RESUMO
Melanoma is an aggressive skin cancer that has become increasingly prevalent in western populations. Current treatments such as surgery, chemotherapy, and high-dose radiation have had limited success, often failing to treat late stage, metastatic melanoma. Alternative strategies such as immunotherapies have been successful in treating a small percentage of patients with metastatic disease, although these treatments to date have not been proven to enhance overall survival. Several melanoma antigens (Ags) proposed as targets for immunotherapeutics include tyrosinase, NY-ESO-1, gp-100, and Mart-1, all of which contain both human leukocyte antigen (HLA) class I and class II-restricted epitopes necessary for immune recognition. We have previously shown that an enzyme, gamma-IFN-inducible lysosomal thiol-reductase (GILT), is abundantly expressed in professional Ag presenting cells (APCs), but absent or expressed at greatly reduced levels in many human melanomas. In the current study, we report that increased GILT expression generates a greater pool of antigenic peptides in melanoma cells for enhanced CD4+ T cell recognition. Our results suggest that the induction of GILT in human melanoma cells could aid in the development of a novel whole-cell vaccine for the enhancement of immune recognition of metastatic melanoma.
Assuntos
Melanoma , Compostos de Sulfidrila , Apresentação de Antígeno , Antígenos de Neoplasias , Antígenos HLA , Humanos , Lisossomos/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , PeptídeosRESUMO
Gamma interferon inducible lysosomal thiol reductase (GILT), is known to be involved in immunity, but its role in hematopoiesis has not been previously reported. Herein, we demonstrate using gilt knockout (-/-) mice that loss of gilt associates with decreased numbers and cycling status of femoral hematopoietic progenitor cells (CFU-GM, BFU-E, and CFU-GEMM) with more modest effects on splenic progenitor cells. Thus, GILT is associated with positive regulation of hematopoietic progenitor cells in mice, mainly in bone marrow.
Assuntos
Regulação Enzimológica da Expressão Gênica , Células-Tronco Hematopoéticas/enzimologia , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/biossíntese , Animais , Camundongos , Camundongos Knockout , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genéticaRESUMO
Heat shock protein 90 (Hsp90) is a protein chaperone that is upregulated and released from pancreatic ß cells under pro-inflammatory conditions. We hypothesized that serum Hsp90 may have utility as a biomarker of type 1 diabetes risk and exhibit elevations before the onset of clinically significant hyperglycemia. To this end, total levels of the alpha cytoplasmic isoform of Hsp90 were assayed in autoantibody-positive progressors to type 1 diabetes using banked serum samples from the TrialNet Pathway to Prevention Cohort that had been collected 12 months prior to diabetes onset, with comparison to age, sex, and BMI-category matched autoantibody-positive nonprogressors and healthy controls. Hsp90 levels were higher in autoantibody-positive progressors and nonprogressors ≤ 18 years of age compared to matched healthy controls. However, Hsp90 levels were not different between progressors and nonprogressors in any age group. Hsp90 was positively correlated with age in control subjects, but this correlation was absent in autoantibody positive individuals. In aggregate these data indicate that elevated Hsp90 levels are present in youth with ß cell autoimmunity, but are not able to distinguish youth or adult type 1 diabetes progressors from nonprogressors in samples collected 12 months prior to diabetes development.
Assuntos
Autoimunidade , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/imunologia , Proteínas de Choque Térmico HSP90/sangue , Células Secretoras de Insulina/metabolismo , Distribuição por Idade , Autoanticorpos/sangue , Biomarcadores/sangue , Criança , Feminino , Humanos , Masculino , Curva ROCRESUMO
OBJECTIVE: Abnormally elevated proinsulin secretion has been reported in type 2 and early type 1 diabetes when significant C-peptide is present. We questioned whether individuals with long-standing type 1 diabetes and low or absent C-peptide secretory capacity retained the ability to make proinsulin. RESEARCH DESIGN AND METHODS: C-peptide and proinsulin were measured in fasting and stimulated sera from 319 subjects with long-standing type 1 diabetes (≥3 years) and 12 control subjects without diabetes. We considered three categories of stimulated C-peptide: 1) C-peptide positive, with high stimulated values ≥0.2 nmol/L; 2) C-peptide positive, with low stimulated values ≥0.017 but <0.2 nmol/L; and 3) C-peptide <0.017 nmol/L. Longitudinal samples were analyzed from C-peptide-positive subjects with diabetes after 1, 2, and 4 years. RESULTS: Of individuals with long-standing type 1 diabetes, 95.9% had detectable serum proinsulin (>3.1 pmol/L), while 89.9% of participants with stimulated C-peptide values below the limit of detection (<0.017 nmol/L; n = 99) had measurable proinsulin. Proinsulin levels remained stable over 4 years of follow-up, while C-peptide decreased slowly during longitudinal analysis. Correlations between proinsulin with C-peptide and mixed-meal stimulation of proinsulin were found only in subjects with high stimulated C-peptide values (≥0.2 nmol/L). Specifically, increases in proinsulin with mixed-meal stimulation were present only in the group with high stimulated C-peptide values, with no increases observed among subjects with low or undetectable (<0.017 nmol/L) residual C-peptide. CONCLUSIONS: In individuals with long-duration type 1 diabetes, the ability to secrete proinsulin persists, even in those with undetectable serum C-peptide.
Assuntos
Diabetes Mellitus Tipo 1/metabolismo , Proinsulina/metabolismo , Adolescente , Adulto , Peptídeo C/sangue , Estudos de Coortes , Diabetes Mellitus Tipo 1/sangue , Jejum/metabolismo , Feminino , Humanos , Insulina/sangue , Masculino , Refeições , Pessoa de Meia-Idade , Proinsulina/sangue , Fatores de Tempo , Adulto JovemRESUMO
Autophagy plays critical but diverse roles in cellular quality control and homeostasis potentially checking tumor development by removing mutated or damaged macromolecules, while conversely fostering tumor survival by supplying essential nutrients during cancer progression. This report documents a novel inhibitory role for a lysosome-associated membrane protein, LAMP-2C in modulating autophagy and melanoma cell growth in vitro and in vivo. Solid tumors such as melanomas encounter a variety of stresses in vivo including inflammatory cytokines produced by infiltrating lymphocytes directed at limiting tumor growth and spread. Here, we report that in response to the anti-tumor, pro-inflammatory cytokine interferon-gamma, melanoma cell expression of LAMP2C mRNA significantly increased. These results prompted an investigation of whether increased melanoma cell expression of LAMP-2C might represent a mechanism to control or limit human melanoma growth and survival. In this study, enhanced expression of human LAMP-2C in melanoma cells perturbed macroautophagy and chaperone-mediated autophagy in several human melanoma lines. In vitro analysis showed increasing LAMP-2C expression in a melanoma cell line, triggered reduced cellular LAMP-2A and LAMP-2B protein expression. Melanoma cells with enhanced LAMP-2C expression displayed increased cell cycle arrest, increased expression of the cell cycle regulators Chk1 and p21, and greater apoptosis and necrosis in several cell lines tested. The increased abundance of Chk1 protein in melanoma cells with increased LAMP-2C expression was not due to higher CHEK1 mRNA levels, but rather an increase in Chk1 protein abundance including Chk1 molecules phosphorylated at Ser345. Human melanoma cell xenografts with increased LAMP-2C expression, displayed reduced growth in immune compromised murine hosts. Melanomas with high LAMP-2C expression showed increased necrosis and reduced cell density upon histological analysis. These results reveal a novel role for LAMP-2C in negatively regulating melanoma growth and survival.
RESUMO
Excessive drinking can lead to the development of immune dysfunction. Our aim is to investigate the effect of alcohol on immune activation from circulating peripheral blood monocytes in excessive drinkers (EDs). Twenty-two EDs and healthy controls were enrolled. Time line follow-back was used to quantify the amount of alcohol consumed in the past 30 days before enrollment. Peripheral blood-derived CD14+ monocytes were isolated for gene expression analyses. Serum interleukin (IL)-6, IL-10 and lipopolysaccharides (LPS) were also measured. We found that serum LPS concentrations were significantly higher in EDs compared with controls (P<0.05). While no differences in the levels of circulating IL-6 and IL-10 were observed, the relative levels of gene transcripts (RQ) for Il6 (an M1-polarizing cytokine) and Il10 (an M2-polarizing cytokine) were significantly higher in peripheral blood-derived monocytes from EDs compared with controls (Il6: P<0.01. Il10: P<0.05). EDs exhibit early immune activation of peripheral blood monocyte mRNA transcripts, notably Il6 and Il10 Future studies are needed to explore the clinical implications of our findings and determine whether the levels of Il6 and Il10 mRNA expression can be used to identify those with excessive drinking and to monitor for alcohol abstinence.
Assuntos
Consumo de Bebidas Alcoólicas/imunologia , Consumo de Bebidas Alcoólicas/patologia , Monócitos/patologia , Adulto , Consumo de Bebidas Alcoólicas/sangue , Estudos de Casos e Controles , Estudos de Coortes , Feminino , Humanos , Imunidade Inata , Interleucina-10/sangue , Interleucina-10/genética , Interleucina-6/sangue , Interleucina-6/genética , Lipopolissacarídeos/sangue , Masculino , Monócitos/metabolismo , Projetos Piloto , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Activation of the lysosomal ceramide-producing enzyme, acid sphingomyelinase (ASM), by various stresses is centrally involved in cell death and has been implicated in autophagy. We set out to investigate the role of the baseline ASM activity in maintaining physiological functions of lysosomes, focusing on the lysosomal nutrient-sensing complex (LYNUS), a lysosomal membrane-anchored multiprotein complex that includes mammalian target of rapamycin (mTOR) and transcription factor EB (TFEB). ASM inhibition with imipramine or sphingomyelin phosphodiesterase 1 (SMPD1) siRNA in human lung cells, or by transgenic Smpd1+/- haploinsufficiency of mouse lungs, markedly reduced mTOR- and P70-S6 kinase (Thr 389)-phosphorylation and modified TFEB in a pattern consistent with its activation. Inhibition of baseline ASM activity significantly increased autophagy with preserved degradative potential. Pulse labeling of sphingolipid metabolites revealed that ASM inhibition markedly decreased sphingosine (Sph) and Sph-1-phosphate (S1P) levels at the level of ceramide hydrolysis. These findings suggest that ASM functions to maintain physiological mTOR signaling and inhibit autophagy and implicate Sph and/or S1P in the control of lysosomal function.
Assuntos
Autofagia/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Complexos Multiproteicos/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Esfingomielina Fosfodiesterase/antagonistas & inibidores , Animais , Células Cultivadas , Inibidores Enzimáticos/química , Humanos , Imipramina/química , Imipramina/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Complexos Multiproteicos/metabolismo , RNA Interferente Pequeno/química , RNA Interferente Pequeno/farmacologia , Esfingomielina Fosfodiesterase/deficiência , Esfingomielina Fosfodiesterase/metabolismoRESUMO
A major obstacle in predicting and preventing the development of autoimmune type 1 diabetes (T1D) in at-risk individuals is the lack of well-established early biomarkers indicative of ongoing beta cell stress during the pre-clinical phase of disease. Recently, serum levels of the α cytoplasmic isoform of heat-shock protein 90 (hsp90) were shown to be elevated in individuals with new-onset T1D. We therefore hypothesized that hsp90α could be released from beta cells in response to cellular stress and inflammation associated with the earliest stages of T1D. Here, human beta cell lines and cadaveric islets released hsp90α in response to stress induced by treatment with a combination of pro-inflammatory cytokines including interleukin-1ß, tumour necrosis factor-α and interferon-γ. Mechanistically, hsp90α release was found to be driven by cytokine-induced endoplasmic reticulum stress mediated by c-Jun N-terminal kinase (JNK), a pathway that can eventually lead to beta cell apoptosis. Cytokine-induced beta cell hsp90α release and JNK activation were significantly reduced by pre-treating cells with the endoplasmic reticulum stress-mitigating chemical chaperone tauroursodeoxycholic acid. The hsp90α release by cells may therefore be a sensitive indicator of stress during inflammation and a useful tool in assessing therapeutic mitigation of cytokine-induced cell damage linked to autoimmunity.
Assuntos
Proteínas de Choque Térmico HSP90/metabolismo , Inflamação/imunologia , Células Secretoras de Insulina/imunologia , Células Secretoras de Insulina/metabolismo , Adulto , Células Cultivadas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estresse Oxidativo/imunologiaRESUMO
Airway epithelial CD55 down-regulation occurs in several hypoxia-associated pulmonary diseases, but the mechanism is unknown. Using in vivo and in vitro assays of pharmacologic inhibition and gene silencing, the current study investigated the role of hypoxia-inducible factor (HIF)-1α in regulating airway epithelial CD55 expression. Hypoxia down-regulated CD55 expression on small-airway epithelial cells in vitro, and in murine lungs in vivo; the latter was associated with local complement activation. Treatment with pharmacologic inhibition or silencing of HIF-1α during hypoxia-recovered CD55 expression in small-airway epithelial cells. HIF-1α overexpression or blockade, in vitro or in vivo, down-regulated CD55 expression. Collectively, these data show a key role for HIF-1α in regulating the expression of CD55 on airway epithelium.
Assuntos
Antígenos CD55/metabolismo , Epitélio/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Pulmão/metabolismo , Aminoácidos Dicarboxílicos/farmacologia , Animais , Hipóxia Celular/efeitos dos fármacos , Ativação do Complemento/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Epitélio/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BLRESUMO
OBJECTIVE: We tested whether an elevation in the serum proinsulin-to-C-peptide ratio (PI:C), a biomarker of ß-cell endoplasmic reticulum (ER) dysfunction, was associated with progression to type 1 diabetes. RESEARCH DESIGN AND METHODS: Fasting total PI and C levels were measured in banked serum samples obtained from TrialNet Pathway to Prevention (PTP) participants, a cohort of autoantibody-positive relatives without diabetes of individuals with type 1 diabetes. Samples were obtained â¼12 months before diabetes onset from PTP progressors in whom diabetes developed (n = 60), and were compared with age-, sex-, and BMI-matched nonprogressors who remained normoglycemic (n = 58). PI:C ratios were calculated as molar ratios and were multiplied by 100% to obtain PI levels as a percentage of C levels. RESULTS: Although absolute PI levels did not differ between groups, PI:C ratios were significantly increased in antibody-positive subjects in whom there was progression to diabetes compared with nonprogressors (median 1.81% vs. 1.17%, P = 0.03). The difference between groups was most pronounced in subjects who were ≤10 years old, where the median progressor PI:C ratio was nearly triple that of nonprogressors; 90.0% of subjects in this age group within the upper PI:C quartile progressed to the development of diabetes. Logistic regression analysis, adjusted for age and BMI, demonstrated increased odds of progression for higher natural log PI:C ratio values (odds ratio 1.44, 95% CI 1.02, 2.05). CONCLUSIONS: These data suggest that ß-cell ER dysfunction precedes type 1 diabetes onset, especially in younger children. Elevations in the serum PI:C ratio may have utility in predicting the onset of type 1 diabetes in the presymptomatic phase.
Assuntos
Peptídeo C/sangue , Diabetes Mellitus Tipo 1/sangue , Jejum/sangue , Proinsulina/sangue , Adolescente , Adulto , Autoanticorpos/sangue , Biomarcadores/sangue , Bancos de Sangue , Criança , Diabetes Mellitus Tipo 1/genética , Progressão da Doença , Feminino , Humanos , Estudos Longitudinais , Masculino , Linhagem , Adulto JovemRESUMO
AIMS/HYPOTHESIS: The incidence of type 1 diabetes is increasing at a rate of 3-5% per year. Genetics cannot fully account for this trend, suggesting an influence of environmental factors. The accelerator hypothesis proposes an effect of metabolic factors on type 1 diabetes risk. To test this in the TrialNet Pathway to Prevention (PTP) cohort, we analysed the influence of BMI, weight status and insulin resistance on progression from single to multiple islet autoantibodies (Aab) and progression from normoglycaemia to diabetes. METHODS: HOMA1-IR was used to estimate insulin resistance in Aab-positive PTP participants. Cox proportional hazards models were used to evaluate the effects of BMI, BMI percentile (BMI%), weight status and HOMA1-IR on the progression of autoimmunity or the development of diabetes. RESULTS: Data from 1,310 single and 1,897 multiple Aab-positive PTP participants were included. We found no significant relationships between BMI, BMI%, weight status or HOMA1-IR and the progression from one to multiple Aabs. Similarly, among all Aab-positive participants, no significant relationships were found between BMI, weight status or HOMA1-IR and progression to diabetes. Diabetes risk was modestly increased with increasing BMI% among the entire cohort, in obese participants 13-20 years of age and with increasing HOMA1-IR in adult Aab-positive participants. CONCLUSIONS/INTERPRETATION: Analysis of the accelerator hypothesis in the TrialNet PTP cohort does not suggest a broad influence of metabolic variables on diabetes risk. Efforts to identify other potentially modifiable environmental factors should continue.
Assuntos
Autoanticorpos/imunologia , Índice de Massa Corporal , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/patologia , Resistência à Insulina/imunologia , Resistência à Insulina/fisiologia , Adolescente , Adulto , Criança , Pré-Escolar , Progressão da Doença , Feminino , Humanos , Lactente , Insulina/metabolismo , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
Therapeutic proteins have a propensity for aggregation during manufacturing, shipping, and storage. The presence of aggregates in protein drug products can induce adverse immune responses in patients that may affect safety and efficacy, and so it is of concern to both manufacturers and regulatory agencies. In this vein, there is a lack of understanding of the physicochemical determinants of immunological responses and a lack of standardized analytical methods to survey the molecular properties of aggregates associated with immune activation. In this review, we provide an overview of the basic immune mechanisms in the context of interactions with protein aggregates. We then critically examine the literature with emphasis on the underlying immune mechanisms as they relate to aggregate properties. Finally, we highlight the gaps in our current understanding of this issue and offer recommendations for future research.
Assuntos
Formação de Anticorpos/imunologia , Imunidade Celular/imunologia , Linfócitos/imunologia , Agregados Proteicos/imunologia , Animais , Ensaios Clínicos como Assunto/métodos , Citocinas/imunologia , Citocinas/metabolismo , Humanos , Fenômenos Imunogenéticos , Linfócitos/metabolismoRESUMO
Cells use multiple autophagy pathways to sequester macromolecules, senescent organelles, and pathogens. Several conserved isoforms of the lysosome-associated membrane protein-2 (LAMP-2) regulate these pathways influencing immune recognition and responses. LAMP-2A is required for chaperone-mediated autophagy (CMA), which promotes Ag capture and MHC class II (MHCII) presentation in B cells and signaling in T cells. LAMP-2B regulates lysosome maturation to impact macroautophagy and phagocytosis. Yet, far less is known about LAMP-2C function. Whereas LAMP2A and LAMP2B mRNA were broadly detected in human tissues, LAMP2C expression was more limited. Transcripts for the three LAMP2 isoforms increased with B cell activation, although specific gene induction varied depending on TLR versus BCR engagement. To examine LAMP-2C function in human B cells and specifically its role in Ag presentation, we used ectopic gene expression. Increased LAMP-2C expression in B cells did not alter MHCII expression or invariant chain processing, but did perturb cytoplasmic Ag presentation via CMA. MHCII presentation of epitopes from exogenous and membrane Ags was not affected by LAMP-2C expression in B cells. Similarly, changes in B cell LAMP-2C expression did not impact macroautophagy. The gene expression of other LAMP2 isoforms and proteasome and lysosomal proteases activities were unperturbed by LAMP-2C ectopic expression. LAMP-2C levels modulated the steady-state expression of several cytoplasmic proteins that are targeted for degradation by CMA and diminished peptide translocation via this pathway. Thus, LAMP-2C serves as a natural inhibitor of CMA that can selectively skew MHCII presentation of cytoplasmic Ags.
Assuntos
Apresentação de Antígeno/imunologia , Autofagia/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Proteína 2 de Membrana Associada ao Lisossomo/imunologia , Linfócitos B/imunologia , Separação Celular , Citoplasma/imunologia , Eletroporação , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Immunoblotting , Imunoprecipitação , Isoformas de Proteínas/imunologia , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
Rapid evaluation of therapies designed to preserve ß cells in persons with type 1 diabetes (T1D) is hampered by limited availability of sensitive ß-cell health biomarkers. In particular, biomarkers elucidating the presence and degree of ß-cell stress are needed. We characterized ß-cell secretory activity and stress in 29 new-onset T1D subjects (10.6 ± 3.0 years, 55% male) at diagnosis and then 8.2 ± 1.2 weeks later at first clinic follow-up. We did comparisons with 16 matched healthy controls. We evaluated hemoglobin A1c (HbA1c), ß-cell function (random C-peptide [C] and proinsulin [PI]), ß-cell stress (PI:C ratio), and the ß-cell stress marker heat shock protein (HSP)90 and examined these parameters' relationships with clinical and laboratory characteristics at diagnosis. Mean diagnosis HbA1c was 11.3% (100 mmol/mol) and 7.6% (60 mmol/mol) at follow-up. C-peptide was low at diagnosis (P < 0.001 vs controls) and increased at follow-up (P < 0.001) to comparable with controls. PI did not differ from controls at diagnosis but increased at follow-up (P = 0.003) signifying increased release of PI alongside improved insulin secretion. PI:C ratios and HSP90 concentrations were elevated at both time points. Younger subjects had lower C-peptide and greater PI, PI:C, and HSP90. We also examined islets isolated from prediabetic nonobese diabetic mice and found that HSP90 levels were increased â¼4-fold compared with those in islets isolated from matched CD1 controls, further substantiating HSP90 as a marker of ß-cell stress in T1D. Our data indicate that ß-cell stress can be assessed using PI:C and HSP90. This stress persists after T1D diagnosis. Therapeutic approaches to reduce ß-cell stress in new-onset T1D should be considered.
Assuntos
Diabetes Mellitus Tipo 1/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Células Secretoras de Insulina/metabolismo , Proinsulina/metabolismo , Estresse Fisiológico/fisiologia , Adolescente , Envelhecimento , Animais , Biomarcadores , Glicemia , Peptídeo C/sangue , Peptídeo C/metabolismo , Estudos de Casos e Controles , Criança , Diabetes Mellitus Tipo 1/genética , Feminino , Proteínas de Choque Térmico HSP90/genética , Humanos , Masculino , Camundongos , Proinsulina/genéticaRESUMO
Lysosomal acid lipase (LAL) is a key enzyme controlling neutral lipid metabolic signaling in myeloid-derived suppressor cells (MDSCs). MDSCs from LAL-deficient (lal-/-) mice directly stimulate cancer cell proliferation. PPARγ ligand treatment inhibited lal-/- MDSCs stimulation of tumor cell growth and metastasis in vivo, and tumor cell proliferation and migration in vitro. In addition, PPARγ ligand treatment impaired lal-/- MDSCs transendothelial migration, and differentiation from lineage-negative cells. The corrective effects of PPARγ ligand on lal-/- MDSCs functions were mediated by regulating the mammalian target of rapamycin (mTOR) pathway, and subsequently blocking MDSCs ROS overproduction. Furthermore, in the myeloid-specific dominant-negative PPARγ (dnPPARγ) overexpression bitransgenic mouse model, tumor growth and metastasis were enhanced, and MDSCs from these mice stimulated tumor cell proliferation and migration. MDSCs with dnPPARγ overexpression showed increased transendothelial migration, overactivation of the mTOR pathway, and ROS overproduction. These results indicate that PPARγ plays a critical role in neutral lipid metabolic signaling controlled by LAL, which provides a mechanistic basis for clinically targeting MDSCs to reduce the risk of cancer proliferation, growth and metastasis.
Assuntos
Carcinoma Pulmonar de Lewis/metabolismo , Comunicação Celular , Movimento Celular , Proliferação de Células , Melanoma Experimental/metabolismo , Células Mieloides/metabolismo , PPAR gama/metabolismo , Neoplasias Cutâneas/metabolismo , Animais , Antígenos Ly/metabolismo , Carcinoma Pulmonar de Lewis/genética , Carcinoma Pulmonar de Lewis/secundário , Comunicação Celular/efeitos dos fármacos , Diferenciação Celular , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Técnicas de Cocultura , Predisposição Genética para Doença , Ácidos Linoleicos Conjugados/farmacologia , Melanoma Experimental/genética , Melanoma Experimental/secundário , Potencial da Membrana Mitocondrial , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Mieloides/efeitos dos fármacos , PPAR gama/agonistas , PPAR gama/genética , Fenótipo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Esterol Esterase/deficiência , Esterol Esterase/genética , Serina-Treonina Quinases TOR/metabolismo , Fatores de Tempo , Migração Transendotelial e Transepitelial , Transfecção , Carga TumoralRESUMO
Chronic granulomatous disease (CGD) is an inherited immunodeficiency linked with mutations in the multi-subunit leucocyte NADPH oxidase. Myeloid-derived phagocytic cells deficient in NADPH oxidase fail to produce sufficient levels of reactive oxygen species to clear engulfed pathogens. In this study we show that oxidase also influences B-cell functions, including responses to single-stranded RNA or unmethylated DNA by endosomal Toll-like receptors (TLRs) 7 and 9. In response to TLR7/9 ligands, B-cell lines derived from patients with CGD with mutations in either the NADPH oxidase p40(phox) or p47(phox) subunits produced only low levels of reactive oxygen species. Remarkably, cytokine secretion and p38 mitogen-activated protein kinase activation by these oxidase-deficient B cells was significantly increased upon TLR7/9 activation when compared with oxidase-sufficient B cells. Increased TLR responsiveness was also detected in B cells from oxidase-deficient mice. NADPH oxidase-deficient patient-derived B cells also expressed enhanced levels of TLR7 and TLR9 mRNA and protein compared with the same cells reconstituted to restore oxidase activity. These data demonstrate that the loss of oxidase function associated with CGD can significantly impact B-cell TLR signalling in response to nucleic acids with potential repercussions for auto-reactivity in patients.
Assuntos
Linfócitos B/metabolismo , NADPH Oxidases/deficiência , Receptor 7 Toll-Like/metabolismo , Receptor Toll-Like 9/metabolismo , Animais , Linfócitos B/patologia , Linhagem Celular Transformada , Citocinas/biossíntese , Expressão Ectópica do Gene , Expressão Gênica , Doença Granulomatosa Crônica/genética , Doença Granulomatosa Crônica/imunologia , Doença Granulomatosa Crônica/metabolismo , Humanos , Camundongos , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , NADPH Oxidases/química , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Fosforilação , Subunidades Proteicas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Receptor 7 Toll-Like/agonistas , Receptor 7 Toll-Like/genética , Receptor Toll-Like 9/agonistas , Receptor Toll-Like 9/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Elevated ratios of circulating unmethylated to methylated preproinsulin (INS) DNA have been suggested to reflect ß-cell death in type 1 diabetes (T1D). We tested the hypothesis that absolute levels (rather than ratios) of unmethylated and methylated INS DNA differ between subjects with new-onset T1D and control subjects and assessed longitudinal changes in these parameters. We used droplet digital PCR to measure levels of unmethylated and methylated INS DNA in serum from subjects at T1D onset and at 8 weeks and 1 year post-onset. Compared with control subjects, levels of both unmethylated and methylated INS DNA were elevated at T1D onset. At 8 weeks post-onset, methylated INS DNA remained elevated, but unmethylated INS DNA fell. At 1 year postonset, both unmethylated and methylated INS DNA returned to control levels. Subjects with obesity, type 2 diabetes, and autoimmune hepatitis exhibited lower levels of unmethylated and methylated INS compared with subjects with T1D at onset and no differences compared with control subjects. Our study shows that elevations in both unmethylated and methylated INS DNA occurs in new-onset T1D and that levels of these DNA species change during T1D evolution. Our work emphasizes the need to consider absolute levels of differentially methylated DNA species as potential biomarkers of disease.
Assuntos
Metilação de DNA , DNA/sangue , Diabetes Mellitus Tipo 1/sangue , Insulina/sangue , Precursores de Proteínas/sangue , Adolescente , Criança , Pré-Escolar , Feminino , Humanos , Insulina/genética , Masculino , Precursores de Proteínas/genéticaRESUMO
The alarming increase in type 2 diabetes mellitus (T2DM) underscores the need for efficient screening and preventive strategies. Select protein biomarker profiles emerge over time during T2DM development. Periodic evaluation of these markers will increase the predictive ability of diabetes risk scores. Noninvasive methods for frequent measurements of biomarkers are increasingly being investigated. Application of salivary diagnostics has gained importance with the establishment of significant similarities between the salivary and serum proteomes. The objective of this study is to identify T2DM-specific salivary biomarkers by literature-based discovery. A serial interrogation of the PubMed database was performed using MeSH terms of specific T2DM pathological processes in primary and secondary iterations to compile cohorts of T2DM-specific serum markers. Subsequent search consisted of mining for the identified serum markers in human saliva. More than 60% of T2DM-associated serum proteins have been measured in saliva. Nearly half of these proteins have been reported in diabetic saliva. Measurements of salivary lipids and oxidative stress markers that can exhibit correlated saliva plasma ratio could constitute reliable factors for T2DM risk assessment. We conclude that a high percentage of T2DM-associated serum proteins can be measured in saliva, which offers an attractive and economical strategy for T2DM screening.
RESUMO
Cells rely on multiple intracellular trafficking pathways to capture antigens for proteolysis. The resulting peptides bind to MHC class II molecules to promote CD4(+) T cell recognition. Endocytosis enhances the capture of extracellular and cell surface bound antigens for processing and presentation, while autophagy pathways shunt cytoplasmic and nuclear antigens for presentation in the context of MHC class II molecules. Understanding how physiological changes and cellular stress alter antigen trafficking and the repertoire of peptides presented by class II molecules remains challenging, yet important in devising novel approaches to boost immune responses to pathogens and tumors. An abundant, constitutively expressed cytoplasmic chaperone, HSC70 plays a central role in modulating antigen transport within cells to control MHC class II presentation during nutrient stress. HSC70 may serve as a molecular switch to modulate endocytic and autophagy pathways, impacting the source of antigens delivered for MHC class II presentation during cellular stress.