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We have shown that activin A (activin), a TGF-ß superfamily member, has pro-metastatic effects in colorectal cancer (CRC). In lung cancer, activin activates pro-metastatic pathways to enhance tumor cell survival and migration while augmenting CD4+ to CD8+ communications to promote cytotoxicity. Here, we hypothesized that activin exerts cell-specific effects in the tumor microenvironment (TME) of CRC to promote anti-tumoral activity of immune cells and the pro-metastatic behavior of tumor cells in a cell-specific and context-dependent manner. We generated an Smad4 epithelial cell specific knockout (Smad4-/-) which was crossed with TS4-Cre mice to identify SMAD-specific changes in CRC. We also performed IHC and digital spatial profiling (DSP) of tissue microarrays (TMAs) obtained from 1055 stage II and III CRC patients in the QUASAR 2 clinical trial. We transfected the CRC cells to reduce their activin production and injected them into mice with intermittent tumor measurements to determine how cancer-derived activin alters tumor growth in vivo. In vivo, Smad4-/- mice displayed elevated colonic activin and pAKT expression and increased mortality. IHC analysis of the TMA samples revealed increased activin was required for TGF-ß-associated improved outcomes in CRC. DSP analysis identified that activin co-localization in the stroma was coupled with increases in T-cell exhaustion markers, activation markers of antigen presenting cells (APCs), and effectors of the PI3K/AKT pathway. Activin-stimulated PI3K-dependent CRC transwell migration, and the in vivo loss of activin lead to smaller CRC tumors. Taken together, activin is a targetable, highly context-dependent molecule with effects on CRC growth, migration, and TME immune plasticity.
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Increased tumor infiltrating lymphocytes (TIL) are associated with improved patient responses to immunotherapy. As a result, there is interest in enhancing lymphocyte trafficking particularly to colon cancers since the majority are checkpoint blockade-resistant and microsatellite stable. Here, we demonstrate that activated T-cells (ATC) armed with anti-CD3 x anti-EGFR bispecific antibody increases TIL and mediate anti-tumor cytotoxicity while decreasing tumor cell viability. Furthermore, treatment induces endogenous anti-tumor immunity that resisted tumor rechallenge and increased memory T-cell subsets in the tumor. When combined with targeted tumor expression of the tumor necrosis factor superfamily member LIGHT, activated T-cell proliferation and infiltration were further enhanced, and human colorectal tumor regressions were observed. Our data indicate that tumor-targeted armed bispecific antibody increases TIL trafficking and is a potentially potent strategy that can be paired with combination immunotherapy to battle microsatellite stable colon cancer. SIGNIFICANCE: Enhancing trafficking of tumor infiltrating lymphocytes (TILs) to solid tumors has been shown to improve outcomes. Unfortunately, few strategies have been successful in the clinical setting for solid tumors, particularly for "cold" microsatellite stable colon cancers. In order to address this gap in knowledge, this study combined TNFSF14/LIGHT immunomodulation with a bispecific antibody armed with activated T-cells targeted to the tumor. This unique T-cell trafficking strategy successfully generated anti-tumor immunity in a microsatellite stable colon cancer model, stimulated T-cell infiltration, and holds promise as a combination immunotherapy for treating advanced and metastatic colorectal cancer.
Assuntos
Anticorpos Biespecíficos , Neoplasias do Colo , Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/uso terapêutico , Complexo CD3 , Neoplasias do Colo/tratamento farmacológico , Humanos , Imunoterapia , Linfócitos TRESUMO
BACKGROUND: The generation of long-term durable tumor immunity and prolonged disease-free survival depends on the ability to generate and support CD8+ central memory T-cells. Microsatellite-stable colon cancer is resistant to currently available immunotherapies; thus, development of novel mechanisms to increase both lymphocyte infiltration and central memory formation are needed to improve outcomes in these patients. We have previously demonstrated that both interleukin-2 (IL-2) and LIGHT (TNFSF14) independently enhance antitumor immune responses and hypothesize that combination immunotherapy may increase the CD8+ central memory T-cell response. METHODS: Murine colorectal cancer tumors were established in syngeneic mice. Tumors were treated with control, soluble, or liposomal IL-2 at established intervals. A subset of animal tumors overexpressed tumor necrosis superfamily factor LIGHT (TNFSF14). Peripheral blood, splenic, and tumor-infiltrating lymphocytes were isolated for phenotypic studies and flow cytometry. RESULTS: Tumors exposed to a combination of LIGHT and IL-2 experienced a decrease in tumor size compared with IL-2 alone that was not demonstrated in wild-type tumors or between other treatment groups. Combination exposure also increased splenic central memory CD8+ cells compared with IL-2 administration alone, while not increasing tumor-infiltrating lymphocytes. In the periphery, the combination enhanced levels of circulating CD8 T-cells and central memory T-cells, while also increasing circulating T-regulatory cells. CONCLUSIONS: Combination of IL-2, whether soluble or liposomal, with exposure to LIGHT results in increased CD8+ central memory cells in the spleen and periphery. New combination immunotherapy strategies that support both effector and memory T-cell functions are critical to enhancing durable antitumor responses and warrant further investigation.
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Linfócitos T CD8-Positivos/imunologia , Neoplasias do Colo/terapia , Imunoterapia/métodos , Interleucina-2/administração & dosagem , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral/genética , Animais , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linhagem Celular Tumoral/transplante , Neoplasias do Colo/genética , Neoplasias do Colo/imunologia , Neoplasias do Colo/patologia , Modelos Animais de Doenças , Feminino , Regulação Neoplásica da Expressão Gênica/imunologia , Humanos , Memória Imunológica/efeitos dos fármacos , Injeções Intralesionais , Lipossomos , Linfócitos do Interstício Tumoral/imunologia , Camundongos , Proteínas Recombinantes/administração & dosagem , Microambiente Tumoral/genética , Microambiente Tumoral/imunologiaRESUMO
ATC is an aggressive disease with limited therapeutic options due to drug resistance. TRAIL is an attractive anti-cancer therapy that can trigger apoptosis in a cancer cell-selective manner. However, TRAIL resistance is a major clinical obstacle for its use as a therapeutic drug. Previously, we demonstrated that MADD is a cancer cell pro-survival factor that can modulate TRAIL resistance. However, its role, if any, in overcoming TRAIL resistance in ATC is unknown. First, we characterized ATC cell lines as either TRAIL resistant, TRAIL sensitive or moderately TRAIL sensitive and evaluated MADD expression/cellular localization. We determined the effect of MADD siRNA on cellular growth and investigated its effect on TRAIL treatment. We assessed the effect of combination treatment (MADD siRNA and TRAIL) on mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) levels. The effect of combination treatment on tumor growth was assessed in vivo. We found increased levels of MADD in ATC cells relative to Nthy-ori 3-1. MADD protein localizes in the cytosol (endoplasmic reticulum and Golgi body) and membrane. MADD knockdown resulted in spontaneous cell death that was synergistically enhanced when combined with TRAIL treatment in otherwise resistant ATC cells. Combination treatment resulted in a significant reduction in MMP and enhanced generation of ROS indicating the putative mechanism of action. In an orthotopic mouse model of TRAIL-resistant ATC, treatment with MADD siRNA alone reduced tumor growth that, when combined with TRAIL, resulted in significant tumor regressions. We demonstrated the potential clinical utility of MADD knockdown in sensitizing cells to TRAIL-induced apoptosis in ATC.
Assuntos
Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/genética , Fatores de Troca do Nucleotídeo Guanina/genética , RNA Interferente Pequeno/administração & dosagem , Ligante Indutor de Apoptose Relacionado a TNF/uso terapêutico , Carcinoma Anaplásico da Tireoide/tratamento farmacológico , Neoplasias da Glândula Tireoide/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Inativação Gênica , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos Nus , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Carcinoma Anaplásico da Tireoide/genética , Carcinoma Anaplásico da Tireoide/metabolismo , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/metabolismoRESUMO
BACKGROUND: Colorectal cancer remains a leading cause of cancer-related mortality worldwide. Metastases to the liver are often present at initial presentation and will form in most patients during their course of disease. We have previously demonstrated that enhanced trafficking and activation of tumor-infiltrating lymphocytes in colorectal liver metastases (CRLM) may improve antitumor immune responses. Thus, development of novel mechanisms to increase lymphocyte infiltration and activation are needed to improve patient outcomes. METHODS: CT26 murine colorectal cancer cells were treated with physiologic levels of the potent inducer of immunogenic cell death mitoxantrone (MTX). An in situ vaccine was created with treated cells in an established model of CRLM. Cells were evaluated by flow cytometry for cell cycle evaluation and calreticulin expression. Splenic and tumor-infiltrating lymphocytes were isolated for phenotypic studies. RESULTS: MTX-treatment of colon cancer cells resulted in a sub-G1 peak, inhibition of G1 cell cycle progression, and increased G2/M cell fractions while simultaneously increasing dynamic exposure of calreticulin on the cell surface (P < 0.05). Vaccination with MTX-treated cells resulted in significant decreases in CRLM formation associated with increased tumor-infiltrating leukocytes that displayed increased expression of the T cell surface activation marker CD69. CONCLUSIONS: Vaccination with MTX-treated primary colon cancer cells enhances tumor-infiltrating lymphocytes and clinical responses in CRLM.
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Antineoplásicos/administração & dosagem , Vacinas Anticâncer/administração & dosagem , Neoplasias Colorretais/patologia , Neoplasias Hepáticas/terapia , Mitoxantrona/administração & dosagem , Animais , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/imunologia , Linhagem Celular Tumoral/transplante , Modelos Animais de Doenças , Feminino , Humanos , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/secundário , Ativação Linfocitária/efeitos dos fármacos , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/imunologia , Camundongos , Resultado do TratamentoRESUMO
The majority of patients with colon cancer will develop advanced disease, with the liver being the most common site of metastatic disease. Patients with increased numbers of tumor-infiltrating lymphocytes in primary colon tumors and liver metastases have improved outcomes. However, the molecular factors that could empower antitumor immune responses in this setting remain to be elucidated. We reported that the immunostimulatory cytokine LIGHT (TNFSF14) in the microenvironment of colon cancer metastases associates with improved patient survival, and here we demonstrate in an immunocompetent murine model that colon tumors expressing LIGHT stimulate lymphocyte proliferation and tumor cell-specific antitumor immune responses. In this model, increasing LIGHT expression in the microenvironment of either primary tumors or liver metastases triggered regression of established tumors and slowed the growth of liver metastases, driven by cytotoxic T-lymphocyte-mediated antitumor immunity. These responses corresponded with significant increases in tumor-infiltrating lymphocytes and increased expression of lymphocyte-homing signals in the metastatic tumors. Furthermore, we demonstrated evidence of durable tumor-specific antitumor immunity. In conclusion, increasing LIGHT expression increased T-cell proliferation, activation, and infiltration, resulting in enhanced tumor-specific immune-mediated tumor regressions in primary tumors and colorectal liver metastases. Mechanisms to increase LIGHT in the colon cancer microenvironment warrant further investigation and hold promise as an immunotherapeutic strategy. Cancer Res; 77(8); 1880-91. ©2017 AACR.
Assuntos
Neoplasias do Colo/imunologia , Neoplasias do Colo/metabolismo , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral/biossíntese , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral/imunologia , Animais , Linhagem Celular Tumoral , Neoplasias do Colo/patologia , Feminino , Células HEK293 , Humanos , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/secundário , Ativação Linfocitária , Linfócitos do Interstício Tumoral/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Linfócitos T/imunologiaRESUMO
Immunotherapeutic approaches to manage patients with advanced gastrointestinal malignancies are desired; however, mechanisms to incite tumor-specific immune responses remain to be elucidated. Rose bengal (RB) is toxic at low concentrations to malignant cells and may induce damage-associated molecular patterns; therefore, we investigated its potential as an immunomodulator in colon cancer. Murine and human colon cancer lines were treated with RB (10% in saline/PV-10) for cell cycle, cell death, and apoptosis assays. Damage-associated molecular patterns were assessed with western blot, ELISA, and flow cytometry. In an immunocompetent murine model of colon cancer, we demonstrate that tumors regress upon RB treatment, and that RB induces cell death in colon cancer cells through G2/M growth arrest and predominantly necrosis. RB-treated colon cancer cells expressed distinct hallmarks of immunogenic cell death (ICD), including enhanced expression of calreticulin and heat-shock protein 90 on the cell surface, a decrease in intracellular ATP, and the release of HMGB1. To confirm the ICD phenotype, we vaccinated immunocompetent animals with syngeneic colon cancer cells treated with RB. RB-treated tumors served as a vaccine against subsequent challenge with the same CT26 colon cancer tumor cells, and vaccination with in vitro RB-treated cells resulted in slower tumor growth following inoculation with colon cancer cells, but not with syngeneic non-CT26 cancer cells, suggesting a specific antitumor immune response. In conclusion, RB serves as an inducer of ICD that contributes to enhanced specific antitumor immunity in colorectal cancer.
Assuntos
Antineoplásicos/imunologia , Antineoplásicos/farmacologia , Morte Celular/efeitos dos fármacos , Morte Celular/imunologia , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/imunologia , Rosa Bengala/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Calreticulina/metabolismo , Linhagem Celular Tumoral , Neoplasias do Colo/metabolismo , Células HCT116 , Proteína HMGB1/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Células HT29 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Necrose/tratamento farmacológico , Necrose/imunologia , Necrose/metabolismoRESUMO
Translating in vitro biological data into actionable information related to human health holds the potential to improve disease treatment and risk assessment of chemical exposures. While genomics has identified regulatory pathways at the cellular level, translation to the organism level requires a multiscale approach accounting for intra-cellular regulation, inter-cellular interaction, and tissue/organ-level effects. Tissue-level effects can now be probed in vitro thanks to recently developed systems of three-dimensional (3D), multicellular, "organotypic" cell cultures, which mimic functional responses of living tissue. However, there remains a knowledge gap regarding interactions across different biological scales, complicating accurate prediction of health outcomes from molecular/genomic data and tissue responses. Systems biology aims at mathematical modeling of complex, non-linear biological systems. We propose to apply a systems biology approach to achieve a computational representation of tissue-level physiological responses by integrating empirical data derived from organotypic culture systems with computational models of intracellular pathways to better predict human responses. Successful implementation of this integrated approach will provide a powerful tool for faster, more accurate and cost-effective screening of potential toxicants and therapeutics. On September 11, 2015, an interdisciplinary group of scientists, engineers, and clinicians gathered for a workshop in Research Triangle Park, North Carolina, to discuss this ambitious goal. Participants represented laboratory-based and computational modeling approaches to pharmacology and toxicology, as well as the pharmaceutical industry, government, non-profits, and academia. Discussions focused on identifying critical system perturbations to model, the computational tools required, and the experimental approaches best suited to generating key data.
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Técnicas de Cultura de Células , Simulação por Computador , Biologia de Sistemas , Alternativas aos Testes com Animais , Animais , Técnicas de Cultura de Células/métodos , Substâncias Perigosas/toxicidade , Humanos , Dispositivos Lab-On-A-Chip , Medição de RiscoRESUMO
This work aimed to characterize the mammary gland pharmacokinetics of cefquinome after an intramammary administration and integrate pharmacokinetic/pharmacodynamic model. The pharmacokinetic profiles of cefquinome in gland tissue were measured using high performance liquid chromatograph. Therapeutic regimens covered various dosages ranging from 25 to 800 µg/gland and multiple dosing intervals of 8, 12, and 24 h. The in vivo bacterial killing activity elevated when dosage increased or when dosing intervals were shortened. The best antibacterial effect was demonstrated by a mean 1.5 log10CFU/gland visible count reduction. On the other hand, the results showed that the percentage of time duration of drug concentration exceeding the MIC during a dose interval (%T > MIC) was generally 100% because of the influence of drug distribution caused by the blood-milk barrier. Therefore, pharmacokinetic/pharmacodynamic parameter of the ratio of area under the concentration-time curve over 24 h to the MIC (AUC0-24/MIC) was used to describe the efficacy of cefquinome instead of %T > MIC. When the magnitude of AUC0-24/MIC exceeding 16571.55 hâ mL/g, considerable activity of about 1.5 log10CFU/g gland bacterial count reduction was observed in vivo. Based on the Monte Carlo simulation, the clinical recommended regimen of three infusions of 75 mg per quarter every 12 h can achieve a 76.67% cure rate in clinical treatment of bovine mastitis caused by Staphylococcus aureus infection.
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Colon cancer is a leading cause of cancer-related mortality for which targeted therapy is needed; however, trials using apoptosis-inducing ligand monotherapy to overcome resistance to apoptosis have not shown clinical responses. Since colon cancer cells selectively uptake and rapidly metabolize glucose, a property utilized for clinical staging, we investigated mechanisms to alter glucose metabolism in order to selectively target the cancer cells and to overcome evasion of apoptosis. We demonstrate TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) resistance in the majority of human colon cancers tested and utilize the glucose analog 2-deoxy-d-glucose to sensitize TRAIL-resistant gastrointestinal adenocarcinoma cells, and not normal gastrointestinal epithelial cells, to TRAIL-induced apoptosis through enhanced death receptor 5 expression, downstream modulation of MAPK signaling and subsequent miRNA expression modulation by increasing the expression of miR-494 via MEK activation. Further, established human colon cancer xenografts treated with this strategy experience anti-tumor responses. These findings in colon adenocarcinoma support further investigation of manipulation of cellular energetics to selectively overcome resistance to apoptosis and to impart tumor regressions in established colon cancer tumors.
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Program death-1 (PD-1) is a co-inhibitory receptor inducibly expressed on activated T cells. PD-1 has been reported to be associated with the development of several autoimmune diseases including rheumatoid arthritis, but the precise cellular and molecular mechanisms have not been fully elucidated. To study the role of PD-1 in the pathogenesis of rheumatoid arthritis and the possible underlying mechanisms, we performed collagen-induced arthritis (CIA) in C57BL/6 mice. Here, we show that PD-1 deficiency leads to the development of severe CIA in mice. When analyzing T cells from CIA mice ex vivo, we noticed aberrant antigen-specific Th17 responses in mice lacking PD-1. This is possibly due to deregulated activation of PKC-θ and Akt. In support of this notion, treating Pdcd1 (-/-) mice with an inhibitor of PI3-kinase that is upstream of PKC-θ and Akt significantly suppressed the disease severity. Therefore, our data indicate that PD-1 dampens antigen-specific Th17 response, thus inhibiting the disease.
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Artrite Reumatoide/imunologia , Doenças Autoimunes/imunologia , Receptor de Morte Celular Programada 1/metabolismo , Células Th17/citologia , Animais , Artrite/imunologia , Artrite/metabolismo , Artrite Experimental , Artrite Reumatoide/metabolismo , Doenças Autoimunes/metabolismo , Diferenciação Celular , Colágeno/química , Modelos Animais de Doenças , Citometria de Fluxo , Humanos , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosfatidilinositol 3-Quinases/metabolismo , FosforilaçãoRESUMO
Staphylococcus aureus remains the major cause of morbidity of bovine mastitis worldwide leading to massive economic losses. Cefquinome is a fourth generation cephalosporin, which preserves susceptibility and antibacterial activity against S. aureus. This work aims to study the pharmacokinetic (PK) and pharmacodynamic (PD) modeling following intramammary administration of cefquinome against S. aureus mastitis. The mouse model of S. aureus mastitis was developed for the PK/PD experiments. The plasma PK characteristics after intramammary injection of cefquinome at various single doses of 25, 50, 100, 200, 400 µg per gland (both fourth pairs of glands: L4 and R4) were calculated using one-compartment and first-order absorption model. PD study was investigated based on twenty-one intermittent dosing regimens, of which total daily dose ranged from 25 to 4800 µg per mouse and dosage intervals included 8, 12 or 24 h. The sigmoid Emax model of inhibitory effect was employed for PK/PD modeling. The results of PK/PD integration of cefquinome against S. aureus suggested that the percentage of duration that drug concentration exceeded the minimal inhibitory concentration (%T>MIC) and the ratio of area under time-concentration curve over MIC (AUC/MIC) are important indexes to evaluate the antibacterial activity. The PK/PD parameters of %T>MIC and AUC0-24/MIC were 35.98% and 137.43 h to obtain a 1.8 logCFU/gland reduction of bacterial colony counts in vivo, against S. aureus strains with cefquinome MIC of 0.5µg/ml.
Assuntos
Antibacterianos/administração & dosagem , Cefalosporinas/administração & dosagem , Mastite/microbiologia , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacos , Animais , Antibacterianos/farmacocinética , Área Sob a Curva , Bovinos , Cefalosporinas/farmacocinética , Modelos Animais de Doenças , Feminino , Injeções , Mastite/tratamento farmacológico , Camundongos , Testes de Sensibilidade Microbiana , Resultado do TratamentoRESUMO
Previously, we demonstrated that CD28 and CTLA-4 signaling control Casitas-B-lineage lymphoma (Cbl)-b protein expression, which is critical for T cell activation and tolerance induction. However, the molecular mechanism(s) of this regulation remains to be elucidated. In this study, we found that Cbl-b fails to undergo tyrosine phosphorylation upon CD3 stimulation because SHP-1 is recruited to and dephosphorylates Cbl-b, whereas CD28 costimulation abrogates this interaction. In support of this finding, T cells lacking SHP-1 display heightened tyrosine phosphorylation and ubiquitination of Cbl-b upon TCR stimulation, which correlates with decreased levels of Cbl-b protein. The aberrant Th2 phenotype observed in T cell-specific Shp1(-/-) mice is reminiscent of heightened Th2 response in Cblb(-/-) mice. Indeed, overexpressing Cbl-b in T cell-specific Shp1(-/-) T cells not only inhibits heightened Th2 differentiation in vitro, but also Th2 responses and allergic airway inflammation in vivo. Therefore, SHP-1 regulates Cbl-b-mediated T cell responses by controlling its tyrosine phosphorylation and ubiquitination.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/fisiologia , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Linfócitos T/imunologia , Animais , Antígenos CD28/fisiologia , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 6/química , Células Th2/imunologia , UbiquitinaçãoRESUMO
BACKGROUND: Haemophilus parasuis (H. parasuis) causes Glässer's disease and multisystem infectious disease. It is one of the major causes of nursery mortality in swine herds. Cefquinome (CEQ) is proposed for the treatment of pigs against respiratory tract infection. However, few studies have investigated the PK/PD characteristics and PK/PD cutoff of this drug against H. parasuis. RESULTS: A total of 213 H. parasuis strains were isolated from diseased pigs in China. The minimal inhibitory concentrations (MICs) of CEQ against these isolates were determined. The MIC(50) and MIC(90) values were 0.125 and 8 mg/L, respectively. An in vitro dynamic PK/PD infection model was used to investigate the antimicrobial effect of CEQ against H. parasuis strain of serotype 5. The target values of CEQ for 3-log(10)-unit and 4-log10-unit decreases effects were the percent time that CEQ concentrations were above the minimum inhibitory concentration (T% > MIC) of 61 and 71 respectively. According to Monte Carlo simulation, the PK/PD cutoff for CEQ against H. parasuis was 0.06 mg/L. The suggested dose regimen was 4 mg/kg/12 h BW. CONCLUSIONS: The value of PK/PD surrogate marker T% > MIC is of great utility in CEQ clinical usage. The very first CEQ PK/PD cutoff provide fundamental data for CEQ breakpoint determination. A more desirable dose regimen against H. parasuis was provided for CEQ using in China district.
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Antibacterianos/farmacologia , Cefalosporinas/farmacocinética , Haemophilus parasuis/efeitos dos fármacos , Antibacterianos/administração & dosagem , Cefalosporinas/administração & dosagem , Técnicas In Vitro , Testes de Sensibilidade Microbiana/veterinária , Método de Monte CarloRESUMO
Lupus nephritis (LN) is a challenging problem that affects 50% of patients with systemic lupus erythematosus (SLE) without effective therapy. Here, we report that A77 1726, the active metabolite of leflunomide, effectively inhibits development of LN and attenuates the generalized autoimmune features. A77 1726 suppresses the expansion of double negative (DN) T cells, and inhibits T and B cell activation. Intriguingly, A77 1726 treatment significantly increases CD4(+)Foxp3(+) regulatory T cells but suppresses potential "pathogenic" IL-17-producing DN T cells in lymph nodes. In vitro experiment shows that A77 1726 potentiates the conversion of naive CD4(+)CD25(-) T cells into CD4(+)CD25(+)Foxp3(+) inducible regulatory T cells (iTregs) by inhibiting Akt. Taken together, our data indicate that the therapeutic effects of A77 1726 in murine LN are mediated, at least in part, by augmenting iTregs which suppress pathogenic IL-17-producing DN T cells through an Akt-dependent mechanism.
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Compostos de Anilina/farmacologia , Hidroxibutiratos/farmacologia , Imunossupressores/farmacologia , Interleucina-17/metabolismo , Rim/efeitos dos fármacos , Nefrite Lúpica/imunologia , Linfócitos T Reguladores/efeitos dos fármacos , Animais , Anticorpos Antinucleares/imunologia , Crotonatos , Modelos Animais de Doenças , Interleucina-17/imunologia , Rim/imunologia , Rim/patologia , Nefrite Lúpica/patologia , Camundongos , Nitrilas , Proteínas Proto-Oncogênicas c-akt/metabolismo , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Reguladores/imunologia , ToluidinasRESUMO
CD28 costimulation is essential for the development of thymic-derived CD4(+)CD25(+)Foxp3(+) regulatory T cells ("tTregs"). E3 ubiquitin ligase Cbl-b has been shown to regulate CD28 dependence of T cell activation. In this paper, we report that the loss of Cbl-b partially but significantly rescues the defective development of tTregs in Cd28(-/-) mice. This partial rescue is independent of IL-2. Mechanistically, Cbl-b binds to Foxp3 upon TCR stimulation and, together with Stub1, targets Foxp3 for ubiquitination and subsequently degradation in the proteasome. As Cbl-b self-ubiquitination and proteasomal degradation is impaired in Cd28(-/-) T cells, the defective development of tTregs in Cd28(-/-) mice may in part be due to increased Foxp3 ubiquitination and degradation targeted by Stub1 and Cbl-b. Treating Cd28(-/-) mice with a proteasome inhibitor completely rescues defective tTreg development in these mice. Therefore, Cbl-b, together with Stub1, ubiquitinate Foxp3, and regulate tTreg development.
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Proteínas Adaptadoras de Transdução de Sinal/imunologia , Fatores de Transcrição Forkhead/imunologia , Ativação Linfocitária/imunologia , Proteínas Proto-Oncogênicas c-cbl/imunologia , Linfócitos T Reguladores/imunologia , Animais , Fatores de Transcrição Forkhead/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Timo/citologia , Timo/imunologia , Transfecção , Ubiquitina-Proteína Ligases/imunologia , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
It has been documented that caspase-8, a central player in apoptosis, is also crucial for TCR-mediated NF-κB activation. However, whether other caspases are also involved this process is unknown. In this report, we showed that in addition to caspase-8, caspase-9 is required for TCR-mediated NF-κB activation. Caspase-9 induces activation of PKC-θ, phosphorylation of Bcl10 and NF-κB activation in a caspase-3-dependent manner, but it appears that Bcl10 phosphorylation is uncoupled from NF-κB activation. Furthermore, caspase-8 lies upstream of caspase-9 during T cell activation. Therefore, TCR ligation elicits a caspase cascade involving caspase-8, caspase-9 and caspase-3 which initiates PKC-θ-dependent pathway leading to NF-κB activation and PKC-θ-independent Bcl10 phosphorylation which limits NF-kB activity.
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Caspases/metabolismo , Isoenzimas/metabolismo , Ativação Linfocitária/fisiologia , NF-kappa B/metabolismo , Proteína Quinase C/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Animais , Células Cultivadas , Humanos , Células Jurkat , Camundongos , Camundongos Endogâmicos C57BL , Complexos Multienzimáticos/metabolismo , Proteína Quinase C-theta , Linfócitos TRESUMO
The antibacterial efficacy of valnemulin against Staphylococcus aureus was studied ex vivo in broiler chickens after intravenous and intramuscular administration at a dose of 10 mg/kg bodyweight (BW). The minimum inhibitory concentrations (MICs) of valnemulin against S. aureus strains ATCC 25923 in broth and serum were 0.12 and 1 µg/mL, respectively. The MIC50 and MIC90 of valnemulin against all susceptible S. aureus strains isolated from chickens in the test population were 0.06 and 0.12 µg/mL, respectively. Protein binding, which greatly influences the efficacy of valnemulin, was assayed by equilibrium dialysate in vitro. A high binding fraction of 86.2% was found, which seems in good agreement with the difference of bacterial susceptibility tests observed in broth and serum. The surrogate index of AUC0-24/MIC required for the lowest bacteriostatic effect, and 2 log10CFU reduction in bacterial count were 24.4 h and 38.0 h, respectively. The required daily dose of valnemulin for a bacteriostatic activity was calculated to be 15 mg/kg BW based on the MIC90 of 0.12 µg/mL. Considering the slow disposition process of valnemulin and an AUC0-24 h value of more than 10-fold obtained from diseased animals, a suggested dose of 3 mg/kg BW is sufficient to achieve a satisfactory therapeutic efficacy in infected broilers. Due to the time-dependent antibacterial characteristics of valnemulin, the recommended daily dose should be split into two or three sub-doses to achieve the highest effectiveness while diminishing the risk of development of bacterial resistance.
Assuntos
Antibacterianos/farmacologia , Antibacterianos/farmacocinética , Galinhas , Administração Oral , Animais , Antibacterianos/efeitos adversos , Antibacterianos/sangue , Área Sob a Curva , Diterpenos/efeitos adversos , Diterpenos/sangue , Diterpenos/farmacocinética , Diterpenos/farmacologia , Injeções Intramusculares/veterinária , Testes de Sensibilidade Microbiana/veterinária , Doenças das Aves Domésticas/tratamento farmacológico , Doenças das Aves Domésticas/microbiologia , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/veterinária , Staphylococcus aureus/efeitos dos fármacosRESUMO
E3 ubiquitin ligase Cbl-b has emerged as a gatekeeper that controls the activation threshold of the T cell antigen receptor and maintains the balance between tolerance and autoimmunity. Here, we report that the loss of Cbl-b facilitates T helper 2 (Th2) and Th9 cell differentiation in vitro. In a mouse model of asthma, the absence of Cbl-b results in severe airway inflammation and stronger Th2 and Th9 responses. Mechanistically, Cbl-b selectively associates with Stat6 upon IL-4 ligation and targets Stat6 for ubiquitination and degradation. These processes are heightened in the presence of T cell receptor (TCR)/CD28 costimulation. Furthermore, we identify K108 and K398 as Stat6 ubiquitination sites. Intriguingly, introducing Stat6 deficiency into Cblb(-/-) mice abrogates hyper-Th2 responses but only partially attenuates Th9 responses. Therefore, our data reveal a function for Cbl-b in the regulation of Th2 and Th9 cell differentiation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Asma/metabolismo , Diferenciação Celular , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Células Th2/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Asma/imunologia , Antígenos CD28/metabolismo , Interleucina-4/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteólise , Proteínas Proto-Oncogênicas c-cbl/genética , Fator de Transcrição STAT6/genética , Fator de Transcrição STAT6/metabolismo , Células Th2/citologia , Células Th2/imunologia , UbiquitinaçãoRESUMO
E3 ubiquitin ligase Casitas-B-lineage lymphoma protein-b (Cbl-b) is critical for establishing the threshold for T cell activation and is essential for induction of T cell anergy. Recent studies suggest that Cbl-b is involved in the development of CD4(+)CD25(+) inducible regulatory T cells (iTregs). In this study, we report that the optimal induction of Foxp3 by naive CD4(+)CD25(-) T cells requires suboptimal TCR triggering. In the absence of Cbl-b, the TCR strength for optimal Foxp3 induction is downregulated in vitro. Using TCR-transgenic Rag(-/-) mice in combination with Cbl-b deficiency, we show that in vivo iTreg development is also controlled by Cbl-b via tuning the TCR strength. Furthermore, we show that Akt-2 but not Akt-1 regulates Foxp3 expression downstream of Cbl-b. Therefore, we demonstrate that Cbl-b regulates the fate of iTregs via controlling the threshold for T cell activation.