RESUMO
Whether interleukin-17A (IL-17A) has pathogenic and/or protective roles in the gut mucosa is controversial and few studies have analyzed specific cell populations for protective functions within the inflamed colonic tissue. Here we have provided evidence for IL-17A-dependent regulation of the tight junction protein occludin during epithelial injury that limits excessive permeability and maintains barrier integrity. Analysis of epithelial cells showed that in the absence of signaling via the IL-17 receptor adaptor protein Act-1, the protective effect of IL-17A was abrogated and inflammation was enhanced. We have demonstrated that after acute intestinal injury, IL-23R(+) γδ T cells in the colonic lamina propria were the primary producers of early, gut-protective IL-17A, and this production of IL-17A was IL-23 independent, leaving protective IL-17 intact in the absence of IL-23. These results suggest that IL-17-producing γδ T cells are important for the maintenance and protection of epithelial barriers in the intestinal mucosa.
Assuntos
Colite/fisiopatologia , Interleucina-17/fisiologia , Interleucina-23/fisiologia , Mucosa Intestinal/fisiopatologia , Doença Aguda , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Animais , Linhagem Celular Tumoral , Polaridade Celular , Colite/induzido quimicamente , Neoplasias do Colo/patologia , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Epitélio/fisiopatologia , Proteínas de Homeodomínio/fisiologia , Humanos , Interleucina-17/deficiência , Interleucina-17/farmacologia , Subpopulações de Linfócitos/metabolismo , Camundongos , Camundongos Knockout , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/deficiência , Ocludina/metabolismo , Permeabilidade , Transporte Proteico , Receptores de Antígenos de Linfócitos T gama-delta/análise , Proteínas Recombinantes/farmacologia , Junções Íntimas/fisiologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Two common missense variants in APOL1 (G1 and G2) have been definitively linked to CKD in black Americans. However, not all individuals with the renal-risk genotype develop CKD, and little is known about how APOL1 variants drive disease. Given the association of APOL1 with HDL particles, which are cleared by the kidney, differences in the level or quality of mutant APOL1HDL particles could be causal for disease and might serve as a useful risk stratification marker. We measured plasma levels of G0 (low risk), G1, and G2 APOL1 in 3450 individuals in the Dallas Heart Study using a liquid chromatography-MS method that enabled quantitation of the different variants. Additionally, we characterized native APOL1HDL from donors with no or two APOL1 risk alleles by size-exclusion chromatography and analysis of immunopurified APOL1HDL particles. Finally, we identified genetic loci associated with plasma APOL1 levels and tested for APOL1-dependent association with renal function. Although we replicated the previous association between APOL1 variant status and renal function in nondiabetic individuals, levels of circulating APOL1 did not associate with microalbuminuria or GFR. Furthermore, the size or known components of APOL1HDL did not consistently differ in subjects with the renal-risk genotype. Genetic association studies implicated variants in loci harboring haptoglobin-related protein (HPR), APOL1, and ubiquitin D (UBD) in the regulation of plasma APOL1 levels, but these variants did not associate with renal function. Collectively, these data demonstrate that the risk of renal disease associated with APOL1 is probably not related to circulating levels of the mutant protein.
Assuntos
Apolipoproteínas/sangue , Lipoproteínas HDL/sangue , Insuficiência Renal Crônica/sangue , Adulto , Apolipoproteína L1 , Apolipoproteínas/genética , Estudos de Coortes , Estudos Transversais , Feminino , Variação Genética , Genótipo , Humanos , Lipoproteínas HDL/genética , Masculino , Insuficiência Renal Crônica/epidemiologia , Insuficiência Renal Crônica/genética , Fatores de RiscoRESUMO
BACKGROUND: Proglucagon-derived peptides (PGDPs), which include glucagon-like peptide (GLP)-1, glucagon, and oxyntomodulin, are key regulators of glucose homeostasis and satiety. These peptide hormones are typically measured with immuno-based assays (e.g., ELISA, RIA), which often suffer from issues of selectivity. METHODS: We developed a multiplexed assay for measuring PGDPs including GLP-1 (7-36) amide, GLP-1 (9-36) amide, glucagon, and oxyntomodulin by mass spectrometry and used this assay to examine the effect of a meal tolerance test on circulating concentrations of these hormones. Participants fasted overnight and were either given a meal (n = 8) or continued to fast (n = 4), with multiple blood collections over the course of 3 h. Plasma samples were analyzed by microflow immunoaffinity (IA)-LC-MS/MS with an isotope dilution strategy. RESULTS: Assay performance characteristics were examined and established during analytical validation for all peptides. Intra- and interassay imprecision were found to be 2.2%-10.7% and 6.8%-22.5%, respectively. Spike recovery was >76%, and dilution linearity was established up to a 16-fold dilution. Immediately after the meal tolerance test, GLP-1 and oxyntomodulin concentrations increased and had an almost identical temporal relationship, and glucagon concentrations increased with a slight delay. CONCLUSIONS: IA-LC-MS/MS was used for the simultaneous and selective measurement of PGDPs. This work includes the first indication of the physiological concentrations and modulation of oxyntomodulin after a meal.
Assuntos
Jejum , Peptídeo 1 Semelhante ao Glucagon/sangue , Glucagon/sangue , Imunoensaio , Oxintomodulina/sangue , Espectrometria de Massas em Tandem , Animais , Cromatografia Líquida de Alta Pressão , Glucagon/imunologia , Peptídeo 1 Semelhante ao Glucagon/imunologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Oxintomodulina/imunologiaRESUMO
Elevated levels of proinflammatory cytokines associated with infection and inflammation can modulate cytochrome P450 enzymes, leading to potential disease-drug interactions and altered small-molecule drug disposition. We established a human-derived hepatocyte-Kupffer cell (Hep:KC) coculture model to assess the indirect cytokine impact on hepatocytes through stimulation of KC-mediated cytokine release and compared this model with hepatocytes alone. Characterization of Hep:KC cocultures showed an inflammation response after treatment with lipopolysaccharide and interleukin (IL)-6 (indicated by secretion of various cytokines). Additionally, IL-6 exposure upregulated acute-phase proteins (C-reactive protein, alpha-1-acid glycoprotein, and serum amyloid A2) and downregulated CYP3A4. Compared with hepatocytes alone, Hep:KC cocultures showed enhanced IL-1ß-mediated effects but less impact from both IL-2 and IL-23. Hep:KC cocultures treated with IL-1ß exhibited a higher release of proinflammatory cytokines, an increased upregulation of acute-phase proteins, and a larger extent of metabolic enzyme and transporter suppression. IC50 values for IL-1ß-mediated CYP3A4 suppression were lower in Hep:KC cocultures (98.0-144 pg/ml) compared with hepatocytes alone (IC50 > 5000 pg/ml). Cytochrome suppression was preventable by blocking IL-1ß interaction with IL-1R1 using an antagonist cytokine or an anti-IL-1ß antibody. Unlike IL-1ß, IL-6-mediated effects were comparable between hepatocyte monocultures and Hep:KC cocultures. IL-2 and IL-23 caused a negligible inflammation response and a minimal inhibition of CYP3A4. In both hepatocyte monocultures and Hep:KC cocultures, IL-2RB and IL-23R were undetectable, whereas IL-6R and IL-1R1 levels were higher in Hep:KC cocultures. In summary, compared with hepatocyte monocultures, the Hep:KC coculture system is a more robust in vitro model for studying the impact of proinflammatory cytokines on metabolic enzymes.
Assuntos
Proteínas de Transporte/metabolismo , Hepatócitos/metabolismo , Inflamação/metabolismo , Interleucinas/metabolismo , Células de Kupffer/metabolismo , Células 3T3 , Adulto , Animais , Transporte Biológico/fisiologia , Proteína C-Reativa/metabolismo , Linhagem Celular , Técnicas de Cocultura/métodos , Citocromo P-450 CYP3A/metabolismo , Regulação para Baixo/fisiologia , Glicoproteínas/metabolismo , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Proteína Amiloide A Sérica/metabolismo , Regulação para Cima/fisiologiaRESUMO
RATIONALE: Human genetics studies in African Americans have shown a strong correlation between polymorphisms in the ApoL1 gene and chronic kidney disease (CKD). To gain further insight into the etiology of ApoL1-associated kidney diseases, the determination of circulating levels of both wild type as well as ApoL1 variants could be of significant use. To date, antibodies that discriminate between all three ApoL1 variant forms (wild type, G1 and G2) are not available. We aimed to develop a rapid method for detecting and quantifying ApoL1 variants and total levels in plasma. METHODS: Ultra-performance liquid chromatography (UPLC) and tandem mass spectrometry (MS/MS) in multiple-reaction monitoring acquisition mode was used to quantify ApoL1. RESULTS: We demonstrated that it is feasible to detect and quantify ApoL1 variants (wild type, G1 and G2), and total ApoL1 concentrations in plasma. ApoL1 genotypes determined by LC/MS agreed perfectly with the traditional method DNA sequencing for 74 human subjects. The method exhibited at least three orders of linearity with a lower limit of quantification of 10 nM. Moreover, the method can readily be multiplexed for the quantification of a panel of protein markers in a single sample. CONCLUSIONS: The method reported herein obviates the need to perform DNA genotyping of ApoL1 variants, which is of significant value in cases where stored samples are unsuitable for DNA analysis. More importantly, the method could potentially be of use in the early identification of individuals at risk of developing CKD, and for the stratification of patients for treatment with future ApoL1-modifying therapies.
Assuntos
Apolipoproteínas/sangue , Apolipoproteínas/genética , Cromatografia Líquida de Alta Pressão/métodos , Variação Genética , Nefropatias/sangue , Lipoproteínas HDL/sangue , Lipoproteínas HDL/genética , Espectrometria de Massas em Tandem/métodos , Sequência de Aminoácidos , Animais , Apolipoproteína L1 , Genótipo , Humanos , Nefropatias/diagnóstico , Nefropatias/genética , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência MolecularRESUMO
Interleukin-10 (IL-10) is an immunosuppressive cytokine that signals through STAT3 to regulate T follicular helper (Tfh) cell differentiation and germinal center formation. In SIV-infected macaques, levels of IL-10 in plasma and lymph nodes (LNs) were induced by infection and not normalized with antiretroviral therapy (ART). During chronic infection, plasma IL-10 and transcriptomic signatures of IL-10 signaling were correlated with the cell-associated SIV-DNA content within LN CD4+ memory subsets, including Tfh cells, and predicted the frequency of CD4+ Tfh cells and their cell-associated SIV-DNA content during ART, respectively. In ART-treated rhesus macaques, cells harboring SIV-DNA by DNAscope were preferentially found in the LN B cell follicle in proximity to IL-10. Finally, we demonstrated that the in vivo neutralization of soluble IL-10 in ART-treated, SIV-infected macaques reduced B cell follicle maintenance and, by extension, LN memory CD4+ T cells, including Tfh cells and those expressing PD-1 and CTLA-4. Thus, these data support a role for IL-10 in maintaining a pool of target cells in lymphoid tissue that serve as a niche for viral persistence. Targeting IL-10 signaling to impair CD4+ T cell survival and improve antiviral immune responses may represent a novel approach to limit viral persistence in ART-suppressed people living with HIV.
Assuntos
Infecções por HIV , Síndrome de Imunodeficiência Adquirida dos Símios , Vírus da Imunodeficiência Símia , Animais , Linfócitos T CD4-Positivos , Infecções por HIV/tratamento farmacológico , Humanos , Interleucina-10/genética , Macaca mulatta , Síndrome de Imunodeficiência Adquirida dos Símios/tratamento farmacológicoRESUMO
Purpose: To evaluate the feasibility of using the Proximity Extension Assay (PEA) platform to detect biomarkers in vitreous and to compare the findings with results obtained with an electrochemiluminescent (ECL) sandwich immunoassay. Methods: Vitreous samples from patients with proliferative diabetic retinopathy (PDR) and non-diabetic controls were tested using two different proteomics platforms. Forty-one assays were completed with the ECL platform and 459 with the PEA platform. Spearman's rank correlation coefficient (rs ) was used to determine the direction and strength of the relationship between protein levels detected by both platforms. Results: Three hundred sixty-six PEA assays detected the tested protein in at least 25% of samples, and the difference in protein abundance between PDR and controls was statistically significant for 262 assays. Seventeen ECL assays yielded a detection rate ≥ 25%, and the difference in protein concentration between PDR and controls was statistically significant for 13 proteins. There was a subset of proteins that were detected by both platforms, and for those the Spearman's correlation coefficient was higher than 0.8. Conclusions: PEA is suitable for the analysis of vitreous samples, showing a strong correlation with the ECL platform. The detection rate of PEA panels was higher than the panels tested with ECL. The levels of several proinflammatory and angiogenic cytokines were significantly higher in PDR vitreous compared to controls. Translational Relevance: This study provides new information on the yields of small-volume assays that can detect proteins of interest in ocular specimens, and it identifies patterns of cytokine dysregulation in PDR.
Assuntos
Diabetes Mellitus , Retinopatia Diabética , Biomarcadores , Citocinas , Retinopatia Diabética/diagnóstico , Humanos , Proteômica , Corpo VítreoRESUMO
The programmed cell death 1 (PD-1) pathway represents a major immune checkpoint, which may be engaged by cells in the tumor microenvironment to overcome active T-cell immune surveillance. Pembrolizumab (Keytruda®, MK-3475) is a potent and highly selective humanized mAb of the IgG4/kappa isotype designed to directly block the interaction between PD-1 and its ligands, PD-L1 and PD-L2. This blockade enhances the functional activity of T cells to facilitate tumor regression and ultimately immune rejection. Pembrolizumab binds to human and cynomolgus monkey PD-1 with picomolar affinity and blocks the binding of human and cynomolgus monkey PD-1 to PD-L1 and PD-L2 with comparable potency. Pembrolizumab binds both the C'D and FG loops of PD-1. Pembrolizumab overcomes human and cynomolgus monkey PD-L1-mediated immune suppression in T-cell cultures by enhancing IL2 production following staphylococcal enterotoxin B stimulation of healthy donor and cancer patient cells, and IFNγ production in human primary tumor histoculture. Ex vivo and in vitro studies with human and primate T cells show that pembrolizumab enhances antigen-specific T-cell IFNγ and IL2 production. Pembrolizumab does not mediate FcR or complement-driven effector function against PD-1-expressing cells. Pembrolizumab displays dose-dependent clearance and half-life in cynomolgus monkey pharmacokinetic and toxicokinetic studies typical for human IgG4 antibodies. In nonhuman primate toxicology studies, no findings of toxicologic significance were observed. The preclinical data for pembrolizumab are consistent with the clinical anticancer activity and safety that has been demonstrated in human clinical trials.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais Humanizados/farmacocinética , Leucócitos Mononucleares/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Linfócitos T/efeitos dos fármacos , Animais , Antineoplásicos Imunológicos/farmacocinética , Antineoplásicos Imunológicos/farmacologia , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/imunologia , Antígeno B7-H1/metabolismo , Feminino , Humanos , Inibidores de Checkpoint Imunológico/farmacocinética , Inibidores de Checkpoint Imunológico/farmacologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/patologia , Macaca fascicularis , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias/imunologia , Neoplasias/patologia , Proteína 2 Ligante de Morte Celular Programada 1/antagonistas & inibidores , Proteína 2 Ligante de Morte Celular Programada 1/imunologia , Proteína 2 Ligante de Morte Celular Programada 1/metabolismo , Receptor de Morte Celular Programada 1/imunologia , Linfócitos T/imunologia , Linfócitos T/patologia , Distribuição Tecidual , Testes de ToxicidadeRESUMO
BACKGROUND: Thymic stromal lymphopoietin (TSLP) is an attractive therapeutic target for the treatment of allergic diseases, and plasma TSLP is a potential patient selection marker in the development of therapeutic agents. RESULTS: We developed and validated an ultrasensitive electrochemiluminescence assay for measurement of TSLP in plasma with a lower limit of quantitation of 0.12 pg/ml, which allowed the quantitation of TSLP in approximately 90% of human plasma samples tested. The assay demonstrated excellent performance characteristics, including precision, accuracy, sensitivity and dilution linearity. Stability and biological variability of TSLP in plasma were also assessed for clinical sample analysis and data interpretation. CONCLUSION: The validated TSLP assay enables assessment of circulating TSLP as a patient selection marker in the development of therapeutics to treat atopic diseases.
Assuntos
Citocinas/sangue , Hipersensibilidade/tratamento farmacológico , Biomarcadores , Citocinas/uso terapêutico , Humanos , Linfopoietina do Estroma do TimoRESUMO
Tumor immune surveillance and cancer immunotherapies are thought to depend on the intratumoral infiltration of activated CD8(+) T cells. Intratumoral CD8(+) T cells are rare and lack activity. IL-10 is thought to contribute to the underlying immune suppressive microenvironment. Defying those expectations we demonstrate that IL-10 induces several essential mechanisms for effective antitumor immune surveillance: infiltration and activation of intratumoral tumor-specific cytotoxic CD8(+) T cells, expression of the Th1 cytokine interferon-γ (IFNγ) and granzymes in CD8(+) T cells, and intratumoral antigen presentation molecules. Consequently, tumor immune surveillance is weakened in mice deficient for IL-10 whereas transgenic overexpression of IL-10 protects mice from carcinogenesis. Treatment with pegylated IL-10 restores tumor-specific intratumoral CD8(+) T cell function and controls tumor growth.
Assuntos
Interferon gama/metabolismo , Interleucina-10/metabolismo , Neoplasias Experimentais/imunologia , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Neoplasias da Mama/metabolismo , Linfócitos T CD8-Positivos/enzimologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Feminino , Granzimas/metabolismo , Humanos , Interferon gama/genética , Interleucina-10/genética , Interleucina-10/imunologia , Subunidade alfa de Receptor de Interleucina-10/genética , Subunidade alfa de Receptor de Interleucina-10/metabolismo , Neoplasias Mamárias Experimentais/irrigação sanguínea , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transplante de Neoplasias , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/patologia , Perforina/metabolismo , Neoplasias Cutâneas/induzido quimicamente , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/patologia , Baço/metabolismo , Transplante Heterólogo , Carga Tumoral , Evasão TumoralRESUMO
Bone erosion is a clinical endpoint for various diseases including rheumatoid arthritis. In this paper, we used rodent arthritis models with severe bone erosion to examine the structural, cellular, and molecular aspects of the inflammation-driven bone resorption process. Our data show that bone loss is observed only in chronically, severely inflamed joints. The most severely affected anatomic sites were the metatarsal phalangeal joint and tarsal bones of the paw. The magnitude of the inflammation-driven bone erosion was dependent on both the duration of inflammatory response and the severity of the joint swelling response. The application of micro-computed tomography well demonstrated the therapeutic benefit of anti-IL-17A in protection of bones from erosion. Alterations in the cellular profile of the joint occurred prior to any major structural deterioration of the bone. Receptor activator for nuclear factor κB ligand, a potent inducer of osteoclast differentiation and bone resorption, was elevated in animals coincident with severe arthritis initiation. The experimental approaches and concepts outlined in this paper provide a valuable process to evaluate and quantify therapies that modulate rodent arthritis-associated bone-erosion models.