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1.
Anticancer Res ; 40(11): 6473-6484, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33109586

RESUMO

BACKGROUND/AIM: Glioblastoma multiforme (GBM) is an intractable tumor that has a very poor prognosis despite intensive treatment with temozolomide plus radiotherapy. PATIENTS AND METHODS: Sixteen newly diagnosed patients with high-grade gliomas were enrolled in a phase II study of the α-type-1 DC vaccine. Briefly, DCs obtained from the culture of enriched monocytes in the presence of a cytokine cocktail, were pulsed with a cocktail of 5 synthetic peptides and cryopreserved until injection into patients. RESULTS: The amount of IL-12 produced by activated DCs was higher than that previously reported. Among 15 evaluable patients, 10 showed positive CTL responses to any peptides in an ELISPOT assay. After 6 years of observation, five patients were still alive, and two of these patients were relapse-free. Moreover, a significant survival-prolonging effect was verified in DC-treated glioma patients. CONCLUSION: Peptide-cocktail-pulsed α-type-1 DC vaccines have a potential therapeutic effect on survival when used in combination with the standard regimen, which is partly based on IL-12-IFN-γ-mediated T-cell activation.


Assuntos
Vacinas Anticâncer/administração & dosagem , Células Dendríticas/imunologia , Glioma/tratamento farmacológico , Recidiva Local de Neoplasia/tratamento farmacológico , Adulto , Vacinas Anticâncer/imunologia , Polaridade Celular/imunologia , Intervalo Livre de Doença , Feminino , Glioma/imunologia , Glioma/patologia , Humanos , Interferon gama/genética , Interleucina-12/genética , Masculino , Pessoa de Meia-Idade , Monócitos/imunologia , Monócitos/patologia , Recidiva Local de Neoplasia/imunologia , Recidiva Local de Neoplasia/patologia , Linfócitos T/imunologia , Vacinação/métodos
2.
Mol Immunol ; 126: 120-128, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32823236

RESUMO

The interleukin 12 (IL-12) family of cytokines regulates T cell functions and is key for the orchestration of immune responses. Each heterodimeric IL-12 family member is a glycoprotein. However, the impact of glycosylation on biogenesis and function of the different family members has remained incompletely defined. Here, we identify glycosylation sites within human IL-12 family subunits that become modified upon secretion. Building on these insights, we show that glycosylation is dispensable for secretion of human IL-12 family cytokines except for IL-35. Furthermore, our data show that glycosylation differentially influences IL-12 family cytokine functionality, with IL-27 being most strongly affected. Taken together, our study provides a comprehensive analysis of how glycosylation affects biogenesis and function of a key human cytokine family and provides the basis for selectively modulating their secretion via targeting glycosylation.


Assuntos
Interleucina-12/metabolismo , Interleucinas/metabolismo , Glicosilação , Células HEK293 , Humanos , Interleucina-12/genética , Interleucina-12/imunologia , Interleucina-23/genética , Interleucina-23/imunologia , Interleucina-23/metabolismo , Interleucinas/genética , Interleucinas/imunologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
3.
Nat Commun ; 11(1): 3366, 2020 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-32632165

RESUMO

CD4+ T lymphocytes consist of naïve, antigen-specific memory, and memory-phenotype (MP) cell compartments at homeostasis. We recently showed that MP cells exert innate-like effector function during host defense, but whether MP CD4+ T cells are functionally heterogeneous and, if so, what signals specify the differentiation of MP cell subpopulations under homeostatic conditions is still unclear. Here we characterize MP lymphocytes as consisting of T-bethigh, T-betlow, and T-bet- subsets, with innate, Th1-like effector activity exclusively associated with T-bethigh cells. We further show that the latter population depends on IL-12 produced by CD8α+ type 1 dendritic cells (DC1) for its differentiation. Finally, our data demonstrate that this tonic IL-12 production requires TLR-MyD88 signaling independent of foreign agonists, and is further enhanced by CD40-CD40L interactions between DC1 and CD4+ T lymphocytes. We propose that optimal differentiation of T-bethigh MP lymphocytes at homeostasis is driven by self-recognition signals at both the DC and Tcell levels.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular/imunologia , Homeostase/imunologia , Memória Imunológica/imunologia , Proteínas com Domínio T/imunologia , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Antígenos CD40/imunologia , Antígenos CD40/metabolismo , Ligante de CD40/genética , Ligante de CD40/imunologia , Ligante de CD40/metabolismo , Antígenos CD8/imunologia , Antígenos CD8/metabolismo , Comunicação Celular/imunologia , Células Dendríticas/citologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Interleucina-12/genética , Interleucina-12/imunologia , Interleucina-12/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Transdução de Sinais/imunologia , Proteínas com Domínio T/metabolismo , Células Th1/citologia , Células Th1/imunologia , Células Th1/metabolismo
4.
BMC Med Genet ; 21(1): 143, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620160

RESUMO

BACKGROUND: Intervertebral disc degeneration (IVDD) is an age-related degenerative disease, presenting with low back pain or radicular pain. The inflammatory changes would occur in discs in the process of IVDD. Therefore, the inflammatory and anti-inflammatory cytokines, as well as their respective genes, have been proposed to play roles in pathophysiology of disease. This study has been conducted to elucidate the role of IL-2, IL-12, and IFN-γ single nucleotide polymorphisms (SNP) in this disease. METHOD: Seventy-six patients who were diagnosed with IVDD and 140 healthy controls who complied with eligibility criteria were included. A total volume of 5 cc peripheral blood was obtained from each participant to investigate the IL-2 + 166G/T, IL-2 -330G/T, IL-12 - 1188A/C, and IFN-γ +847A/T SNPs through PCR-SSP method. RESULTS: The 'TG' and 'TT' genotypes of IL-2 - 330G/T polymorphism were significantly more common among patients and healthy controls respectively. The 'GT' and 'TT' haplotypes of IL-2 (comprised of -330G/T, and + 166G/T SNPs) were also more common among patients and controls respectively. CONCLUSION: This study indicated the significant role of IL-2 genotypes and haplotypes in IVDD. These SNPs were differently distributed in patients and controls. Therefore, alteration in the structure of IL-2 gene could play an important role in pathophysiology of IVDD.


Assuntos
Estudos de Associação Genética , Interferon gama/genética , Interleucina-12/genética , Interleucina-2/genética , Adulto , Estudos de Casos e Controles , Avaliação da Deficiência , Frequência do Gene/genética , Predisposição Genética para Doença , Humanos , Degeneração do Disco Intervertebral/genética , Irã (Geográfico) , Polimorfismo de Nucleotídeo Único , Escala Visual Analógica
5.
PLoS Pathog ; 16(5): e1008572, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32413093

RESUMO

The apicomplexan Toxoplasma gondii induces strong protective immunity dependent upon recognition by Toll-like receptors (TLR)11 and 12 operating in conjunction with MyD88 in the murine host. However, TLR11 and 12 proteins are not present in humans, inspiring us to investigate MyD88-independent pathways of resistance. Using bicistronic IL-12-YFP reporter mice on MyD88+/+ and MyD88-/- genetic backgrounds, we show that CD11c+MHCII+F4/80- dendritic cells, F4/80+ macrophages, and Ly6G+ neutrophils were the dominant cellular sources of IL-12 in both wild type and MyD88 deficient mice after parasite challenge. Parasite dense granule protein GRA24 induces p38 MAPK activation and subsequent IL-12 production in host macrophages. We show that Toxoplasma triggers an early and late p38 MAPK phosphorylation response in MyD88+/+ and MyD88-/- bone marrow-derived macrophages. Using the uracil auxotrophic Type I T. gondii strain cps1-1, we demonstrate that the late response does not require active parasite proliferation, but strictly depends upon GRA24. By i. p. inoculation with cps1-1 and cps1-1:Δgra24, we identified unique subsets of chemokines and cytokines that were up and downregulated by GRA24. Finally, we demonstrate that cps1-1 triggers a strong host-protective GRA24-dependent Th1 response in the absence of MyD88. Our data identify GRA24 as a major mediator of p38 MAPK activation, IL-12 induction and protective immunity that operates independently of the TLR/MyD88 cascade.


Assuntos
Interleucina-12/imunologia , Sistema de Sinalização das MAP Quinases/imunologia , Macrófagos/imunologia , Fator 88 de Diferenciação Mieloide/imunologia , Proteínas de Protozoários/imunologia , Toxoplasma/imunologia , Toxoplasmose/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/imunologia , Animais , Ativação Enzimática/genética , Ativação Enzimática/imunologia , Interleucina-12/genética , Sistema de Sinalização das MAP Quinases/genética , Macrófagos/parasitologia , Macrófagos/patologia , Camundongos , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Proteínas de Protozoários/genética , Toxoplasma/genética , Toxoplasmose/genética , Toxoplasmose/patologia , Proteínas Quinases p38 Ativadas por Mitógeno/genética
6.
PLoS One ; 15(5): e0233096, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32421725

RESUMO

Congenital cytomegalovirus (cCMV) infection is the most common intrauterine infection. A non-specific immune response is the first line of host defense mechanism against human cytomegalovirus (HCMV). There is limited data on associations between Single Nucleotide Polymorphisms (SNPs) in genes involving innate immunity and the risk and clinical manifestation of cCMV infection. The aim of the study was to investigate association between selected SNPs in genes encoding cytokines and cytokine receptors, and predisposition to cCMV infection including symptomatic course of disease and symptoms. A panel of eight SNPs: IL1B rs16944, IL12B rs3212227, IL28B rs12979860, CCL2 rs1024611, DC-SIGN rs735240, TLR2 rs5743708, TLR4 rs4986791, TLR9 rs352140 was analyzed in 233 infants (92 cCMV-infected and 141 healthy controls). Associations between genotyped SNPs and predisposition to cCMV infection and symptoms were analyzed. The association analysis was performed using SNPStats software. No statistically significant association was found between any genotyped SNPs and predisposition to cCMV infection and symptomatic course of disease. In relation to particular symptoms, polymorphism of IL12B rs3212227 was linked to decreased risk of prematurity (OR = 0.37;95%CI,0.14-0.98;p = 0.025), while polymorphism of IL1B rs16944 was linked to reduced risk of splenomegaly (OR = 0.36;95%CI,0.14-0.98; p = 0.034) in infants with cCMV infection. An increased risk of thrombocytopenia was associated with IL28B rs12979860 polymorphism (OR = 2.55;95%CI,1.03-6.32;p = 0.042), while hepatitis was associated with SNP of TLR4rs4986791 (OR = 7.80;95%CI,1.49-40,81; p = 0.024). This is the first study to demonstrate four new associations between SNPs in selected genes (IL1B, IL12B, IL28B, TLR4) and particular symptoms in cCMV disease. Further studies on the role of SNPs in the pathogenesis of cCMV infection and incorporation of selected SNPs in the clinical practice might be considered in the future.


Assuntos
Infecções por Citomegalovirus/genética , Interferons/genética , Interleucina-12/genética , Interleucina-1beta/genética , Polimorfismo de Nucleotídeo Único/genética , Receptor 4 Toll-Like/genética , Adulto , Infecções por Citomegalovirus/virologia , Feminino , Frequência do Gene/genética , Genótipo , Técnicas de Genotipagem , Humanos , Masculino , Trombocitopenia/genética , Trombocitopenia/virologia
7.
Sci Rep ; 10(1): 7823, 2020 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-32385413

RESUMO

This study investigates the role of NLRP3 inflammasome and its main effector Caspase-1 in inflammation and alveolar bone resorption associated with periodontitis. Heat-killed Aggregatibacter actinomycetemcomitans (Aa) was injected 3x/week (4 weeks) into gingival tissues of wild-type (WT), Nlrp3-KO and Caspase1-KO mice. Bone resorption was measured by µCT and osteoclast number was determined by tartrate-resistant acid phosphatase (TRAP) staining. Inflammation was assessed histologically (H/E staining and immunofluorescence of CD45 and Ly6G). In vitro studies determined the influence of Nlrp3 and Caspase-1 in Rankl-induced osteoclast differentiation and activity and on LPS-induced expression of inflammation-associated genes. Bone resorption was significantly reduced in Casp1-KO but not in Nlrp3-KO mice. Casp1-KO mice had increased in osteoclast numbers, whereas the inflammatory infiltrate or on gene expression were similar to those of WT and Nlrp3-KO mice. Strikingly, osteoclasts differentiated from Nlrp3-deficient macrophages had increased resorbing activity in vitro. LPS-induced expression of Il-10, Il-12 and Tnf-α was significantly reduced in Nlrp3- and Casp1-deficient macrophages. As an inceptive study, these results suggest that Nlrp3 inflammasome does not play a significant role in inflammation and bone resorption in vivo and that Caspase-1 has a pro-resorptive role in experimental periodontal disease.


Assuntos
Perda do Osso Alveolar/genética , Caspase 1/genética , Inflamação/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Periodontite/genética , Aggregatibacter actinomycetemcomitans , Perda do Osso Alveolar/microbiologia , Perda do Osso Alveolar/patologia , Animais , Diferenciação Celular/efeitos dos fármacos , Modelos Animais de Doenças , Gengiva/crescimento & desenvolvimento , Gengiva/microbiologia , Humanos , Inflamação/microbiologia , Inflamação/patologia , Interleucina-10/genética , Interleucina-12/genética , Camundongos , Camundongos Knockout , Osteoclastos/microbiologia , Osteoclastos/patologia , Periodontite/microbiologia , Periodontite/patologia , Ligante RANK/genética , Fator de Necrose Tumoral alfa/genética
8.
Oncogene ; 39(16): 3292-3304, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32071396

RESUMO

GPR81 is a G-protein-coupled receptor for lactate, which is upregulated in breast cancer and plays an autocrine role to promote tumor growth by tumor cell-derived lactate. Here we asked whether lactate has any paracrine role via activation of GPR81 in cells present in tumor microenvironment to help tumor growth. First, we showed that deletion of Gpr81 suppresses breast cancer growth in a constitutive breast cancer mouse model (MMTV-PyMT-Tg). We then used a syngeneic transplant model by monitoring tumor growth from a mouse breast cancer cell line (AT-3, Gpr81-negative) implanted in mammary fat pad of wild-type mice and Gpr81-null mice. Tumor growth was suppressed in Gpr81-null mice compared with wild-type mice. There were more tumor-infiltrating T cells and MHCIIhi-immune cells in tumors from Gpr81-null mice compared with tumors from wild-type mice. RNA-seq analysis of tumors indicated involvement of immune cells and antigen presentation in Gpr81-dependent tumor growth. Antigen-presenting dendritic cells expressed Gpr81 and activation of this receptor by lactate suppressed cell-surface presentation of MHCII. Activation of Gpr81 in dendritic cells was associated with decreased cAMP, IL-6 and IL-12. These findings suggest that tumor cell-derived lactate activates GPR81 in dendritic cells and prevents presentation of tumor-specific antigens to other immune cells. This paracrine mechanism is complementary to the recently discovered autocrine mechanism in which lactate induces PD-L1 in tumor cells via activation of GPR81 in tumor cells, thus providing an effective means for tumor cells to evade immune system. As such, blockade of GPR81 signaling could boost cancer immunotherapy.


Assuntos
Células Apresentadoras de Antígenos/imunologia , Neoplasias da Mama/genética , Linfócitos do Interstício Tumoral/imunologia , Receptores Acoplados a Proteínas-G/genética , Animais , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Proliferação de Células/genética , AMP Cíclico/genética , Células Dendríticas/imunologia , Modelos Animais de Doenças , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/imunologia , Humanos , Interleucina-12/genética , Interleucina-6/genética , Ácido Láctico/metabolismo , Comunicação Parácrina/genética , Comunicação Parácrina/imunologia , Microambiente Tumoral/imunologia
9.
J Immunother Cancer ; 8(1)2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31959727

RESUMO

BACKGROUND: Interleukin-12 (IL-12) is a potent, proinflammatory cytokine that holds promise for cancer immunotherapy, but its clinical use has been limited by its toxicity. To minimize systemic exposure and potential toxicity while maintaining the beneficial effects of IL-12, we developed a novel IL-12-based therapeutic system that combines tumor-specific T-cell-mediated delivery of IL-12 with membrane-restricted IL-12 localization and inducible IL-12 expression. METHODS: Therapeutic T cells targeting a tumor antigen were genetically engineered to express membrane-anchored IL-12 (aIL-12). Expression, function, and shedding of the aIL-12 molecule was assessed in vitro. Tumor treatment efficacy was assessed in vivo with T cell receptor (TCR) transgenic murine tumor models and a tumor xenograft model. Key outcomes were change in tumor size, circulating levels of IL-12 and other cytokines, and survival. Toxicity was assessed via change in body weight. Tumor growth curve measurements were compared using repeated-measures two-way analyses of variance. RESULTS: Retroviral gene transfer resulted in cell membrane expression of aIL-12 by transduced T cells. In each of two transgenic murine tumor models, tumor-specific T cells constitutively expressing aIL-12 demonstrated increased antitumor efficacy, low circulating IL-12 and interferon-γ, and no weight loss. Expression of aIL-12 via a NFAT-inducible promoter resulted in coordinate expression of aIL-12 with T cell activation. In an OT-I TCR transgenic murine tumor model, the NFAT-inducible aIL-12 molecule improved tumor treatment and did not result in detectable levels of IL-12 in serum or in weight loss. In a human tumor xenograft model, the NFAT-inducible aIL-12 molecule improved antitumor responses by human T cells coexpressing a tumor-specific engineered TCR. Serum IL-12 levels were undetectable with the NFAT-inducible construct in both models. CONCLUSION: Expression of aIL-12 by tumor-targeting therapeutic T cells demonstrated low systemic exposure and improved efficacy. This treatment strategy may have broad applications to cellular therapy with tumor-infiltrating lymphocytes, chimeric antigen receptor T cells, and TCR T cells.


Assuntos
Antígenos de Neoplasias/imunologia , Imunoterapia Adotiva/métodos , Interleucina-12/imunologia , Linfócitos do Interstício Tumoral/imunologia , Melanoma Experimental/imunologia , Melanoma Experimental/terapia , Receptores de Antígenos de Linfócitos T/imunologia , Animais , Linhagem Celular Tumoral , Membrana Celular/genética , Membrana Celular/metabolismo , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Humanos , Interferon gama/metabolismo , Interleucina-12/administração & dosagem , Interleucina-12/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ensaios Antitumorais Modelo de Xenoenxerto
10.
PLoS Negl Trop Dis ; 14(1): e0008021, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31961868

RESUMO

Domestic dogs are the main reservoir of Leishmania infantum, a causative agent of visceral leishmaniasis (VL). The number of human disease cases is associated with the rate of canine infection. Currently available drugs are not efficient at treating canine leishmaniasis (CanL) and months after the treatment most dogs show disease relapse, therefore the development of new drugs or new therapeutic strategies should be sought. In CanL, dogs lack the ability to mount a specific cellular immune response suitable for combating the parasite and manipulation of cytokine signaling pathway has the potential to form part of effective immunotherapeutic methods. In this study, recombinant canine cytokines (rcaIL-12, rcaIL-2, rcaIL-15 and rcaIL-7) and soluble receptor IL-10R1 (rcasIL-10R1), with antagonistic activity, were evaluated for the first time in combination (rcaIL-12/rcaIL-2, rcaIL-12/rcaIL-15, rcaIL-12/rcasIL-10R1, rcaIL-15/rcaIL-7) or alone (rcasIL-10R1) to evaluate their immunomodulatory capacity in peripheral blood mononuclear cells (PBMCs) from dogs with leishmaniasis. All the combinations of recombinant proteins tested were shown to improve lymphoproliferative response. Further, the combinations rcaIL-12/rcaIL-2 and rcaIL-12/rcaIL-15 promoted a decrease in programmed cell death protein 1 (PD-1) expression in lymphocytes. These same combinations of cytokines and rcaIL-12/rcasIL-10R1 induced IFN-γ and TNF-α production in PBMCs. Furthermore, the combination IL-12/IL-15 led to an increased in T-bet expression in lymphocytes. These findings are encouraging and indicate the use of rcaIL-12 and rcaIL-15 in future in vivo studies aimed at achieving polarization of cellular immune responses in dogs with leishmaniasis, which may contribute to the development of an effective treatment against CanL.


Assuntos
Doenças do Cão/tratamento farmacológico , Doenças do Cão/imunologia , Interleucina-12/administração & dosagem , Interleucina-15/administração & dosagem , Leishmaniose Visceral/imunologia , Animais , Doenças do Cão/genética , Doenças do Cão/parasitologia , Cães , Imunidade Celular , Interferon gama/genética , Interferon gama/imunologia , Interleucina-12/genética , Interleucina-12/imunologia , Interleucina-15/genética , Interleucina-15/imunologia , Leishmania infantum/fisiologia , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/veterinária , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/parasitologia , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/imunologia , Receptores de Citocinas/genética , Receptores de Citocinas/imunologia
11.
Methods Mol Biol ; 2052: 229-251, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31452166

RESUMO

Cryptosporidiosis threatens life of young children in developing countries and newborn calves around the world. No vaccine or therapy can prevent or cure this diarrhea-inducing enteric disease caused by Cryptosporidium spp. protozoan parasites. There is an essential need to discover new therapeutic drugs efficient in reducing parasite burden in infected individuals. Research therefore relies on reliable small animal models of cryptosporidiosis. Here, we present excellent mouse models which can efficiently mimic pathogenesis of human and bovine cryptosporidiosis. We also describe methods to purify C. parvum oocysts from stool and intestine of infected mice to facilitate oocyst quantification. Moreover, we present protocols using flow cytometry, quantitative polymerase chain reaction, and histopathology to accurately quantify parasite burden in stool or intestine samples.


Assuntos
Criptosporidiose/parasitologia , Cryptosporidium parvum/isolamento & purificação , Oocistos/isolamento & purificação , Animais , Criptosporidiose/patologia , Cryptosporidium , Cryptosporidium parvum/crescimento & desenvolvimento , Modelos Animais de Doenças , Fezes/parasitologia , Citometria de Fluxo/métodos , Íleo/citologia , Íleo/parasitologia , Íleo/patologia , Interleucina-12/genética , Camundongos , Camundongos Knockout , Camundongos SCID , Oocistos/crescimento & desenvolvimento , Reação em Cadeia da Polimerase em Tempo Real/métodos , Receptores de Interferon/genética , Fluxo de Trabalho
12.
Methods Mol Biol ; 2060: 153-168, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31617177

RESUMO

In the previous chapter, we describe the engineering of a HSV-BAC genome by galK recombineering. Here we describe the procedures to reconstitute, or regenerate, the replicating recombinant virus, and the methods to purify it and characterize it for the correct expression of the transgene. We present the example of R-115, a recombinant expressing murine interleukin 12 (mIL12) from the US1-US2 intergenic region. A specific method for the production of highly purified virions by iodixanol gradient, suitable for in vivo applications, is also detailed.


Assuntos
Cromossomos Artificiais Bacterianos/genética , Expressão Gênica , Herpesvirus Humano 1 , Interleucina-12 , Recombinação Genética , Animais , Linhagem Celular Tumoral , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/metabolismo , Interleucina-12/biossíntese , Interleucina-12/genética , Camundongos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética
13.
Mol Med Rep ; 21(1): 304-310, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31746382

RESUMO

Dioscin has been shown to affect the regulation of metabolic diseases, including diabetes; however, the mechanism of action is still unclear. Under high glucose (HG) conditions, the expression of scavenger receptors and the uptake of oxidized low­density lipoprotein (oxLDL) are upregulated in dendritic cells (DCs), which are critical steps in atherogenesis and inflammation. In this study, the focus was on the impact of dioscin on the function of DCs. Immature DCs were cultured with: 5.5 mM glucose medium (control group); 30 mM glucose medium (HG group); HG + 10 mM dioscin; HG + 20 mM dioscin; HG + 30 mM dioscin; and HG + 40 mM dioscin. For subsequent experiments, 30 mM dioscin was used as the experimental concentration. Dichlorodihydrofluorescein fluorescence was used to measure the intracellular production of reactive oxygen species (ROS) in DCs. The expression levels of the scavenger receptors, including class A scavenger receptors (SR­A), CD36 and lectin­like oxidized low­density lipoprotein receptor­1 (LOX­1) were determined via quantitative PCR. The protein expression of p38 mitogen­activated protein kinase (MAPK) was determined by western blotting. Furthermore, ELISA was used to detect the levels of interleukin (IL)­6, IL­10 and IL­12. Finally, DCs were incubated with diOlistic (Dil)­labeled oxLDL, and flow cytometry analysis was used to investigate the Dil­oxLDL­incorporated fraction. The incubation of DCs with dioscin inhibited the induction of ROS production, in a dose­dependent manner, under HG conditions. The upregulation of SR­A, CD36 and LOX­1 genes was partially abolished by dioscin, which also partially reversed p38 MAPK protein upregulation. Furthermore, increased secretion of IL­6 and IL­12, and decreased secretion of IL­10 in DCs, induced by HG, was also reversed by dioscin. To conclude, dioscin could attenuate the production of ROS, inflammatory cytokine secretion and oxLDL uptake by DCs in HG conditions by preventing the expression of scavenger receptors and p38 MAPK, thus playing a positive role in preventing atherogenesis.


Assuntos
Antígenos CD36/genética , Diosgenina/análogos & derivados , Inflamação/tratamento farmacológico , Receptores Depuradores Classe E/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Adulto , Aterosclerose/genética , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Diosgenina/farmacologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Humanos , Inflamação/genética , Inflamação/patologia , Interleucina-10/genética , Interleucina-12/genética , Interleucina-6/genética , Lipoproteínas LDL/genética , Masculino , Receptores Depuradores Classe A/genética
14.
Parasit Vectors ; 12(1): 600, 2019 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-31870416

RESUMO

BACKGROUND: The chemotherapeutic arsenal available to treat visceral leishmaniasis is currently limited, in view of many drawbacks such as high cost, toxicity or emerging resistance. New therapeutic strategies are particularly needed to improve the management and the outcome in immunosuppressed patients. The combination of an immunomodulatory drug to a conventional anti-Leishmania treatment is an emerging concept to reverse the immune bias from Th2 to Th1 response to boost healing and prevent relapses. METHODS: Here, immunostimulating and leishmanicidal properties of octyl-ß-D-galactofuranose (Galf) were assessed in human monocyte-derived macrophages (HM) and in a murine model, after challenge with Leishmania donovani promastigotes. We recorded parasite loads and expression of various cytokines and immune effectors in HM and mouse organs (liver, spleen, bone marrow), following treatment with free (Galf) and liposomal (L-Galf) formulations. RESULTS: Both treatments significantly reduced parasite proliferation in HM, as well as liver parasite burden in vivo (Galf, P < 0.05). Consistent with in vitro results, we showed that Galf- and L-Galf-treated mice displayed an enhanced Th1 immune response, particularly in the spleen where pro-inflammatory cytokines TNF-α, IL-1ß and IL-12 were significantly overexpressed compared to control group. The hepatic recruitment of myeloid cells was also favored by L-Galf treatment as evidenced by the five-fold increase of myeloperoxidase (MPO) induction, which was associated with a higher number of MPO-positive cells within granulomas. By contrast, the systemic level of various cytokines such as IL-1ß, IL-6, IL-17A or IL-27 was drastically reduced at the end of treatment. CONCLUSIONS: Overall, these results suggest that Galf could be tested as an adjuvant in combination with current anti-parasitic drugs, to restore an efficient immune response against infection in a model of immunosuppressed mice.


Assuntos
Adjuvantes Imunológicos/administração & dosagem , Dissacarídeos/administração & dosagem , Leishmania donovani/efeitos dos fármacos , Leishmaniose Visceral/tratamento farmacológico , Animais , Feminino , Humanos , Interleucina-12/genética , Interleucina-12/imunologia , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Leishmania donovani/genética , Leishmania donovani/metabolismo , Leishmaniose Visceral/genética , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/parasitologia , Fígado/efeitos dos fármacos , Fígado/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Baço/efeitos dos fármacos , Baço/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia
15.
Pol J Microbiol ; 68(4): 439-447, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31880888

RESUMO

Salmonella infection is most common in patients with infected aortic aneurysm, especially in Asia. When the aortic wall is heavily atherosclerotic, the intima is vulnerable to invasion by Salmonella, leading to the development of infected aortic aneurysm. By using THP-1 macrophage-derived foam cells to mimic atherosclerosis, we investigated the role of three Salmonella enterica serotypes - Typhimurium, Enteritidis, and Choleraesuis - in foam cell autophagy and inflammasome formation. Herein, we provide possible pathogenesis of Salmonella-associated infected aortic aneurysms. Three S. enterica serotypes with or without virulence plasmid were studied. Through Western blotting, we investigated cell autophagy induction and inflammasome formation in Salmonella-infected THP-1 macrophage-derived foam cells, detected CD36 expression after Salmonella infection through flow cytometry, and measured interleukin (IL)-1ß, IL-12, and interferon (IFN)-α levels through enzyme-linked immunosorbent assay. At 0.5 h after infection, plasmid-bearing S. Enteritidis OU7130 induced the highest foam cell autophagy - significantly higher than that induced by plasmid-less OU7067. However, plasmid-bearing S. Choleraesuis induced less foam cell autophagy than did its plasmid-less strain. In foam cells, plasmid-less Salmonella infection (particularly S. Choleraesuis OU7266 infection) led to higher CD36 expression than did plasmid-bearing strains infection. OU7130 and OU7266 infection induced the highest IL-1ß secretion. OU7067-infected foam cells secreted the highest IL-12p35 level. Plasmid-bearing S. Typhimurium OU5045 induced a higher IFN-α level than did other Salmonella serotypes. Salmonella serotypes are correlated with foam cell autophagy and IL-1ß secretion. Salmonella may affect the course of foam cells formation, or even aortic aneurysm, through autophagy.Salmonella infection is most common in patients with infected aortic aneurysm, especially in Asia. When the aortic wall is heavily atherosclerotic, the intima is vulnerable to invasion by Salmonella, leading to the development of infected aortic aneurysm. By using THP-1 macrophage-derived foam cells to mimic atherosclerosis, we investigated the role of three Salmonella enterica serotypes ­ Typhimurium, Enteritidis, and Choleraesuis ­ in foam cell autophagy and inflammasome formation. Herein, we provide possible pathogenesis of Salmonella-associated infected aortic aneurysms. Three S. enterica serotypes with or without virulence plasmid were studied. Through Western blotting, we investigated cell autophagy induction and inflammasome formation in Salmonella-infected THP-1 macrophage-derived foam cells, detected CD36 expression after Salmonella infection through flow cytometry, and measured interleukin (IL)-1ß, IL-12, and interferon (IFN)-α levels through enzyme-linked immunosorbent assay. At 0.5 h after infection, plasmid-bearing S. Enteritidis OU7130 induced the highest foam cell autophagy ­ significantly higher than that induced by plasmid-less OU7067. However, plasmid-bearing S. Choleraesuis induced less foam cell autophagy than did its plasmid-less strain. In foam cells, plasmid-less Salmonella infection (particularly S. Choleraesuis OU7266 infection) led to higher CD36 expression than did plasmid-bearing strains infection. OU7130 and OU7266 infection induced the highest IL-1ß secretion. OU7067-infected foam cells secreted the highest IL-12p35 level. Plasmid-bearing S. Typhimurium OU5045 induced a higher IFN-α level than did other Salmonella serotypes. Salmonella serotypes are correlated with foam cell autophagy and IL-1ß secretion. Salmonella may affect the course of foam cells formation, or even aortic aneurysm, through autophagy.


Assuntos
Aneurisma Aórtico/microbiologia , Infecções por Salmonella/microbiologia , Salmonella typhimurium/patogenicidade , Animais , Aneurisma Aórtico/genética , Aneurisma Aórtico/imunologia , Linhagem Celular , Humanos , Interferon-alfa/genética , Interferon-alfa/imunologia , Interleucina-12/genética , Interleucina-12/imunologia , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Antígeno Ki-1/genética , Antígeno Ki-1/imunologia , Macrófagos/imunologia , Macrófagos/microbiologia , Monócitos/imunologia , Monócitos/microbiologia , Plasmídeos/genética , Plasmídeos/metabolismo , Infecções por Salmonella/genética , Infecções por Salmonella/imunologia , Salmonella typhimurium/genética , Salmonella typhimurium/fisiologia , Sorogrupo , Virulência
16.
Cancer Cell ; 36(6): 613-629.e7, 2019 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-31761658

RESUMO

Retroviral gene transfer of interleukin-12 (IL-12) into T cells markedly enhances antitumor efficacy upon adoptive transfer but has clinically shown unacceptable severe side effects. To overcome the toxicity, we engineered tumor-specific CD8+ T cells to transiently express IL-12. Engineered T cells injected intratumorally, but not intravenously, led to complete rejections not only of the injected lesion but also of distant concomitant tumors. Efficacy was further enhanced by co-injection with agonist anti-CD137 mAb or by transient co-expression of CD137 ligand. This treatment induced epitope spreading of the endogenous CD8+ T cell immune response in a manner dependent on cDC1 dendritic cells. Mouse and human tumor-infiltrating T lymphocyte cultures can be transiently IL-12 engineered to attain marked immunotherapeutic effects.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Imunoterapia Adotiva , Interleucina-12/genética , Linfócitos do Interstício Tumoral/imunologia , Transferência Adotiva/métodos , Animais , Anticorpos Monoclonais/farmacologia , Linhagem Celular Tumoral , Células Dendríticas/imunologia , Humanos , Imunoterapia Adotiva/métodos , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/imunologia , Camundongos , Linfócitos T Citotóxicos/imunologia
17.
Nat Commun ; 10(1): 5100, 2019 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-31723132

RESUMO

Endowing chimeric antigen receptor (CAR) T cells with additional potent functionalities holds strong potential for improving their antitumor activity. However, because potency could be deleterious without control, these additional features need to be tightly regulated. Immune pathways offer a wide array of tightly regulated genes that can be repurposed to express potent functionalities in a highly controlled manner. Here, we explore this concept by repurposing TCR, CD25 and PD1, three major players of the T cell activation pathway. We insert the CAR into the TCRα gene (TRACCAR), and IL-12P70 into either IL2Rα or PDCD1 genes. This process results in transient, antigen concentration-dependent IL-12P70 secretion, increases TRACCAR T cell cytotoxicity and extends survival of tumor-bearing mice. This gene network repurposing strategy can be extended to other cellular pathways, thus paving the way for generating smart CAR T cells able to integrate biological inputs and to translate them into therapeutic outputs in a highly regulated manner.


Assuntos
Sistema Imunitário/metabolismo , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/imunologia , Animais , Linhagem Celular Tumoral , Edição de Genes , Humanos , Interleucina-12/genética , Ativação Linfocitária/imunologia , Camundongos , Neoplasias/imunologia , Neoplasias/patologia , Receptores de Antígenos de Linfócitos T/metabolismo , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/metabolismo
18.
Viruses ; 11(10)2019 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-31623390

RESUMO

Tumor-targeted immunomodulation using oncolytic viral vectors is currently being investigated as a promising strategy in cancer therapy. In a previous study, we showed that a measles virus Schwarz vaccine strain (MeVac) vector encoding an interleukin-12 fusion protein (FmIL-12) is an effective immunotherapy in the MC38cea murine colon adenocarcinoma model. We hypothesized that MeVac encoding interleukin-15 may mediate enhanced T and NK cell responses and thus increase the therapeutic efficacy, especially in NK cell-controlled tumors. Therefore, we generated MeVac vectors encoding an interleukin-15 superagonist, FmIL-15. Replication and oncolytic capacity, transgene expression, and functionality of MeVac FmIL-15 vectors were validated in vitro. Effects on the tumor immune landscape and therapeutic efficacy of both FmIL-12 and FmIL-15 vectors were studied in the MC38cea and B16hCD46 tumor models. Treatment with MeVac FmIL-15 increased T and NK cell infiltration in both models. However, MeVac FmIL-12 showed more robust viral gene expression and immune activation, resulting in superior anti-tumor efficacy. Based on these results, MeVac encoding a human IL-12 fusion protein was developed for future clinical translation.


Assuntos
Regulação Viral da Expressão Gênica , Interleucina-12/agonistas , Interleucina-15/agonistas , Vacina contra Sarampo/imunologia , Adenocarcinoma , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Colo , Modelos Animais de Doenças , Feminino , Genes Virais , Imunoterapia , Interleucina-12/genética , Interleucina-15/genética , Células Matadoras Naturais/imunologia , Sarampo , Camundongos , Camundongos Endogâmicos C57BL , Vírus Oncolíticos , Transcriptoma , Vacinas Sintéticas , Proteínas Virais de Fusão/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Sci Rep ; 9(1): 14204, 2019 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-31578370

RESUMO

Canadian Indigenous peoples (First Nations and Inuit) exhibit a high burden of infectious diseases including tuberculosis influenced by societal factors, and biological determinants. Toll-like receptor (TLR)-mediated innate immune responses are the first line of defence against infections. We examined the production of a panel of 30 cytokines in peripheral blood-derived mononuclear cells (PBMC) isolated from Indigenous and non-Indigenous participants, following stimulation with five different TLR ligands. The levels of TLR-induced pro-inflammatory cytokines such as IL-12/23p40, IL-16, and IFN-γ, and chemokines (MCP-4, MDC and eotaxin) were different between Indigenous compared to non-Indigenous participants. Antimicrobial cationic host defence peptides (CHDP) induced by TLR activation are critical for resolution of infections and modulate the TLR-to-NFκB pathway to alter downstream cytokine responses. Therefore, we examined the expression of human CHDP defensins and cathelicidin in PBMC. mRNA expression of genes encoding for def-A1 and def-B1 were significantly higher following stimulation with TLR ligands in Indigenous compared to non-Indigenous participants. The purinergic receptor P2X7 known to be activated by ATP released following TLR stimulation, is a receptor for CHDP. Therefore, we further examined single nucleotide polymorphisms (SNP) in P2X7. Indigenous participants had a significantly higher percentage of a P2X7 SNP which is associated with reduced function and lower ability to clear infections. These results suggest that a higher frequency of non-functional P2X7 receptors may influence the activity of downstream immune mediators required for resolution of infections such as pro-inflammatory cytokines and CHDP defensins, thus contributing to higher burden of infections in Indigenous population.


Assuntos
Povos Indígenas/genética , Polimorfismo Genético/genética , Receptores Purinérgicos P2X7/genética , Receptores Toll-Like/genética , Canadá/epidemiologia , Citocinas/genética , Defensinas/genética , Humanos , Imunidade Inata/genética , Interleucina-12/genética , Leucócitos Mononucleares/metabolismo , Macrófagos/metabolismo , Polimorfismo de Nucleotídeo Único/genética , Fatores de Risco , Transdução de Sinais/genética
20.
Free Radic Biol Med ; 145: 61-66, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31525456

RESUMO

Over the past years, systemic derived cues that regulate cellular metabolism have been implicated in the regulation of immune responses. Ghrelin is an orexigenic hormone produced by enteroendocrine cells in the gastric mucosa with known immunoregulatory roles. The mechanism behind the function of ghrelin in immune cells, such as macrophages, is still poorly understood. Here, we explored the hypothesis that ghrelin leads to alterations in macrophage metabolism thus modulating macrophage function. We demonstrated that ghrelin exerts an immunomodulatory effect over LPS-activated peritoneal macrophages, as evidenced by inhibition of TNF-α and IL-1ß secretion and increased IL-12 production. Concomitantly, ghrelin increased mitochondrial membrane potential and increased respiratory rate. In agreement, ghrelin prevented LPS-induced ultrastructural damage in the mitochondria. Ghrelin also blunted LPS-induced glycolysis. In LPS-activated macrophages, glucose deprivation did not affect ghrelin-induced IL-12 secretion, whereas the inhibition of pyruvate transport and mitochondria-derived ATP abolished ghrelin-induced IL-12 secretion, indicating a dependence on mitochondrial function. Ghrelin pre-treatment of metabolic activated macrophages inhibited the secretion of TNF-α and enhanced IL-12 levels. Moreover, ghrelin effects on IL-12, and not on TNF-α, are dependent on mitochondria elongation, since ghrelin did not enhance IL-12 secretion in metabolic activated mitofusin-2 deficient macrophages. Thus, ghrelin affects macrophage mitochondrial metabolism and the subsequent macrophage function.


Assuntos
Grelina/farmacologia , Interleucina-12/genética , Interleucina-1beta/genética , Macrófagos Peritoneais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/genética , Trifosfato de Adenosina/genética , Animais , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Grelina/química , Glicólise/efeitos dos fármacos , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Inflamação/genética , Inflamação/patologia , Lipopolissacarídeos/toxicidade , Macrófagos Peritoneais/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Óxido Nítrico/genética , Transdução de Sinais/genética
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