RESUMEN
Developing an effective Staphylococcus aureus (S. aureus) vaccine has been a challenging endeavor, as demonstrated by numerous failed clinical trials over the years. In this study, we formulated a vaccine containing a highly conserved moonlighting protein, the pyruvate dehydrogenase complex E2 subunit (PDHC), and showed that it induced strong protective immunity against epidemiologically relevant staphylococcal strains in various murine disease models. While antibody responses contributed to bacterial control, they were not essential for protective immunity in the bloodstream infection model. Conversely, vaccine-induced systemic immunity relied on γδ T cells. It has been suggested that prior S. aureus exposure may contribute to the reduction of vaccine efficacy. However, PDHC-induced protective immunity still facilitated bacterial clearance in mice previously exposed to S. aureus. Collectively, our findings indicate that PDHC is a promising serotype-independent vaccine candidate effective against both methicillin-sensitive and methicillin-resistant S. aureus isolates.
Asunto(s)
Infecciones Estafilocócicas , Vacunas Estafilocócicas , Staphylococcus aureus , Animales , Infecciones Estafilocócicas/prevención & control , Infecciones Estafilocócicas/inmunología , Infecciones Estafilocócicas/microbiología , Ratones , Staphylococcus aureus/inmunología , Staphylococcus aureus/enzimología , Vacunas Estafilocócicas/inmunología , Complejo Piruvato Deshidrogenasa/metabolismo , Complejo Piruvato Deshidrogenasa/inmunología , Femenino , Anticuerpos Antibacterianos/inmunología , Modelos Animales de Enfermedad , Humanos , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , Ratones Endogámicos C57BL , Staphylococcus aureus Resistente a Meticilina/inmunología , Piruvato Deshidrogenasa (Lipoamida)/inmunología , Piruvato Deshidrogenasa (Lipoamida)/metabolismo , Piruvato Deshidrogenasa (Lipoamida)/genéticaRESUMEN
Primary biliary cholangitis (PBC) is characterized by the presence of serum anti-mitochondrial autoantibodies (AMAs). To date, four antigens among the 2-oxo-acid dehydrogenase complex family, which commonly have lipoyl domains as an epitope, have been identified as AMA-corresponding antigens (AMA-antigens). It has recently been reported that AMAs react more strongly with certain chemically modified mimics than with the native lipoyl domains in AMA-antigens. Moreover, high concentrations of circulating immune complexes (ICs) in PBC patients have been reported. However, the existence of ICs formed by AMAs and their antigens has not been reported to date. We hypothesized that AMAs and their antigens formed ICs in PBC sera, and analyzed sera of PBC and four autoimmune diseases (Sjögren's syndrome, systemic lupus erythematosus, systemic scleroderma, and rheumatoid arthritis) using immune complexome analysis, in which ICs are separated from serum and are identified by nano-liquid chromatography-tandem mass spectrometry. To correctly assign MS/MS spectra to peptide sequences, we used a protein-search algorithm that including lipoylation and certain xenobiotic modifications. We found three AMA-antigens, the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2), the E2 subunit of the 2-oxo-glutarate dehydrogenase complex (OGDC-E2) and dihydrolipoamide dehydrogenase binding protein (E3BP), by detecting peptides containing lipoylation and xenobiotic modifications from PBC sera. Although the lipoylated sites of these peptides were different from the well-known sites, abnormal lipoylation and xenobiotic modification may lead to production of AMAs and the formation ICs. Further investigation of the lipoylated sites, xenobiotic modifications, and IC formation will lead to deepen our understanding of PBC pathogenesis.
Asunto(s)
Complejo Antígeno-Anticuerpo/inmunología , Autoantígenos/inmunología , Lipoilación/inmunología , Cirrosis Hepática Biliar/inmunología , Mitocondrias/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Autoanticuerpos/inmunología , Epítopos/inmunología , Femenino , Humanos , Persona de Mediana Edad , Complejo Piruvato Deshidrogenasa/inmunología , Espectrometría de Masas en Tándem/métodos , Adulto JovenRESUMEN
The genus Listeria comprises ubiquitous bacteria, commonly present in foods and food production facilities. In this study, three different phage display technologies were employed to discover targets, and to generate and characterize novel antibodies against Listeria: antibody display for biomarker discovery and antibody generation; ORFeome display for target identification; and single-gene display for epitope characterization. With this approach, pyruvate dehydrogenase complex-enzyme 2 (PDC-E2) was defined as a new detection target for Listeria, as confirmed by immunomagnetic separation-mass spectrometry (IMS-MS). Immunoblot and fluorescence microscopy showed that this protein is accessible on the bacterial cell surface of living cells. Recombinant PDC-E2 was produced in E. coli and used to generate 16 additional antibodies. The resulting set of 20 monoclonal scFv-Fc was tested in indirect ELISA against 17 Listeria and 16 non-Listeria species. Two of them provided 100% sensitivity (CI 82.35-100.0%) and specificity (CI 78.20-100.0%), confirming PDC-E2 as a suitable target for the detection of Listeria. The binding region of 18 of these antibodies was analyzed, revealing that ≈ 90% (16/18) bind to the lipoyl domains (LD) of the target. The novel target PDC-E2 and highly specific antibodies against it offer new opportunities to improve the detection of Listeria.
Asunto(s)
Bacteriófagos/inmunología , Listeria/inmunología , Complejo Piruvato Deshidrogenasa/inmunología , Secuencia de Aminoácidos , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos/inmunología , Técnicas de Visualización de Superficie Celular/métodos , Epítopos/inmunología , Escherichia coli/inmunología , Immunoblotting/métodos , Biblioteca de Péptidos , Anticuerpos de Cadena Única/inmunologíaRESUMEN
Metabolic reprogramming during macrophage polarization supports the effector functions of these cells in health and disease. Here, we demonstrate that pyruvate dehydrogenase kinase (PDK), which inhibits the pyruvate dehydrogenase-mediated conversion of cytosolic pyruvate to mitochondrial acetyl-CoA, functions as a metabolic checkpoint in M1 macrophages. Polarization was not prevented by PDK2 or PDK4 deletion but was fully prevented by the combined deletion of PDK2 and PDK4; this lack of polarization was correlated with improved mitochondrial respiration and rewiring of metabolic breaks that are characterized by increased glycolytic intermediates and reduced metabolites in the TCA cycle. Genetic deletion or pharmacological inhibition of PDK2/4 prevents polarization of macrophages to the M1 phenotype in response to inflammatory stimuli (lipopolysaccharide plus IFN-γ). Transplantation of PDK2/4-deficient bone marrow into irradiated wild-type mice to produce mice with PDK2/4-deficient myeloid cells prevented M1 polarization, reduced obesity-associated insulin resistance, and ameliorated adipose tissue inflammation. A novel, pharmacological PDK inhibitor, KPLH1130, improved high-fat diet-induced insulin resistance; this was correlated with a reduction in the levels of pro-inflammatory markers and improved mitochondrial function. These studies identify PDK2/4 as a metabolic checkpoint for M1 phenotype polarization of macrophages, which could potentially be exploited as a novel therapeutic target for obesity-associated metabolic disorders and other inflammatory conditions.
Asunto(s)
Activación de Macrófagos/inmunología , Macrófagos/inmunología , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora/inmunología , Complejo Piruvato Deshidrogenasa/inmunología , Acetilcoenzima A/inmunología , Acetilcoenzima A/metabolismo , Animales , Citosol/inmunología , Citosol/metabolismo , Dieta Alta en Grasa/efectos adversos , Resistencia a la Insulina/genética , Resistencia a la Insulina/inmunología , Macrófagos/clasificación , Macrófagos/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/inmunología , Mitocondrias/metabolismo , Obesidad/etiología , Obesidad/genética , Obesidad/inmunología , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora/deficiencia , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora/genética , Complejo Piruvato Deshidrogenasa/metabolismo , Ácido Pirúvico/inmunología , Ácido Pirúvico/metabolismoRESUMEN
The virulence factors of pathogenic microbes often have single functions that permit immune suppression. However, a proportion possess multiple activities and are considered moonlighting proteins. By examining secreted virulence factors of Staphylococcus aureus, we determine that the bacterial lipoic acid synthetase LipA suppresses macrophage activation. LipA is known to modify the E2 subunit of the metabolic enzyme complex pyruvate dehydrogenase (E2-PDH) with a fatty acid derivative, lipoic acid, yielding the metabolic protein lipoyl-E2-PDH. We demonstrate that lipoyl-E2-PDH is also released by S. aureus and moonlights as a macrophage immunosuppressant by reducing Toll-like receptor 1/2 (TLR1/2) activation by bacterial lipopeptides. A LipA-deficient strain induces heightened pro-inflammatory cytokine production, which is diminished in the absence of TLR2. During murine systemic infection, LipA suppresses pro-inflammatory macrophage activation, rendering these cells inefficient at controlling infection. These observations suggest that bacterial metabolism and immune evasion are linked by virtue of this moonlighting protein.
Asunto(s)
Proteínas Bacterianas/antagonistas & inhibidores , Activación de Macrófagos/efectos de los fármacos , Activación de Macrófagos/inmunología , Infecciones Estafilocócicas/inmunología , Staphylococcus aureus/inmunología , Staphylococcus aureus/metabolismo , Factores de Virulencia/antagonistas & inhibidores , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Médula Ósea , Técnicas de Cultivo de Célula , Citocinas/metabolismo , ADN Bacteriano/aislamiento & purificación , Dihidrolipoamida Deshidrogenasa/inmunología , Modelos Animales de Enfermedad , Femenino , Interacciones Huésped-Parásitos/inmunología , Evasión Inmune , Inmunidad Innata , Macrófagos/inmunología , Ratones , Complejo Piruvato Deshidrogenasa/inmunología , Proteínas Recombinantes , Eliminación de Secuencia , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidad , Ácido Tióctico/metabolismo , Receptor Toll-Like 1/metabolismo , Receptor Toll-Like 2/metabolismo , Factores de Virulencia/inmunologíaRESUMEN
The immune system is a powerful resource for the eradication of cancer, but to overcome the low immunogenicity of tumor cells, a sufficiently strong CD8(+) T cell-mediated adaptive immune response is required. Nanoparticulate biomaterials represent a potentially effective delivery system for cancer vaccines, as they can be designed to mimic viruses, which are potent inducers of cellular immunity. We have been exploring the non-viral pyruvate dehydrogenase E2 protein nanoparticle as a biomimetic platform for cancer vaccine delivery. Simultaneous conjugation of a melanoma-associated gp100 epitope and CpG to the E2 nanoparticle (CpG-gp-E2) yielded an antigen-specific increase in the CD8(+) T cell proliferation index and IFN-γ secretion by 1.5-fold and 5-fold, respectively, compared to an unbound peptide and CpG formulation. Remarkably, a single nanoparticle immunization resulted in a 120-fold increase in the frequency of melanoma epitope-specific CD8(+) T cells in draining lymph nodes and a 30-fold increase in the spleen, relative to free peptide with free CpG. Furthermore, in the very aggressive B16 melanoma murine tumor model, prophylactic immunization with CpG-gp-E2 delayed the onset of tumor growth by approximately 5.5 days and increased animal survival time by approximately 40%, compared to PBS-treated animals. These results show that by combining optimal particle size and simultaneous co-delivery of molecular vaccine components, antigen-specific anti-tumor immune responses can be significantly increased.
Asunto(s)
Vacunas contra el Cáncer/uso terapéutico , Islas de CpG , Melanoma Experimental/prevención & control , Nanopartículas/uso terapéutico , Complejo Piruvato Deshidrogenasa/uso terapéutico , Antígeno gp100 del Melanoma/uso terapéutico , Animales , Biomimética , Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/administración & dosificación , Vacunas contra el Cáncer/inmunología , Células Cultivadas , Sistemas de Liberación de Medicamentos , Epítopos/administración & dosificación , Epítopos/inmunología , Epítopos/uso terapéutico , Femenino , Humanos , Inmunización , Interferón gamma/inmunología , Melanoma Experimental/inmunología , Ratones , Ratones Endogámicos C57BL , Nanopartículas/administración & dosificación , Complejo Piruvato Deshidrogenasa/administración & dosificación , Complejo Piruvato Deshidrogenasa/inmunología , Antígeno gp100 del Melanoma/administración & dosificación , Antígeno gp100 del Melanoma/inmunologíaRESUMEN
Primary biliary cirrhosis is an immune-mediated chronic liver disease whose diagnosis relies on the detection of serum antimitochondrial antibodies directed against a complex set of proteins, among which pyruvate dehydrogenase complex is considered the main autoantigen. We studied the immunological role of the lipoyl domain of this protein using synthetic lipoylated peptides, showing that the lipoyl chain chirality does not affect autoantibody recognition and, most importantly, confirming that both lipoylated and unlipoylated peptides are able to recognize specific autoantibodies in patients sera. In fact, 74% of patients sera recognize at least one of the tested peptides but very few positive sera recognized exclusively the lipoylated peptide, suggesting that the lipoamide moiety plays a marginal role within the autoreactive epitope. These results are supported by a conformational analysis showing that the lipoyl moiety of pyruvate dehydrogenase complex appears to be involved in hydrophobic interactions, which may limit its exposition and thus its contribution to the complex antigenic epitope. A preliminary analysis of the specificity of the two most active peptides indicates that they could be part of a panel of synthetic antigens collectively able to mimic in a simple immunoenzymatic assay the complex positivity pattern detected in immunofluorescence.
Asunto(s)
Epítopos Inmunodominantes/inmunología , Cirrosis Hepática Biliar/diagnóstico , Péptidos , Complejo Piruvato Deshidrogenasa/química , Complejo Piruvato Deshidrogenasa/inmunología , Antígenos/química , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente , Humanos , Técnicas para Inmunoenzimas , Cirrosis Hepática Biliar/sangre , Cirrosis Hepática Biliar/enzimología , Conformación Molecular , Péptidos/síntesis química , Péptidos/química , Piruvato Deshidrogenasa (Lipoamida)/sangre , Piruvato Deshidrogenasa (Lipoamida)/química , Piruvato Deshidrogenasa (Lipoamida)/inmunología , Complejo Piruvato Deshidrogenasa/sangre , Relación Estructura-ActividadRESUMEN
BACKGROUND: Pyruvate dehydrogenase (PDH) activity is altered in many human disorders. Current methods require tissue samples and yield inconsistent results. We describe a modified method for measuring PDH activity from isolated human peripheral blood mononuclear cells (PBMCs). RESULTS/METHODOLOGY: We found that PDH activity and quantity can be successfully measured in human PBMCs. Freeze-thaw cycles cannot efficiently disrupt the mitochondrial membrane. Processing time of up to 20 h does not affect PDH activity with proteinase inhibitor addition and a detergent concentration of 3.3% showed maximum yield. Sample protein concentration is correlated to PDH activity and quantity in human PBMCs from healthy subjects. CONCLUSION: Measuring PDH activity from PBMCs is a novel, easy and less invasive way to further understand the role of PDH in human disease.
Asunto(s)
Leucocitos Mononucleares/inmunología , Complejo Piruvato Deshidrogenasa/inmunología , HumanosRESUMEN
Our understanding of primary biliary cirrhosis has been rapidly growing over the past decade and the disease is now regarded as a model for other female-predominant, organ-specific autoimmune conditions. Primary biliary cirrhosis ensues from a multi-lineage loss of tolerance to the E2 component of the pyruvate dehydrogenase complex. One of the major unanswered questions in the pathogenesis of primary biliary cirrhosis is the specificity of small intrahepatic bile ducts attack while PDC-E2 is present in mitochondria of all nucleated cells. Recent findings suggest that the uniqueness of the primary target tissue, biliary epithelium, may be of considerable importance for understanding primary biliary cirrhosis and that the biliary epithelial cell is more than an innocent victim. Rather, it attracts an immune attack by virtue of the unique apoptotic mechanisms and by the way it handles PDC-E2. Moreover, recent evidence suggests that apoptotic bodies of biliary epithelial cell are able to activate the innate immune system in the presence of anti-mitochondrial antibodies. This review article is intended to provide a critical overview of the role of apoptosis in biliary epithelial cells, the activation of the innate immune system, and its biological and clinical significance in primary biliary cirrhosis.
Asunto(s)
Apoptosis/inmunología , Inmunidad Innata/inmunología , Cirrosis Hepática Biliar/inmunología , Autoanticuerpos/inmunología , Autoantígenos/inmunología , Células Epiteliales/inmunología , Epitelio/inmunología , Medicina Basada en la Evidencia , Humanos , Factores Inmunológicos/inmunología , Cirrosis Hepática Biliar/enzimología , Cirrosis Hepática Biliar/patología , Mitocondrias/inmunología , Complejo Piruvato Deshidrogenasa/inmunología , Complejo Piruvato Deshidrogenasa/metabolismoRESUMEN
Environmental stimulation is a major factor in the initiation and perpetuation of autoimmune diseases. We have addressed this issue and focused on primary biliary cirrhosis (PBC), an autoimmune disease of the liver. Immunologically, PBC is distinguished by immune mediated destruction of the intra hepatic bile ducts and the presence of high titer antimitochondrial autoantibodies (AMA) directed against a highly specific epitope within the lipoic acid binding domain of the pyruvate dehydrogenase E2 subunit (PDC-E2). We submit that the uniqueness of AMA epitope specificity and the conformational changes of the PDC-E2 lipoyl domain during physiological acyl transfer could be the lynchpin to the etiology of PBC and postulate that chemical xenobiotics modification of the lipoyl domain of PDC-E2 is sufficient to break self-tolerance, with subsequent production of AMA in patients with PBC. Indeed, using quantitative structure activity relationship (QSAR) analysis on a peptide-xenobiotic conjugate microarray platform, we have demonstrated that when the lipoyl domain of PDC-E2 was modified with specific synthetic small molecule lipoyl mimics, the ensuing structures displayed highly specific reactivity to PBC sera, at levels often higher than the native PDC-E2 molecule. Hereby, we discuss our recent QSAR analysis data on specific AMA reactivity against a focused panel of lipoic acid mimic in which the lipoyl di-sulfide bond are modified. Furthermore, data on the immunological characterization of antigen and Ig isotype specificities against one such lipoic acid mimic; 6,8-bis(acetylthio)octanoic acid (SAc), when compared with rPDC-E2, strongly support a xenobiotic etiology in PBC. This observation is of particular significance in that approximately one third of patients who have taken excessive acetaminophen (APAP) developed AMA with same specificity as patients with PBC, suggesting that the lipoic domain are a target of APAP electrophilic metabolites such as NAPQI. We submit that in genetically susceptible hosts, electrophilic modification of lipoic acid in PDC-E2 by acetaminophen or similar drugs can facilitate loss of tolerance and lead to the development of PBC.
Asunto(s)
Autoanticuerpos/inmunología , Cirrosis Hepática Biliar/inmunología , Complejo Piruvato Deshidrogenasa/inmunología , Xenobióticos/inmunología , Acetaminofén/efectos adversos , Acetaminofén/inmunología , Acetaminofén/metabolismo , Analgésicos no Narcóticos/efectos adversos , Analgésicos no Narcóticos/inmunología , Analgésicos no Narcóticos/metabolismo , Sitios de Unión/genética , Sitios de Unión/inmunología , Epítopos/química , Epítopos/inmunología , Epítopos/metabolismo , Humanos , Cirrosis Hepática Biliar/sangre , Cirrosis Hepática Biliar/etiología , Modelos Inmunológicos , Modelos Moleculares , Estructura Molecular , Unión Proteica/inmunología , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/inmunología , Subunidades de Proteína/metabolismo , Complejo Piruvato Deshidrogenasa/química , Complejo Piruvato Deshidrogenasa/metabolismo , Ácido Tióctico/química , Ácido Tióctico/inmunología , Ácido Tióctico/metabolismo , Xenobióticos/efectos adversos , Xenobióticos/metabolismoRESUMEN
Primary biliary cirrhosis (PBC) has been often coined a model autoimmune disease based on the homogeneity amongst patients, the frequency and similarity of antimitochondrial antibodies, including the highly directed immune response to pyruvate dehydrogenase (PDC-E2). A significant number of patients with PBC suffer from sicca and amongst these, there are patients who also have classic Sjögren's syndrome. Indeed, both PBC and Sjögren's syndrome are characterized by inflammation of target epithelial elements. Both diseases can be considered on the basis of a number of other related clinical aspects, including proposed unique apoptotic features of the target tissue, the role of secretory IgA, and the frequency with which both diseases overlap with each other. Indeed, PBC may be considered a Sjögren's syndrome of the liver, whereas Sjögren's syndrome can be equally discussed as PBC of the salivary glands. Dissection of the genetic predispositions for both diseases and especially the molecular basis of effector mechanisms, will become critical elements in developing new therapies.
Asunto(s)
Autoanticuerpos/inmunología , Cirrosis Hepática Biliar/inmunología , Hígado/inmunología , Glándulas Salivales/inmunología , Síndrome de Sjögren/inmunología , Autoanticuerpos/sangre , Autoinmunidad , Humanos , Inmunoglobulina A Secretora/inmunología , Cirrosis Hepática Biliar/complicaciones , Cirrosis Hepática Biliar/genética , Cirrosis Hepática Biliar/metabolismo , Mitocondrias/inmunología , Complejo Piruvato Deshidrogenasa/inmunología , Síndrome de Sjögren/complicaciones , Síndrome de Sjögren/genética , Síndrome de Sjögren/metabolismoRESUMEN
Autoantibodies to intracellular targets in mitochondria and nuclei are serological hallmarks of primary biliary cirrhosis (PBC). One of the most recently identified cellular targets of PBC autoantibodies is a novel cytoplasmic structure referred to as GW bodies [GWB, G (glycine) W (tryptophan)-containing bodies (GWB)]. GWB are indentified as discrete cytoplasmic domains that are involved in mRNA processing via the RNA interference (RNAi) pathway. Key components of GWB include the proteins GW182, Ago2, RNA-associated protein 55 (RAP55) and Ge-1/Hedls. The primary objective was to study the frequency and clinical association of antibodies directed to GWB components, in 109 PBC patients. Autoantibodies to mitochondrial antigen-pyruvate dehydrogenase complex (M2), branched-chain 2-oxo-acid dehydrogenase complex and 2-oxo glutarate dehydrogenase complex (3E-BPO), gp210, sp100, promyelocytic leukaemia cell antigen (PML) and liver kidney microsomal-1 antigen (LKM-1) were detected by a line immunoassay and antibodies to GWB (GW182, RAP55, Ge-1, GW2, GW3) and glutamate receptor interacting protein (GRIP)-associated protein-1 (GRASP-1), by an addressable laser bead immunoassay (ALBIA). The most common GWB autoantigen targets were: RAP55-28%, GW182-12%, GW2-2% and antibodies to GRASP-1-17%. By comparison, the frequency of reactivity to established PBC autoantigens was: gp210, 27%; sp100, 27% and PML, 17%. None of the autoantibodies were associated with differences in Mayo risk score or liver decompensation. This study is the first study to show that antibodies to RAP55, GW182 and GRASP-1 are the most common GWB targets in PBC.
Asunto(s)
Autoanticuerpos/inmunología , Autoantígenos/inmunología , Estructuras Citoplasmáticas/inmunología , Cirrosis Hepática Biliar/inmunología , Adulto , Anciano , Antígenos Nucleares/inmunología , Femenino , Humanos , Complejo Cetoglutarato Deshidrogenasa/inmunología , Masculino , Persona de Mediana Edad , Proteínas de Complejo Poro Nuclear/inmunología , Proteínas Nucleares/inmunología , Proteína de la Leucemia Promielocítica , Proteínas/inmunología , Complejo Piruvato Deshidrogenasa/inmunología , Proteínas de Unión al ARN/inmunología , Estudios Retrospectivos , Ribonucleoproteínas/inmunología , Factores de Transcripción/inmunología , Proteínas Supresoras de Tumor/inmunología , Adulto JovenRESUMEN
BACKGROUND: In primary biliary cirrhosis (PBC), autoreactivity mainly targets members of the pyruvate dehydrogenase complex (PDC). Because PDC subunits are expressed on the surface of mycoplasma and molecular mimicry may be one aetiological factor, we analysed the presence of mammalian and mycoplasma PDC-specific antibodies in PBC patients. METHODS: Antibodies to porcine PDC and Mycoplasma pneumoniae (mp) antigens mpPDH-C (to be designated mpPDC-E2 chain), mpPDH-B (to be designated mpPDC-E1beta chain), mpCARDS TX and mpP1 were investigated in sera from 43 PBC patients, 19 patients with autoimmune hepatitis and 11 healthy controls by an enzyme-linked immunosorbent assay and Western blotting. To study the rate of acute mycoplasma infection, an adhesin P1-specific polymerase chain reaction (PCR) was performed. RESULTS: Immune reactivity to the mpPDC-E2 antigen was significantly enhanced in PBC patients (83.7%) as compared with controls (overall frequency of 36.7%), while antibodies to the porcine PDC-E2 chain were found only in PBC patients (88%) excluding a simple cross-reactivity of PDC-related antibodies. This observation was confirmed by inhibition studies demonstrating that porcine PDC did not inhibit mycoplasma PDC-specific antibodies and vice versa. The occurrence of antibodies to mpPDC seems to precede the occurrence of antibodies to porcine PDC. Infection with mycoplasma was equally distributed in the groups as evidenced by an antibody frequency comparable to CARDS TX and P1 and PCR reactivity. CONCLUSION: Because PBC patients show a significantly enhanced frequency of mpPDC-E2-related antibodies, besides other factors, molecular mimicry between surface molecules of mycoplasma and epitopes of the autoantigen may play a central role in the aetiopathology of PBC.
Asunto(s)
Cirrosis Hepática Biliar/inmunología , Cirrosis Hepática Biliar/microbiología , Proteínas Mitocondriales/metabolismo , Imitación Molecular/fisiología , Mycoplasma/metabolismo , Complejo Piruvato Deshidrogenasa/metabolismo , Autotolerancia/fisiología , Adulto , Anciano , Animales , Anticuerpos/sangre , Western Blotting , Cartilla de ADN/genética , Ensayo de Inmunoadsorción Enzimática , Femenino , Técnica del Anticuerpo Fluorescente , Alemania , Humanos , Masculino , Persona de Mediana Edad , Proteínas Mitocondriales/inmunología , Mycoplasma/inmunología , Complejo Piruvato Deshidrogenasa/inmunología , Sus scrofaRESUMEN
Infectious agents have been postulated to play a pathogenic role in the loss of immunological tolerance and the induction of primary biliary cirrhosis, an immune-mediated cholestatic liver disease characterized by progressive destruction of the small intrahepatic bile ducts and subsequent cirrhosis and liver failure. This review discusses emerging issues implicating infectious agents such as Escherichia coli, mycobacteria, chlamydia, helicobacter species, lactobacilli, Novosphingobium aromaticivorans, and betaretroviruses in the pathogenesis of primary biliary cirrhosis. We also review the immunopathological mechanisms responsible for the induction of the disease with special emphasis on the role of molecular mimicry and microbial/self immunological cross-reactivity.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/microbiología , Infecciones Bacterianas/inmunología , Cirrosis Hepática Biliar/inmunología , Cirrosis Hepática Biliar/microbiología , Imitación Molecular/inmunología , Aciltransferasas/inmunología , Animales , Autoanticuerpos/inmunología , Enfermedades Autoinmunes/epidemiología , Infecciones Bacterianas/complicaciones , Conductos Biliares Intrahepáticos/inmunología , Conductos Biliares Intrahepáticos/microbiología , Reacciones Cruzadas/inmunología , Humanos , Cirrosis Hepática Biliar/epidemiología , Complejo Piruvato Deshidrogenasa/inmunologíaRESUMEN
Diagnosis and management of patients with SLE (Systemic Lupus Eritematosus), autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC), involves specific diagnostic tests, such as IFA-AMA, IFA anti-dsDNA and immunoblotting for the detection of autoantibodies for specific autoantigens (mitochondria, dsDNA, M2, LKM-1, LC-1, SLA/LP). We established specific correlation between the detected autoantibodies and corresponding clinical findings. The total of 813 serum specimens were probed with IFA-anti-dsDNA, 98 of which tested positive. We also performed dilution analysis to the end point for all the positive specimens. Numerous specimens were tested by IFA, AMA and immunoblotting.
Asunto(s)
Autoanticuerpos/sangre , Enfermedades Autoinmunes/inmunología , ADN/inmunología , Mitocondrias/inmunología , Adulto , Autoanticuerpos/inmunología , Enfermedades Autoinmunes/sangre , Enfermedades Autoinmunes/diagnóstico , Femenino , Técnica del Anticuerpo Fluorescente , Hepatitis Autoinmune/sangre , Hepatitis Autoinmune/diagnóstico , Hepatitis Autoinmune/inmunología , Humanos , Immunoblotting , Cirrosis Hepática Biliar/sangre , Cirrosis Hepática Biliar/diagnóstico , Cirrosis Hepática Biliar/inmunología , Lupus Eritematoso Sistémico/sangre , Lupus Eritematoso Sistémico/diagnóstico , Lupus Eritematoso Sistémico/inmunología , Masculino , Persona de Mediana Edad , Miocitos del Músculo Liso , Complejo Piruvato Deshidrogenasa/inmunologíaRESUMEN
There is growing evidence that the onset of autoimmune disorders can be linked to the inefficient removal of apoptotic cells. Since defects in the elimination of apoptotic cells lead to secondary necrosis and subsequent release of intracellular components, this might explain the generation of autoantibodies against intracellular antigens. Accordingly, we wanted to investigate, whether antibodies from patients with the autoimmune liver disease primary biliary cirrhosis (PBC) recognize self-proteins generated and released during apoptosis. Using Western blot analyses we could detect intracellular antigens with serum IgG from PBC patients but not with serum IgG from healthy donors in lysates of Jurkat T-leukemia, HepG2 hepatoma, and HT-29 colon-carcinoma cells. Interestingly, PBC serum IgG also recognized caspase substrates in cells undergoing apoptosis induced by staurosporine or TRAIL (TNF-related apoptosis inducing ligand). In addition to intracellular antigens, serum IgG from PBC patients detected caspase-dependent antigens in the supernatants of apoptotic (secondary necrotic) cells and antigens on the surface of apoptotic Jurkat cells. Among the caspase substrates recognized by PBC serum IgG we could identify the components PDC-E2 and -E1beta of the known autoantigen PDC (pyruvate dehydrogenase complex). Thus, caspase-mediated processing of intracellular proteins might generate de novo autoantigens that upon release contribute to the generation of autoantibodies and autoimmune diseases as PBC.
Asunto(s)
Apoptosis/inmunología , Autoanticuerpos/inmunología , Enfermedades Autoinmunes/inmunología , Caspasas/metabolismo , Cirrosis Hepática Biliar/inmunología , Complejo Piruvato Deshidrogenasa/inmunología , Autoanticuerpos/sangre , Enfermedades Autoinmunes/metabolismo , Línea Celular Tumoral , Inhibidores Enzimáticos/farmacología , Humanos , Cirrosis Hepática Biliar/etiología , Cirrosis Hepática Biliar/metabolismo , Complejo Piruvato Deshidrogenasa/metabolismo , Estaurosporina/farmacología , Ligando Inductor de Apoptosis Relacionado con TNF/farmacologíaRESUMEN
Primary biliary cirrhosis (PBC) is a chronic autoimmune liver disease characterized by inflammation and destruction of intrahepatic biliary epithelial cells, ultimately leading to liver failure. The serological hallmark of PBC is the presence of high-titer antimitochondrial antibodies (AMA) against the inner lipoyl domain of E2 subunits of 2-oxo-acid dehydrogenase complexes, in particular the E2 component of the pyruvate dehydrogenase complex (PDC-E2). The initiating events triggering the autoimmune response are not yet identified but the hypothesis of molecular mimicry is a widely proposed mechanism for the development of autoimmunity in PBC. Several candidates, including bacteria and viruses, have been suggested as causative agents, but also environmental factors, such as chemical xenobiotics, have been implicated in the pathogenesis of primary biliary cirrhosis. In this review, we will discuss our current knowledge of the immunoreactivity of xenobiotically modified PDC peptide antigens. In addition, we will provide a working hypothesis how xenobiotic modification of antigens might occur that ultimately leads to the breaking of self-tolerance and the induction of PBC.
Asunto(s)
Autoanticuerpos/inmunología , Enfermedades Autoinmunes/inmunología , Cirrosis Hepática Biliar/inmunología , Proteínas Mitocondriales/inmunología , Imitación Molecular/inmunología , Complejo Piruvato Deshidrogenasa/inmunología , Xenobióticos/toxicidad , Animales , Autoanticuerpos/biosíntesis , Humanos , Cirrosis Hepática Biliar/etiología , Cirrosis Hepática Biliar/genética , Cirrosis Hepática Biliar/patología , Imitación Molecular/efectos de los fármacos , Xenobióticos/inmunología , Xenobióticos/metabolismoRESUMEN
Human autoimmune cholangiopathy comprises several intractable liver diseases that ultimately lead to hepatic failure. Primary biliary cirrhosis (PBC), allograft rejection, graft versus host diseases, and, possibly, primary sclerosing cholangitis are representative of immune-mediated cholangiopathies. Among them, PBC is the best-investigated human autoimmune cholangiopathy. The immunological approach to PBC has provided much critical information regarding its pathogenesis. The breakdown of self-tolerance in both B cells and T cells toward E2 components of the pyruvate dehydrogenase complex is evident. However, a number of questions regarding its etiology are unclear, in particular, the mechanisms involved in the selectivity of cholangiocyte destruction. In this brief review, we discuss what we know and we do not know regarding the pathogenesis of PBC.
Asunto(s)
Cirrosis Hepática Biliar/inmunología , Animales , Enfermedades Autoinmunes/inmunología , Linfocitos B/inmunología , Conductos Biliares/citología , Citocinas/sangre , Modelos Animales de Enfermedad , Humanos , Inmunidad Innata , Ratones , Complejo Piruvato Deshidrogenasa/inmunología , Linfocitos T/inmunologíaRESUMEN
Antimitochondrial antibodies (AMA) are the serum hallmark of primary biliary cirrhosis (PBC). However, AMA-positivity can be found in non-PBC sera when lower dilutions are used, thus raising issues about the specificity and sensitivity of the test. AMA reacts primarily with the lipoylated domains of pyruvate dehydrogenase-E2 (PDC-E2) which is highly conserved across species, including bacteria. We studied 77 serum samples, including 24 from patients with anti-PDC-E2-positive PBC and 53 controls (16 with autoimmune hepatitis (AIH), 10 with primary sclerosing cholangitis (PSC), and 27 healthy individuals) for their reactivities at serial dilutions (1:10, 1:20 and 1:40) against Escherichia coli DH5 alpha lysate overexpressing human PDC-E2 using immunoblotting (IB). A murine anti-human PDC-E2 monoclonal antibody (mAB) was used as control. We further studied positive sera using adsorption with a synthetic E. coli peptide sharing similarity with human PDC-E2. Finally, we verified whether a unique buffer for E. coli preparation could reduce non-specific serum reactivity. Results demonstrated that 100% of anti-PDC-E2-positive PBC and up to 38% of control sera at 1:10 dilution recognized E. coli PDC-E2 at IB while dilution tests indicated that the overall potency of PBC reactivity was 100-fold higher compared to controls. In fact, a subgroup (20-38%) of non-PBC sera were positive at low titers but lost the reactivity when absorbed with the synthetic E. coli peptide. Finally, our unique buffer reduced the reactivity of non-PBC sera as measured by ELISA. In conclusion, we demonstrated that weak cross-reactivity with E. coli PDC-E2 occurs in non-PBC sera at lower dilutions and that such reactivity is not due to AMA-positivity. The use of a specific buffer might avoid the risk of false positive AMA determinations when E. coli-expressed recombinant antigens are used.
Asunto(s)
Autoanticuerpos/sangre , Autoantígenos/inmunología , Cirrosis Hepática Biliar/diagnóstico , Mitocondrias/inmunología , Complejo Piruvato Deshidrogenasa/inmunología , Humanos , Cirrosis Hepática Biliar/sangre , Cirrosis Hepática Biliar/inmunologíaRESUMEN
Primary biliary cirrhosis (PBC) is an organ-specific autoimmune disease that predominantly affects women and is characterized by chronic, progressive destruction of small intrahepatic bile ducts with portal inflammation and ultimately fibrosis, leading to liver failure in the absence of treatment. Little is known about the etiology of PBC. PBC is characterized by anti-mitochondrial antibodies and destruction of intra-hepatic bile ducts. The serologic hallmark of PBC is the presence of auto-antibodies to mitochondria, especially to the E2 component of the pyruvate dehydrogenase complex (PDC). Current theories on the pathogenesis of PBC favor the hypothesis that the disease develops as a result of an inappropriate immune response following stimulation by an environmental or infectious agent. Some reports suggest that xenobiotics and viral infections may induce PBC. The pathogenetic mechanism is believed to be caused by a defect in immunologic tolerance, resulting in the activation and expansion of self-antigen specific T and B lymphocyte clones and the production of circulating autoantibodies in addition to a myriad of cytokines and other inflammatory mediators. This leads to ductulopenia and persistent cholestasis, by developing end-stage hepatic-cell failure. In this review are given our own and literary data about mechanisms of development of intrahepatic cholestasis and possible ways of its correction.