expression is sensitive to SARS-CoV-2 and HIV-1 concentration. Considering the key role played by ERAPs in antigen processing and presentation, it is conceivable that these enzymes may be potential targets and modulators of the pathogenicity of infectious diseases and further analyses are needed to define the role played by the different isoforms.


Assuntos
Aminopeptidases/genética , Betacoronavirus/imunologia , Infecções por Coronavirus/genética , Imunização/métodos , Leucócitos Mononucleares/virologia , Macrófagos/virologia , Pneumonia Viral/genética , Isoformas de Proteínas/genética , Apresentação do Antígeno/genética , Doadores de Sangue , Linhagem Celular Tumoral , Infecções por Coronavirus/virologia , Expressão Gênica/imunologia , Genótipo , Infecções por HIV/genética , Infecções por HIV/virologia , HIV-1/imunologia , Humanos , Leucócitos Mononucleares/metabolismo , Macrófagos/metabolismo , Antígenos de Histocompatibilidade Menor/genética , Pandemias , Pneumonia Viral/virologia , Polimorfismo de Nucleotídeo Único , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Genética/imunologia
7.
Oncology ; 98(7): 501-511, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32380498

RESUMO

INTRODUCTION: Anti-programmed cell death 1 (PD-1) therapies have shown promising clinical activity against gastric cancer (GC). We evaluated the clinical significance of immune-related gene expression in GC tissues to better understand the tumor immune microenvironment. METHODS: PD-1, PD-1 ligand 1 (PD-L1) and CD8 mRNA levels and clinicopathological factors, including survival, were examined by quantitative RT-PCR in 155 GC patients who underwent surgery. PD-1 and PD-L1 expression in tumor tissue from 24 GC patients was investigated by immunohistochemical analysis. RESULTS: PD-1, PD-L1 and CD8 mRNA levels were significantly lower in tumor tissue than in normal tissue (p < 0.0001, p < 0.05, and p < 0.0001). GC patients with low PD-1, PD-L1 and CD8 mRNA levels had significantly poorer overall survival (OS) than those with high PD-1, PD-L1 and CD8 mRNA levels, respectively (p < 0.001, p < 0.01 and p < 0.05). Low PD-1, PD-L1 and CD8 mRNA levels were more significantly associated with poor prognosis in undifferentiated-type GC patients than in differentiated-type GC patients (PD-1: differentiated p = 0.0071 vs. undifferentiated p = 0.0024; PD-L1: p = 0.6527 vs. p < 0.0001; CD8: p = 0.4465 vs. p < 0.05). Multivariate analysis showed that lymph node metastasis, peritoneal dissemination, distant metastasis, low PD-1 mRNA levels and low CD8 mRNA levels were independent prognostic factors for worse OS (low PD-1 mRNA level: OR 2.16, 95% CI 1.10-4.58, p < 0.05; low CD8 mRNA level: OR 2.55, 95% CI 1.12-6.90, p < 0.05). PD-1 and PD-L1 mRNA levels in immune cells were significantly associated with PD-1 and PD-L1 protein levels (both p < 0.05), respectively. CONCLUSIONS: PD-1, PD-L1 and CD8 mRNA levels may reflect antitumor immunity in GC, and low PD-1 and CD8 mRNA levels are potential predictive biomarkers for poor prognosis in GC patients who underwent surgery.


Assuntos
Antígeno B7-H1/genética , Antígenos CD8/genética , Expressão Gênica/imunologia , Receptor de Morte Celular Programada 1/genética , Neoplasias Gástricas/genética , Microambiente Tumoral/imunologia , Idoso , Antígeno B7-H1/metabolismo , Biomarcadores Tumorais/genética , Linfócitos T CD8-Positivos/imunologia , Feminino , Humanos , Metástase Linfática/genética , Masculino , Pessoa de Meia-Idade , Prognóstico , Receptor de Morte Celular Programada 1/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real
8.
PLoS One ; 15(5): e0232884, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32384121

RESUMO

BACKGROUND: Obliterative bronchiolitis (OB) is a known issue during minor histocompatibility antigen (mHA) disparity during lung transplantation. This study evaluated gene expression in a murine orthotropic lung transplantation model using microarray analysis. METHODS: Left lungs from C57BL/10(H-2b) donor mice were transplanted into mHA-mismatched C57BL/6(H-2b) recipient mice. Three groups (OB, non-OB, and sham controls) were confirmed pathologically and analyzed. Gene expression changes in the lung grafts were determined by microarray and immunohistochemical staining, and genes were verified by quantitative PCR in the lungs and mediastinal lymph nodes (LNs). RESULTS: A total of 1343 genes were upregulated in the OB lungs compared to the sham group. Significant upregulation was observed for genes related to innate, e.g. Tlr2 and CCL3 and adaptive immunity, e.g. H2-ab1 and Il-21. Positive labeling for MHC class II antigen was observed in the bronchial epithelium of OB accompanied with B cells. We found increased Tlr2, Ccl3, H2-ab1, Il-21, Ighg3, Ifng, and Pdcd1 mRNA expression in the OB lung, and increased Il-21, Ighg3, and Pdcd1 expression in the OB LNs. CONCLUSIONS: Adaptive and innate immune reactions were involved in OB after lung transplantation, and genetic examination of related genes could be used for detection of OB.


Assuntos
Bronquiolite/etiologia , Bronquiolite/imunologia , Transplante de Pulmão , Imunidade Adaptativa , Animais , Bronquiolite/genética , Bronquiolite/patologia , Modelos Animais de Doenças , Expressão Gênica/imunologia , Perfilação da Expressão Gênica , Imunidade Inata , Pulmão/imunologia , Pulmão/patologia , Pulmão/cirurgia , Linfonodos/imunologia , Masculino , Camundongos Endogâmicos C57BL , Antígenos de Histocompatibilidade Menor , RNA Mensageiro/metabolismo , Organismos Livres de Patógenos Específicos , Baço/imunologia , Transcriptoma , Imunologia de Transplantes
9.
Artigo em Inglês | MEDLINE | ID: mdl-32401956

RESUMO

Paracoccidioides species cause paracoccidioidomycosis (PCM), a systemic mycosis highly prevalent in Brazil. Therapy of PCM has some issues that make studies for new therapeutic and vaccine targets relevant, such as the P. brasiliensis 60-kDa-heat-shock protein (PbHsp60), an immunogenic antigen that induces protection in experimental mice infection. Here, we investigated the relative expression of mRNA for PbHsp60 in P. brasiliensis in the different morphotypes of P. brasiliensis and in morphological transition phases. In addition, antibodies to rPbHsp60 were produced and used to analyze the location of PbHsp60 in yeast and hyphae by electron microscopy. The analyses showed a substantial increase in the relative amounts of HSP60 mRNA in yeast when compared to mycelium and an intermediate expression in transitional forms. Regarding the cell location, immunoelectron microscopy analysis revealed that PbHsp60 is within the cell wall. These observations suggest that this protein may be involved in the maintenance of the cell wall integrity and the interaction with the host for colonization, infection and pathogenesis.


Assuntos
Chaperonina 60/imunologia , Paracoccidioides/imunologia , RNA Mensageiro/imunologia , Animais , Antígenos de Fungos/imunologia , Expressão Gênica/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Paracoccidioides/patogenicidade , Reação em Cadeia da Polimerase
10.
PLoS One ; 15(4): e0231003, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32240238

RESUMO

Systematic interrogation of tumor-infiltrating immune cells (TIICs) is key to the prediction of clinical outcome and development of immunotherapies. However, little is known about the TIICs of hepatocellular carcinoma (HCC) and its impact on the prognosis of patients and potential for immunotherapy. We applied CIBERSORT of 1090 tumors to infer the infiltration of 22 subsets of TIICs using gene expression data. Unsupervised clustering analysis by 22 TIICs revealed 4 clusters of tumors, mainly defined by macrophages and T cells, with distinct prognosis and associations with immune checkpoint molecules, including PD-1, CD274, CTLA-4, LAG-3 and IFNG. We found tumors with decreased number of M1 macrophages or increased regulatory T cells were associated with poor prognosis. Based on the multivariate Cox analysis, a nomogram was also established for clinical application. In conclusion, composition of the TIICs in HCC was quite different, which is an important determinant of prognosis with great potential to identify candidates for immunotherapy.


Assuntos
Carcinoma Hepatocelular/imunologia , Neoplasias Hepáticas/imunologia , Linfócitos do Interstício Tumoral/imunologia , Linfócitos T Reguladores/imunologia , Antígeno B7-H1/imunologia , Antígeno CTLA-4/imunologia , Feminino , Expressão Gênica/imunologia , Humanos , Imunoterapia/métodos , Macrófagos/imunologia , Masculino , Pessoa de Meia-Idade , Prognóstico , Receptor de Morte Celular Programada 1/imunologia
11.
Vet Parasitol ; 280: 109063, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32151890

RESUMO

The proliferation of Demodex mites is mainly controlled by host immunity; however, the precised mechanism of host-mite interplay and host immune response in the cutaneous microenvironment of dogs with generalized demodicosis (GD) are not yet established. In the present study, we envisaged the alterations in the expression of toll-like receptors (TLRs) and immuno-regulatory cytokine gene in the skin lesions and peripheral blood mononuclear cells (PBMCs) of dogs with GD. The expression of TLR2, TLR6, IFN-γ, TGF-ß and IL-10 genes in the skin lesions and PBMCs of 15 dogs with GD was quantified by qRT-PCR. Compared to healthy dogs, significantly elevated expression of TLR2 (P = 0.048), TGF-ß (P = 0.04) and IL-10 (P = 0.012) were found in the PBMCs of dogs with GD. Conversely, there was significantly reduced expression of TLR6 gene (P = 0.021) in the PBMCs of these dogs. The infested dogs also revealed significantly elevated expression of TLR2 gene (P = 0.034) in the skin lesions, while, the expression of the TLR6 gene was found to be significantly (P = 0.004) reduced. Interestingly, significant alterations in TGF-ß (P = 0.105) and IL-10 (P = 0.162) genes expression were not observed in the skin lesions of diseased dogs. Our findings suggest that Demodex mites contribute to a different systemic and cutaneous immune response in dogs for their proliferation, and consequently the development of GD. Therefore, Demodex mites might be inducing the immunosuppression through activating the systemic over-expression of immunosuppressive cytokines; however, in the cutaneous lesions, the expression of immunosuppressive cytokines remained unaltered. Both systemic and local over-expression of TLR2 and reduced expression of TLR6 genes might be responsible for the inflammatory signs of canine demodicosis and helping to the mite to escape the host immunity.


Assuntos
Citocinas/genética , Doenças do Cão/genética , Expressão Gênica/imunologia , Infestações por Ácaros/veterinária , Receptores Toll-Like/genética , Animais , Citocinas/imunologia , Doenças do Cão/imunologia , Cães , Infestações por Ácaros/genética , Infestações por Ácaros/imunologia , Dermatopatias Parasitárias/genética , Dermatopatias Parasitárias/imunologia , Dermatopatias Parasitárias/veterinária , Receptores Toll-Like/imunologia
13.
J Immunol ; 204(7): 1760-1769, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32094206

RESUMO

Lymphocyte lineage specification and commitment requires the activation of lineage-specific genes and repression of alternative lineage genes, respectively. The mechanisms governing alternative lineage gene repression and commitment in lymphocytes are largely unknown. In this study, we demonstrate that Ezh2, which represses gene expression through methylation of histone 3 lysine 27, was essential for repression of numerous genes, including genes encoding innate lymphocyte transcription factors, specifically in murine B lymphocyte progenitors, but these cells maintained their B lymphocyte identity. However, adult Ezh2-deficient B lymphocytes expressed Lin28b, which encodes an RNA-binding protein associated with fetal hematopoietic gene expression programs, and these cells acquired a fetal B-1 lymphocyte phenotype in vitro and in vivo. Therefore, Ezh2 coordinates the repression of multiple gene programs in B lymphocytes and maintains the adult B-2 cell fate.


Assuntos
Linfócitos B/imunologia , Proteína Potenciadora do Homólogo 2 de Zeste/imunologia , Imunidade Inata/imunologia , Células Precursoras de Linfócitos B/imunologia , Transcrição Genética/imunologia , Animais , Diferenciação Celular/imunologia , Linhagem da Célula/imunologia , Expressão Gênica/imunologia , Histonas/imunologia , Metilação , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Ligação a RNA/imunologia , Fatores de Transcrição/imunologia
14.
Biochem Pharmacol ; 175: 113867, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32088260

RESUMO

Hepcidin peptide is crucial in the regulation of systemic iron availability controlling its uptake from the diet and its release from the body storage tissues. Hepcidin dysregulation causes different human disorders ranging from iron overload (e.g. hemochromatosis) to iron deficiency (e.g. anemia). Hepcidin excess is common in the Anemia of Chronic Diseases or Anemia of Inflammation and in the genetic form of anemia named IRIDA; the pharmacological downregulation of hepcidin in these disorders could improve the anemia. Commercial heparins were shown to be strong inhibitors of hepcidin expression, by interfering with BMP6/SMAD pathway. The non-anti-coagulant heparins, modified to abolish the anti-thrombin binding site, were equally potent and could be used to improve iron status. To perform its anti-hepcidin activity heparin needs 2O- and 6O-sulfation and an average molecular weight (MW) up to 4000-8000 Dalton, depending on the sulfation level. The pentosane polysulfate (PPS), which shares with heparin a high degree of sulfation, is a compound with low anti-coagulant activity that is already in use for pharmaceutical treatment. In the present work we analyzed the anti-hepcidin activity of PPS in vitro and in vivo. We found that it acts as a strong inhibitor of hepcidin expression in HepG2 cells with an effect already visible after 2-3 h of treatment. It also suppressed hepcidin in mice in a dose dependent manner after 3 h and with a significant redistribution of systemic iron without evident side effects. PPS is also able to abolish the LPS dependent hepcidin upregulation similarly to that showed for heparin derivatives. These results suggest PPS as an interesting compound to control hepcidin in vivo.


Assuntos
Expressão Gênica/efeitos dos fármacos , Hepcidinas/antagonistas & inibidores , Poliéster Sulfúrico de Pentosana/farmacologia , Administração Oral , Animais , Expressão Gênica/imunologia , Células Hep G2 , Hepcidinas/sangue , Hepcidinas/genética , Humanos , Injeções Subcutâneas , Fígado/efeitos dos fármacos , Fígado/imunologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Poliéster Sulfúrico de Pentosana/administração & dosagem
15.
Mol Immunol ; 120: 52-60, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32065987

RESUMO

Avian infectious bronchitis (IB) is an acute, highly infectious and contagious viral disease of chickens caused by avian infectious bronchitis virus (IBV) belonging to the genus Coronavirus and family Coronaviridae. It can affect all age groups of birds. The toll-like receptors (TLRs) are a major class of innate immune pattern recognition receptors that have a key role in immune response and defense against various infections.The TLRs are essential for initiation of innate immune responses and in the development of adaptive immune responses. An in ovo model was employed to study the antiviral activity of TLR ligands (Pam3CSK4, LPS and CpG ODN) on replication of IBV. It was hypothesized that optimum dose and specific timing of TLR ligands may reduce viral load of IBV in specific pathogen free (SPF) embryonated chicken eggs (ECEs). Further, the mechanism involved in the TLR-mediated antiviral response in chorioallantoic membrane (CAM) of ECEs was investigated. The ECEs of 9-11 days old were treated with different doses (high, intermediate and low) of TLR-2 (Pam3CSK4), TLR-4 (LPS) and TLR-21 (CpG ODN) ligands. In addition, to know the timing of TLR ligand treatment, six time intervals were analyzed viz. 36, 24 and 12 h prior to infection, time of infection (co-administration of TLR ligands and avian IBV) and 12 and 24 h post-IBV infection. For studying the relative expression of immuno-stimulatory genes (IFN-α, IFN-ß, IFN-γ, IL-1ß, iNOS and OAS) in CAM, TLR ligands were administered through intra-allantoicroute and CAM were collected at 4, 8 and 16 h post treatment. The results demonstrated that intermediate dose of all the three TLR ligands significantly reduced virus titers and used in the present study. However, the LPS reduced virus titer pre- and post-IBV infection but Pam3CSK4 and CpG ODN reduced only pre-IBV infection. Further analysis showed that TLR ligands induced IFN-γ, IL-1ß and IFN stimulated genes viz. iNOS and OAS genes in CAM. The present study pointed towards the novel opportunities for rational design of LPS as immuno-stimulatory agent in chickens with reference to IBV. It may be speculated that in ovo administration of these TLR ligands may enhance resistance against viral infection in neonatal chicken and may contribute towards the development of more effective and safer vaccines including in ovo vaccines.


Assuntos
Vírus da Bronquite Infecciosa/imunologia , Receptores Toll-Like/agonistas , Adjuvantes Imunológicos/farmacologia , Animais , Antivirais/farmacologia , Proteínas Aviárias/agonistas , Proteínas Aviárias/imunologia , Embrião de Galinha , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/veterinária , Infecções por Coronavirus/virologia , Citocinas/metabolismo , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/imunologia , Imunidade Inata , Vírus da Bronquite Infecciosa/patogenicidade , Vírus da Bronquite Infecciosa/fisiologia , Ligantes , Lipopeptídeos/farmacologia , Lipopolissacarídeos/farmacologia , Oligodesoxirribonucleotídeos/farmacologia , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/prevenção & controle , Doenças das Aves Domésticas/virologia , Receptores Toll-Like/imunologia , Carga Viral/efeitos dos fármacos , Carga Viral/imunologia , Replicação Viral/efeitos dos fármacos , Replicação Viral/imunologia
16.
Immunohorizons ; 4(1): 14-22, 2020 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-31974109

RESUMO

Mucosal-associated invariant T (MAIT) cells acquire effector function in response to proinflammatory signals, which synergize with TCR-mediated signals. We asked if cell-intrinsic regulatory mechanisms exist to curtail MAIT cell effector function akin to the activation-induced expression of inhibitory receptors by conventional T cells. We examined human MAIT cells from blood and oral mucosal tissues by RNA sequencing and found differential expression of immunoregulatory genes, including CTLA-4, by MAIT cells isolated from tissue. Using an ex vivo experimental setup, we demonstrate that inflammatory cytokines were sufficient to induce CTLA-4 expression on the MAIT cell surface in the absence of TCR signals. Even brief exposure to the cytokines IL-12, IL-15, and IL-18 was sufficient for sustained CTLA-4 expression by MAIT cells. These data suggest that control of CTLA-4 expression is fundamentally different between MAIT cells and conventional T cells. We propose that this mechanism serves to limit MAIT cell-mediated tissue damage.


Assuntos
Antígenos de Superfície/imunologia , Linfócitos T CD8-Positivos/imunologia , Antígeno CTLA-4/imunologia , Citocinas/imunologia , Células T Invariáveis Associadas à Mucosa/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Sangue/imunologia , Feminino , Expressão Gênica/imunologia , Humanos , Inflamação/genética , Masculino , Pessoa de Meia-Idade , Membrana Mucosa/imunologia , Receptores de Antígenos de Linfócitos T/imunologia
17.
PLoS One ; 15(1): e0227993, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31990927

RESUMO

OBJECTIVES: The regulatory mechanisms affecting the modulation of the immune system accompanying the progressive effort to exhaustion, particularly associated with T cells, are not fully understood. We analysed the impact of two progressive effort protocols on T helper (Th) cell distribution and selected cytokines. METHODS: Sixty-two male soccer players with a median age of 17 (16-29) years performed different protocols for progressive exercise until exhaustion: YO-YO (YYRL1) and Beep. Blood samples for all analyses were taken three times: at baseline, post-effort, and in recovery. RESULTS: The percentage of Th1 cells increased post-effort and in recovery. The post-effort percentage of Th1 cells was higher in the Beep group compared to the YYRL1 group. Significant post-effort increase in Th17 cells was observed in both groups. The post-effort percentage of regulatory T cells (Treg) increased in the Beep group. An increased post-effort concentration of IL-2, IL-6, IL-8 and IFN-γ in both groups was observed. Post-effort TNF-α and IL-10 levels were higher than baseline in the YYRL1 group, while the post-effort IL-17A concentration was lower than baseline only in the Beep group. The recovery IL-2, IL-4, TNF-α and IFN-γ levels were higher than baseline in the YYRL1 group. The recovery IL-4, IL-6, IL-8, TNF-α and IFN-γ values were higher than baseline in the Beep group. CONCLUSION: The molecular patterns related to cytokine secretion are not the same between different protocols for progressive effort. It seems that Treg cells are probably the key cells responsible for silencing the inflammation and enhancing anti-inflammatory pathways.


Assuntos
Esforço Físico/imunologia , Futebol/fisiologia , Linfócitos T Reguladores/imunologia , Células Th1/imunologia , Células Th17/imunologia , Células Th2/imunologia , Adolescente , Adulto , Atletas , Expressão Gênica/imunologia , Humanos , Interferon gama/genética , Interferon gama/imunologia , Interleucina-10/genética , Interleucina-10/imunologia , Interleucina-17/genética , Interleucina-17/imunologia , Interleucina-2/genética , Interleucina-2/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Interleucina-8/genética , Interleucina-8/imunologia , Masculino , Esforço Físico/genética , Recuperação de Função Fisiológica/imunologia , Linfócitos T Reguladores/citologia , Células Th1/citologia , Células Th17/citologia , Células Th2/citologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia
18.
Fish Shellfish Immunol ; 97: 656-668, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31891812

RESUMO

AquAdvantage Salmon (growth hormone transgenic female triploid Atlantic salmon) are a faster-growing alternative to conventional farmed diploid Atlantic salmon. To investigate optimal rearing conditions for their commercial production, a laboratory study was conducted in a freshwater recirculating aquaculture system (RAS) to examine the effect of rearing temperature (10.5 °C, 13.5 °C, 16.5 °C) on their antiviral immune and stress responses. When each temperature treatment group reached an average weight of 800 g, a subset of fish were intraperitoneally injected with either polyriboinosinic polyribocytidylic acid (pIC, a viral mimic) or an equal volume of sterile phosphate-buffered saline (PBS). Blood and head kidney samples were collected before injection and 6, 24 and 48 h post-injection (hpi). Transcript abundance of 7 antiviral biomarker genes (tlr3, lgp2, stat1b, isg15a, rsad2, mxb, ifng) was measured by real-time quantitative polymerase chain reaction (qPCR) on head kidney RNA samples. Plasma cortisol levels from blood samples collected pre-injection and from pIC and PBS groups at 24 hpi were quantified by ELISA. While rearing temperature and treatment did not significantly affect circulating cortisol, all genes tested were significantly upregulated by pIC at all three temperatures (except for tlr3, which was only upregulated in the 10.5 °C treatment). Target gene activation was generally observed at 24 hpi, with most transcript levels decreasing by 48 hpi in pIC-injected fish. Although a high amount of biological variability in response to pIC was evident across all treatments, rearing temperature significantly influenced transcript abundance and/or fold-changes comparing time- and temperature-matched pIC- and PBS-injected fish for several genes (tlr3, lgp2, stat1b, isg15a, rsad2 and ifng) at 24 hpi. As an example, significantly higher fold-changes of rsad2, isg15a and ifng were found in fish reared at 10.5 °C when compared to 16.5 °C. Multivariate analysis confirmed that rearing temperature modulated antiviral immune response. The present experiment provides novel insight into the relationship between rearing temperature and innate antiviral immune response in AquAdvantage Salmon.


Assuntos
Hormônio do Crescimento/imunologia , Imunidade Inata , Salmo salar/imunologia , Temperatura , Triploidia , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/imunologia , Aquicultura/métodos , Feminino , Expressão Gênica/imunologia , Hormônio do Crescimento/genética , Indutores de Interferon/administração & dosagem , Indutores de Interferon/imunologia , Poli I-C/administração & dosagem , Poli I-C/imunologia , Salmo salar/genética , Estresse Fisiológico/efeitos dos fármacos , Viroses/imunologia , Viroses/veterinária
19.
J Vet Sci ; 21(1): e15, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31940694

RESUMO

The present study evaluated the anti-inflammatory effect of horse oil in 2, 4-dinitrochlorobenzene (DNCB)-treated BALB/c mice. After the application of DNCB, the mice showed atopic dermatitis symptoms, including severe erythema, hemorrhage, and erosion, whereas those symptoms were alleviated by treatment with horse oil. To explain the anti-dermatitis effect of horse oil, the gene expression levels in the healing process in dorsal skin were observed using a cDNA microarray. The cDNA microarray analysis revealed that the expression levels of 30 genes related to the inflammation, including Ccr1, Ccr2, Ccl20, Anxa1, and Hc genes, were up-regulated (higher than 2.0-fold) in the DNCB group compared to the levels in the control group, whereas the levels were restored to the control level in the DNCB + horse oil-treated group. In contrast, the gene expression levels of 28 genes related to inflammation, including chemokine genes Ccl5, Ccl7, Ccl8, Cxcl10, and Cxcl13 genes, were down-regulated (lower than 0.5-fold) in the DNCB group compared to the levels in the control group, whereas the levels were restored to the control level in the DNCB + horse oil-treated group. Overall, the results show that horse oil restores the expression levels of genes related to inflammation that were perturbed by DNCB treatment.


Assuntos
Anti-Inflamatórios/farmacologia , Dermatite Atópica/tratamento farmacológico , Dinitroclorobenzeno/farmacologia , Expressão Gênica/imunologia , Irritantes/farmacologia , Sebo , Administração Tópica , Animais , Dermatite Atópica/induzido quimicamente , Ácidos Graxos Insaturados/administração & dosagem , Ácidos Graxos Insaturados/farmacologia , Cavalos , Masculino , Camundongos , Camundongos Endogâmicos BALB C
20.
Cancer Immunol Immunother ; 69(4): 593-610, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31982940

RESUMO

Despite recent progress in the understanding of γδ T cells' roles and functions, their interaction with αß T cells still remains to be elucidated. In this study, we sought to clarify what precisely endows peripheral Vδ2+ T cells with immunosuppressive function on autologous αß T cells. We found that negatively freshly isolated Vδ2+ T cells do not exhibit suppressive behavior, even after stimulation with IL-12/IL-18/IL-15 or the sheer contact with butyrophilin-3A1-expressing tumor cell lines (U251 or SK-Mel-28). On the other hand, Vδ2+ T cells positively isolated through TCR crosslinking or after prolonged stimulation with isopentenyl pyrophosphate (IPP) mediate strong inhibitory effects on αß T cell proliferation. Stimulation with IPP in the presence of IL-15 induces the most robust suppressive phenotype of Vδ2+ T cells. This indicates that Vδ2+ T cells' suppressive activity is dependent on a TCR signal and that the degree of suppression correlates with its strength. Vδ2+ T cell immunosuppression does not correlate with their Foxp3 expression but rather with their PD-L1 protein expression, evidenced by the massive reduction of suppressive activity when using a blocking antibody. In conclusion, pharmacologic stimulation of Vδ2+ T cells via the Vδ2 TCR for activation and expansion induces Vδ2+ T cells' potent killer activity while simultaneously licensing them to suppress αß T cell responses. Taken together, the study is a further step to understand-in more detail-the suppressive activity of Vδ2+ γδ T cells.


Assuntos
Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/imunologia , Subpopulações de Linfócitos T/imunologia , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Antígeno B7-H1/genética , Antígeno B7-H1/imunologia , Antígeno B7-H1/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/imunologia , Hemiterpenos/farmacologia , Humanos , Tolerância Imunológica/efeitos dos fármacos , Tolerância Imunológica/genética , Tolerância Imunológica/imunologia , Interleucina-15/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Compostos Organofosforados/farmacologia , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Transdução de Sinais/efeitos dos fármacos , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/metabolismo
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