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1.
Transpl Int ; 36: 10749, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-36891519

RESUMEN

We previously reported associations between autoantibodies to the LG3 fragment of perlecan, anti-LG3, and a higher risk of delayed graft function (DGF) in kidney transplant recipients. Here, we aimed to determine whether some factors that modulate ischemia-reperfusion injury (IRI) can modify this association. We performed a retrospective cohort study in kidney transplant recipients in 2 university-affiliated centers. In 687 patients, we show that high pre-transplant anti-LG3 are associated with DGF when the kidney is transported on ice (odds ratio (OR): 1.75, 95% confidence interval 1.02-3.00), but not when placed on hypothermic perfusion pump (OR: 0.78, 95% CI 0.43-1.37). In patients with DGF, high pre-transplant anti-LG3 are associated with a higher risk of graft failure (subdistribution hazard ratio (SHR): 4.07, 95% CI: 1.80, 9.22), while this was not the case in patients with immediate graft function (SHR: 0.50, 95% CI 0.19, 1.29). High anti-LG3 levels are associated with a higher risk of DGF in kidneys exposed to cold storage, but not when hypothermic pump perfusion is used. High anti-LG3 are also associated with a higher risk of graft failure in patients who experience DGF, a clinical manifestation of severe IRI.


Asunto(s)
Funcionamiento Retardado del Injerto , Trasplante de Riñón , Humanos , Funcionamiento Retardado del Injerto/etiología , Trasplante de Riñón/efectos adversos , Estudios Retrospectivos , Riñón , Perfusión , Supervivencia de Injerto , Factores de Riesgo , Donantes de Tejidos
2.
Proteomics ; 13(7): 1108-20, 2013 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-23436686

RESUMEN

The stress status of the apoptotic cell can promote phenotypic changes that have important consequences on the immunogenicity of the dying cell. Autophagy is one of the biological processes activated in response to a stressful condition. It is an important mediator of intercellular communications, both by regulating the unconventional secretion of molecules, including interleukin 1ß, and by regulating the extracellular release of ATP from early stage apoptotic cells. Additionally, autophagic components can be released in a caspase-dependent manner by serum-starved human endothelial cells that have engaged apoptotic and autophagic processes. The nature and the components of the extracellular vesicles released by dying autophagic cells are not known. In this study, we have identified extracellular membrane vesicles that are released by human endothelial cells undergoing apoptosis and autophagy, and characterized their biochemical, ultrastructural, morphological properties as well as their proteome. These extracellular vesicles differ from classical apoptotic bodies because they do not contain nucleus components and are released independently of Rho-associated, coiled-coil containing protein kinase 1 activation. Instead, they are enriched with autophagosomes and mitochondria and convey various danger signals, including ATP, suggesting that they could be involved in the modulation of innate immunity.


Asunto(s)
Autofagia , Exosomas/metabolismo , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Medio de Cultivo Libre de Suero/farmacología , Activación Enzimática/efectos de los fármacos , Exosomas/ultraestructura , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/ultraestructura , Humanos , Necrosis , Fagosomas/efectos de los fármacos , Fagosomas/metabolismo , Fagosomas/ultraestructura , Proteómica , Quinasas Asociadas a rho/metabolismo
3.
Transplantation ; 107(7): 1580-1592, 2023 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-36728359

RESUMEN

BACKGROUND: Potentially harmful nonhuman leukocyte antigen antibodies have been identified in renal transplantation, including natural immunoglobulin G antibodies (Nabs) reactive to varied antigenic structures, including apoptotic cells. METHODS: In this retrospective, multicenter study, we assessed Nabs by reactivity to apoptotic cells in sera collected from 980 kidney transplant recipients across 4 centers to determine their association with graft outcomes. RESULTS: Elevated pretransplant Nabs were associated with graft loss (hazard ratio [HR] 2.71; 95% confidence interval [CI], 1.15-6.39; P = 0.0232), the composite endpoint of graft loss or severe graft dysfunction (HR 2.40; 95% CI, 1.13-5.10; P = 0.0232), and T cell-mediated rejection (odds ratio [OR] 1.77; 95% CI, 1.07-3.02; P = 0.0310). High pretransplant Nabs together with donor-specific antibodies (DSAs) were associated with increased risk of composite outcomes (HR 6.31; 95% CI, 1.81-22.0; P = 0.0039). In patients with high pretransplant Nabs, the subsequent development of posttransplant Nabs was associated with both T cell-mediated rejection (OR 3.64; 95% CI, 1.61-8.36; P = 0.0021) and mixed rejection (OR 3.10; 95% CI, 1.02-9.75; P = 0.0473). Finally, elevated pre- and posttransplant Nabs combined with DSAs were associated with increased risk of composite outcomes (HR 3.97; 95% CI, 1.51-10.43; P = 0.0052) and T cell-mediated rejection (OR 7.28; 95% CI, 2.16-25.96; P = 0.0016). CONCLUSIONS: The presence of pre- and posttransplant Nabs, together with DSAs, was associated with increased risk of poor graft outcomes and rejection after renal transplantation.


Asunto(s)
Trasplante de Riñón , Humanos , Trasplante de Riñón/efectos adversos , Estudios Retrospectivos , Trasplante Homólogo , Inmunoglobulina G , Antígenos HLA , Aloinjertos , Rechazo de Injerto , Supervivencia de Injerto
4.
PLoS One ; 16(12): e0261987, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34972158

RESUMEN

Nanoparticles made of the coat protein of papaya mosaic virus (PapMV) and a single-strand RNA were previously shown to be an efficient antigen presentation system for the trigger of cellular immunity. Engineering of PapMV nano with a cytotoxic T lymphocyte epitope was previously shown activating specific T lymphocytes through a proteasome-independent major histocompatibility complex class I (MHC-I) cross-presentation. In this study, we provide new insights into the mechanism of the MHC-I cross-presentation mediated by PapMV nanoparticles. We demonstrate that PapMV nanoparticles do not require the transporter associated with antigen presentation (TAP), but rather depend on lysosome acidification and cathepsin S protease activity for presentation of the T cell epitope. We have also linked the induction of autophagy with this vacuolar MHC-I cross-presentation process. Interestingly, autophagy is induced in antigen-presenting cells after PapMV nanoparticles exposure and inhibition of autophagy reduce MHC-I cross-presentation. This study demonstrates that autophagy is associated with TAP- and proteasome-independent MHC-I cross-presentation. A deeper understanding of the autophagy-dependent MHC-I cross-presentation will be useful in designing vaccination platforms that aim to trigger an efficient cytotoxic T lymphocyte response.


Asunto(s)
Presentación de Antígeno , Células Presentadoras de Antígenos/inmunología , Autofagia , Reactividad Cruzada/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Catepsinas/química , Cloroquina/química , Epítopos/química , Epítopos de Linfocito T/química , Humanos , Lisosomas/química , Microscopía Confocal , Nanopartículas/química , Potexvirus , Ingeniería de Proteínas , ARN/química
5.
Sci Transl Med ; 7(318): 318ra200, 2015 Dec 16.
Artículo en Inglés | MEDLINE | ID: mdl-26676607

RESUMEN

Autoantibodies to components of apoptotic cells, such as anti-perlecan antibodies, contribute to rejection in organ transplant recipients. However, mechanisms of immunization to apoptotic components remain largely uncharacterized. We used large-scale proteomics, with validation by electron microscopy and biochemical methods, to compare the protein profiles of apoptotic bodies and apoptotic exosome-like vesicles, smaller extracellular vesicles released by endothelial cells downstream of caspase-3 activation. We identified apoptotic exosome-like vesicles as a central trigger for production of anti-perlecan antibodies and acceleration of rejection. Unlike apoptotic bodies, apoptotic exosome-like vesicles triggered the production of anti-perlecan antibodies in naïve mice and enhanced anti-perlecan antibody production and allograft inflammation in mice transplanted with an MHC (major histocompatibility complex)-incompatible aortic graft. The 20S proteasome core was active within apoptotic exosome-like vesicles and controlled their immunogenic activity. Finally, we showed that proteasome activity in circulating exosome-like vesicles increased after vascular injury in mice. These findings open new avenues for predicting and controlling maladaptive humoral responses to apoptotic cell components that enhance the risk of rejection after transplantation.


Asunto(s)
Lesión Renal Aguda/enzimología , Aorta/trasplante , Apoptosis/inmunología , Autoanticuerpos/biosíntesis , Micropartículas Derivadas de Células/enzimología , Exosomas/enzimología , Rechazo de Injerto/enzimología , Isquemia/enzimología , Complejo de la Endopetidasa Proteasomal/metabolismo , Lesión Renal Aguda/inmunología , Lesión Renal Aguda/patología , Aloinjertos , Animales , Aorta/enzimología , Aorta/inmunología , Aorta/patología , Autoanticuerpos/inmunología , Biomarcadores/metabolismo , Micropartículas Derivadas de Células/inmunología , Micropartículas Derivadas de Células/patología , Células Cultivadas , Modelos Animales de Enfermedad , Exosomas/inmunología , Exosomas/patología , Rechazo de Injerto/inmunología , Rechazo de Injerto/patología , Proteoglicanos de Heparán Sulfato/inmunología , Proteoglicanos de Heparán Sulfato/metabolismo , Células Endoteliales de la Vena Umbilical Humana/enzimología , Células Endoteliales de la Vena Umbilical Humana/inmunología , Células Endoteliales de la Vena Umbilical Humana/patología , Humanos , Inmunidad Humoral , Isquemia/inmunología , Isquemia/patología , Túbulos Renales Proximales/enzimología , Túbulos Renales Proximales/inmunología , Túbulos Renales Proximales/patología , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Músculo Liso Vascular/enzimología , Músculo Liso Vascular/inmunología , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/enzimología , Miocitos del Músculo Liso/inmunología , Miocitos del Músculo Liso/patología , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Complejo de la Endopetidasa Proteasomal/inmunología , Proteómica/métodos , Ratas , Factores de Tiempo
6.
Autophagy ; 10(12): 2193-207, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25495560

RESUMEN

Recent evidence suggests that autophagy may favor fibrosis through enhanced differentiation of fibroblasts in myofibroblasts. Here, we sought to characterize the mediators and signaling pathways implicated in autophagy-induced myofibroblast differentiation. Fibroblasts, serum starved for up to 4 d, showed increased LC3-II/-I ratios and decreased SQSTM1/p62 levels. Autophagy was associated with acquisition of markers of myofibroblast differentiation including increased protein levels of ACTA2/αSMA (actin, α 2, smooth muscle, aorta), enhanced gene and protein levels of COL1A1 (collagen, type I, α 1) and COL3A1, and the formation of stress fibers. Inhibiting autophagy with 3 different class I phosphoinositide 3-kinase and class III phosphatidylinositol 3-kinase (PtdIns3K) inhibitors or through ATG7 silencing prevented myofibroblast differentiation. Autophagic fibroblasts showed increased expression and secretion of CTGF (connective tissue growth factor), and CTGF silencing prevented myofibroblast differentiation. Phosphorylation of the MTORC1 target RPS6KB1/p70S6K kinase was abolished in starved fibroblasts. Phosphorylation of AKT at Ser473, a MTORC2 target, was reduced after initiation of starvation but was followed by spontaneous rephosphorylation after 2 d of starvation, suggesting the reactivation of MTORC2 with sustained autophagy. Inhibiting MTORC2 activation with long-term exposure to rapamycin or by silencing RICTOR, a central component of the MTORC2 complex abolished AKT rephosphorylation. Both RICTOR silencing and rapamycin treatment prevented CTGF and ACTA2 upregulation, demonstrating the central role of MTORC2 activation in CTGF induction and myofibroblast differentiation. Finally, inhibition of autophagy with PtdIns3K inhibitors or ATG7 silencing blocked AKT rephosphorylation. Collectively, these results identify autophagy as a novel activator of MTORC2 signaling leading to CTGF induction and myofibroblast differentiation.


Asunto(s)
Autofagia , Diferenciación Celular/fisiología , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Complejos Multiproteicos/metabolismo , Miofibroblastos/citología , Serina-Treonina Quinasas TOR/metabolismo , Células Cultivadas , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Humanos , Diana Mecanicista del Complejo 2 de la Rapamicina , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal , Activación Transcripcional/fisiología , Factor de Crecimiento Transformador beta/metabolismo , Regulación hacia Arriba
7.
Autophagy ; 8(6): 927-37, 2012 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-22692030

RESUMEN

The endothelium plays a central role in the regulation of vascular wall cellularity and tone by secreting an array of mediators of importance in intercellular communication. Nutrient deprivation of human endothelial cells (EC) evokes unconventional forms of secretion leading to the release of nanovesicles distinct from apoptotic bodies and bearing markers of multivesicular bodies (MVB). Nutrient deficiency is also a potent inducer of autophagy and vesicular transport pathways can be assisted by autophagy. Nutrient deficiency induced a significant and rapid increase in autophagic features, as imaged by electron microscopy and immunoblotting analysis of LC3-II/LC3-I ratios. Increased autophagic flux was confirmed by exposing serum-starved cells to bafilomycin A 1. Induction of autophagy was followed by indices of an apoptotic response, as assessed by microscopy and poly (ADP-ribose) polymerase cleavage in absence of cell membrane permeabilization indicative of necrosis. Pan-caspase inhibition with ZVAD-FMK did not prevent the development of autophagy but negatively impacted autophagic vacuole (AV) maturation. Adopting a multidimensional proteomics approach with validation by immunoblotting, we determined that nutrient-deprived EC released AV components (LC3I, LC3-II, ATG16L1 and LAMP2) whereas pan-caspase inhibition with ZVAD-FMK blocked AV release. Similarly, nutrient deprivation in aortic murine EC isolated from CASP3/caspase 3-deficient mice induced an autophagic response in absence of apoptosis and failed to prompt LC3 release. Collectively, the present results demonstrate the release of autophagic components by nutrient-deprived apoptotic human cells in absence of cell membrane permeabilization. These results also identify caspase-3 as a novel regulator of AV release.


Asunto(s)
Autofagia , Caspasas/metabolismo , Espacio Extracelular/metabolismo , Vacuolas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Transporte Biológico/efectos de los fármacos , Caspasa 3/deficiencia , Caspasa 3/metabolismo , Inhibidores de Caspasas/farmacología , Cromatografía Liquida , Medio de Cultivo Libre de Suero , Activación Enzimática/efectos de los fármacos , Espacio Extracelular/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/ultraestructura , Humanos , Espectrometría de Masas , Ratones , Vacuolas/efectos de los fármacos , Vacuolas/ultraestructura
8.
Appl Environ Microbiol ; 73(2): 477-84, 2007 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-17085696

RESUMEN

Although the number of Escherichia coli bacteria in surface waters can differ greatly between locations, relatively little is known about the distribution of E. coli pathotypes in surface waters used as sources for drinking or recreation. DNA microarray technology is a suitable tool for this type of study due to its ability to detect high numbers of virulence and antimicrobial resistance genes simultaneously. Pathotype, phylogenetic group, and antimicrobial resistance gene profiles were determined for 308 E. coli isolates from surface water samples collected from diverse aquatic ecosystems at six different sites in the St. Clair River and Detroit River areas. A higher frequency (48%) of E. coli isolates possessing virulence and antimicrobial resistance genes was observed in an urban site located downstream of wastewater effluent outfalls than in the other examined sites (average of 24%). Most E. coli pathotypes were extraintestinal pathogenic E. coli (ExPEC) pathotypes and belonged to phylogenetic groups B2 and D. The ExPEC pathotypes were found to occur across all aquatic ecosystems investigated, including riverine, estuarine, and offshore lake locations. The results of this environmental study using DNA microarrays highlight the widespread distribution of E. coli pathotypes in aquatic ecosystems and the potential public health threat of E. coli pathotypes originating from municipal wastewater sources.


Asunto(s)
Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana/genética , Ecosistema , Escherichia coli/clasificación , Ríos/microbiología , Factores de Virulencia/genética , Canadá , Escherichia coli/efectos de los fármacos , Escherichia coli/aislamiento & purificación , Escherichia coli/patogenicidad , Infecciones por Escherichia coli/microbiología , Humanos , Michigan , Análisis de Secuencia por Matrices de Oligonucleótidos , Filogenia , Virulencia/genética
9.
Appl Environ Microbiol ; 72(6): 4200-6, 2006 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-16751532

RESUMEN

Escherichia coli is generally described as a commensal species with occasional pathogenic strains. Due to technological limitations, there is currently little information concerning the prevalence of pathogenic E. coli strains in the environment. For the first time, using a DNA microarray capable of detecting all currently described virulence genes and commonly found antimicrobial resistance genes, a survey of environmental E. coli isolates from recreational waters was carried out. A high proportion (29%) of 308 isolates from a beach site in the Great Lakes carried a pathotype set of virulence-related genes, and 14% carried antimicrobial resistance genes, findings consistent with a potential risk for public health. The results also showed that another 8% of the isolates had unusual virulence gene combinations that would be missed by conventional screening. This new application of a DNA microarray to environmental waters will likely have an important impact on public health, epidemiology, and microbial ecology in the future.


Asunto(s)
Farmacorresistencia Bacteriana , Escherichia coli/genética , Escherichia coli/fisiología , Agua Dulce/microbiología , Virulencia/genética , Antibacterianos/farmacología , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , Escherichia coli/efectos de los fármacos , Escherichia coli/patogenicidad , Pruebas de Sensibilidad Microbiana , Análisis de Secuencia por Matrices de Oligonucleótidos , Recreación , Estaciones del Año , Estados Unidos , Microbiología del Agua
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