RESUMO
Neutrophil extracellular traps (NETs) are web-like structures released by activated neutrophils. Recent studies suggest that NETs play an active role in driving autoimmunity and tissue injury in diseases including rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). The purpose of this study was to investigate if celastrol, a triterpenoid compound, can inhibit NET formation induced by inflammatory stimuli associated with RA and SLE. We found that celastrol can completely inhibit neutrophil oxidative burst and NET formation induced by tumor necrosis factor alpha (TNFα) with an IC50 of 0.34 µM and by ovalbumin:anti-ovalbumin immune complexes (Ova IC) with an IC50 of 1.53 µM. Celastrol also completely inhibited neutrophil oxidative burst and NET formation induced by immunoglobulin G (IgG) purified from RA and SLE patient sera. Further investigating into the mechanisms, we found that celastrol treatment downregulated the activation of spleen tyrosine kinase (SYK) and the concomitant phosphorylation of mitogen-activated protein kinase kinase (MAPKK/MEK), extracellular-signal-regulated kinase (ERK), and NFκB inhibitor alpha (IκBα), as well as citrullination of histones. Our data reveals that celastrol potently inhibits neutrophil oxidative burst and NET formation induced by different inflammatory stimuli, possibly through downregulating the SYK-MEK-ERK-NFκB signaling cascade. These results suggest that celastrol may have therapeutic potentials for the treatment of inflammatory and autoimmune diseases involving neutrophils and NETs.
Assuntos
Armadilhas Extracelulares/imunologia , Inflamação/imunologia , Neutrófilos/imunologia , Explosão Respiratória/imunologia , Triterpenos/farmacologia , Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Armadilhas Extracelulares/efeitos dos fármacos , Humanos , Proteínas I-kappa B/metabolismo , Imunoglobulina G/efeitos dos fármacos , Imunoglobulina G/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/patologia , MAP Quinase Quinase Quinases/metabolismo , Inibidor de NF-kappaB alfa , Neutrófilos/efeitos dos fármacos , Ovalbumina/imunologia , Triterpenos Pentacíclicos , Fosforilação/efeitos dos fármacos , Proteínas Tirosina Quinases/metabolismo , Explosão Respiratória/efeitos dos fármacos , Quinase Syk , Tripterygium/metabolismo , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Activation of hepatic stellate cells (HSCs) involves the induction of ECM protein synthesis and rapid cell proliferation. Thus, agents that interfere with either process could potentially mitigate the development of liver disease by reducing the synthesis of proteins associated with fibrosis or by reducing the number of activated HSC. Previously, we described that the non-metabolizable amino acid analog N-(methylamino)isobutyric acid (MeAIB) reduced hepatic collagen content of rats in a model of CCl(4)-induced liver injury, and in vitro studies using CFSC-2G cells indicated that MeAIB directly reduced collagen synthesis. However, the MeAIB-mediated reduction of hepatic collagen, in vivo, following liver injury was associated with a decrease in hepatic alpha-smooth muscle actin (alpha-SMA) which suggested that MeAIB also inhibited the activation of HSCs. Because HSC activation is inseparable from proliferation, the purpose of this study was to examine the effect of MeAIB treatment on the proliferation of HSCs in an in vitro model utilizing CFSC-2G cell cultures. In these studies, MeAIB effectively inhibited the proliferation of CFSC-2G cells by interfering with the progression of the cells through the G(1)-phase of the cell cycle which delayed entry into S-phase. MeAIB prevented the phosphorylation of p70S6 kinase (p70S6K) at Thr389 and reduced the phosphorylation at Thr421/Ser424. Because p70S6K is required for G(1)-cell cycle progression and is known to be regulated by nutrient availability, this correlates well with MeAIB interfering with the proliferation of CFSC-2G HSCs. In addition, the rate of protein synthesis was reduced by MeAIB treatment following mitogenic stimulation, which agrees with a p70S6K-mediated reduction in translation. These data are consistent with MeAIB inhibiting the proliferation of CFSC-2G cells by altering the mitogen activated pathway(s) leading to phosphorylation of p70S6K by a yet to be described mechanism.
Assuntos
Fase G1/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Fase de Repouso do Ciclo Celular/efeitos dos fármacos , beta-Alanina/análogos & derivados , beta-Alanina/farmacologia , Animais , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Hepatócitos/citologia , Fígado , Fosforilação/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Ratos , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismoRESUMO
OBJECTIVE: To compare the efficacy of minocycline with that of a conventional disease-modifying antirheumatic drug (DMARD), hydroxychloroquine, in patients with early seropositive rheumatoid arthritis (RA). METHODS: Sixty patients with seropositive RA of <1 year's duration who had not been previously treated with DMARDs were randomized to receive minocycline, 100 mg twice per day, or hydroxychloroquine, 200 mg twice per day, in a 2-year, double-blind protocol. All patients also received low-dose prednisone. The primary end points of the study were 1) the percentage of patients with an American College of Rheumatology (ACR) 50% improvement (ACR50) response at 2 years, and 2) the dosage of prednisone at 2 years. RESULTS: Minocycline-treated patients were more likely to achieve an ACR50 response at 2 years compared with hydroxychloroquine-treated patients (60% compared with 33%, respectively; P = 0.04). Minocycline-treated patients were also receiving less prednisone at 2 years compared with the hydroxychloroquine group (mean 0.81 mg/day compared with 3.21 mg/day, respectively; P < 0.01). In addition, patients treated with minocycline were more likely to have been completely tapered off prednisone (P = 0.03). Trends favoring the minocycline treatment group were seen when outcomes were assessed according to components of the ACR core criteria set, with the differences reaching statistical significance for patient's global assessment of disease activity (P = 0.004). CONCLUSION: Minocycline is an effective DMARD in patients with early seropositive RA. Patients treated with minocycline were more likely to achieve an ACR50 response and did so while receiving less prednisone. In addition, minocycline-treated patients were more likely to have discontinued treatment with prednisone at 2 years.
Assuntos
Antibacterianos/administração & dosagem , Antirreumáticos/administração & dosagem , Artrite Reumatoide/tratamento farmacológico , Hidroxicloroquina/administração & dosagem , Minociclina/administração & dosagem , Adulto , Idoso , Artrite Reumatoide/imunologia , Artrite Reumatoide/fisiopatologia , Método Duplo-Cego , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Testes Sorológicos , Resultado do TratamentoRESUMO
Two patients with severe rheumatoid arthritis (RA) were treated with high dose chemotherapy and autologous blood stem cell transplantation. Hematopoietic stem cells mobilized readily with cyclophosphamide and granulocyte-colony stimulating factor. Both patients achieved an American College of Rheumatology (ACR) 50% response before starting high dose therapy. The transplantation regimen included 200 mg/kg cyclophosphamide and 6 doses of equine antithymocyte globulin. Transplantation was well tolerated and both patients recovered neutrophils on day 7 post-transplant. At one month post-transplant both patients had an ACR response of 80%. Both individuals relapsed at 6 months and responded well to a combination of disease modifying antirheumatic drugs that was previously ineffective. At 12 months ACR responses were 80% and 60%, respectively. The first patient developed a flare at 18 months when she was found to be hypothyroid; she regained an 80% ACR response at 24 months with therapy of hypothyroidism. The second patient progressed relentlessly 15 months post-transplant. Immunological reconstitution showed a continuous inversion of the ratio of CD4 and CD8 lymphocytes with a predominant expansion of memory T cells.
Assuntos
Artrite Reumatoide/terapia , Ciclofosfamida/uso terapêutico , Transplante de Células-Tronco Hematopoéticas , Imunossupressores/uso terapêutico , Condicionamento Pré-Transplante , Centros Médicos Acadêmicos , Adulto , Artrite Reumatoide/imunologia , Feminino , Humanos , Hospedeiro Imunocomprometido , Leucaférese , Masculino , Pessoa de Meia-Idade , Nebraska , Projetos Piloto , Transplante Autólogo , Resultado do TratamentoRESUMO
There is little information about the clinical course of patients with rheumatoid arthritis (RA) who relapse after autologous blood stem cell transplantation (ASCT). We describe 6 patients with severe RA who received ASCT in 3 US centers. Duration of followup was between 24 and 42 months posttransplant. Five patients achieved major responses but relapsed 3-22 months posttransplant. Two patients with relapse improved remarkably after restarting disease modifying antirheumatic drugs (DMARD). Two patients developed a mild RA flare at 3 and 5 months posttransplant and improved spontaneously. All 4 patients who improved after an initial disease flare remained highly functional at 14-22 months posttransplant. All patients in this study were anti-tumor necrosis factor (TNF) drug naive; all received a TNF blocker as a second line posttransplant salvage therapy, but only 3 responded. Future ASCT strategies need to focus on improving the durability of the early posttransplant responses.
Assuntos
Antirreumáticos/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Imunossupressores/uso terapêutico , Adulto , Artrite Reumatoide/etiologia , Artrite Reumatoide/imunologia , Etanercepte , Feminino , Seguimentos , Humanos , Imunoglobulina G/uso terapêutico , Masculino , Pessoa de Meia-Idade , Receptores do Fator de Necrose Tumoral/uso terapêutico , Recidiva , Transplante Autólogo , Resultado do TratamentoRESUMO
Malondialdehyde and acetaldehyde react together with proteins and form hybrid protein conjugates designated as MAA adducts, which have been detected in livers of ethanol-fed animals. Our previous studies have shown that MAA adducts are comprised of two distinct products. One adduct is composed of two molecules of malondialdehyde and one molecule of acetaldehyde and was identified as the 4-methyl-1,4-dihydropyridine-3,5-dicarbaldehyde derivative of an amino group (MDHDC adduct). The other adduct is a 1:1 adduct of malondialdehyde and acetaldehyde and was identified as the 2-formyl-3-(alkylamino)butanal derivative of an amino group (FAAB adduct). In this study, information on the mechanism of MAA adduct formation was obtained, focusing on whether the FAAB adduct serves as a precursor for the MDHDC adduct. Upon the basis of chemical analysis and NMR spectroscopy, two initial reaction steps appear to be a prerequisite for MDHDC formation. One step involves the reaction of one molecule of malondialdehyde and one of acetaldehyde with an amino group of a protein to form the FAAB product, while the other step involves the generation of a malondialdehyde-enamine. It appears that generation of the MDHDC adduct requires the FAAB moiety to be transferred to the nitrogen of the MDA-enamine. For efficient reaction of FAAB with the enamine to take place, additional experiments indicated that these two intermediates likely must be in positions on the protein of close proximity to each other. Further studies showed that the incubation of liver proteins from ethanol-fed rats with MDA resulted in a marked generation of MDHDC adducts, indicating the presence of a pool of FAAB adducts in the liver of ethanol-fed animals. Overall, these findings show that MDHDC-protein adduct formation occurs via the reaction of the FAAB moiety with a malondialdehyde-enamine, and further suggest that a similar mechanism may be operative in vivo in the liver during prolonged ethanol consumption.
Assuntos
Acetaldeído/química , Malondialdeído/química , Proteínas/química , Acetaldeído/síntese química , Animais , Ensaio de Imunoadsorção Enzimática , Etanol/administração & dosagem , Marcação por Isótopo , Fígado/efeitos dos fármacos , Masculino , Malondialdeído/síntese química , Modelos Moleculares , Ratos , Ratos Wistar , Bases de Schiff/químicaRESUMO
This article represents the proceedings of a workshop at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were Geoffrey M. Thiele and Simon Worrall. The presentations were (1) The chemistry of malondialdehyde-acetaldehyde (MAA) adducts, by Dean J. Tuma; (2) The formation and clearance of MAA adducts in ethanol-fed rats, by Simon Worrall; (3) Immune responses to MAA adducts may play a role in the development of alcoholic liver disease, by Lynell W. Klassen; (4) Unique biological responses to MAA-modified proteins that may play a role in the development and/or progression of alcoholic liver disease, by Geoffrey M. Thiele; (5) MAA-adducted bovine serum albumin activates protein kinase C and stimulates interleukin-8 release in bovine bronchial epithelial cells, by Todd A. Wyatt; and (6) An enzyme immune assay for serum antiacetaldehyde adduct antibody using low-density lipoprotein-adduct and its significance in alcoholic liver injury and ALDH2 heterozygotes, by Naruhiko Nagata.
Assuntos
Acetaldeído/metabolismo , Depressores do Sistema Nervoso Central/farmacologia , Adutos de DNA/efeitos dos fármacos , Etanol/farmacologia , Hepatopatias Alcoólicas/metabolismo , Malondialdeído/metabolismo , Aldeído Desidrogenase/efeitos dos fármacos , Aldeído Desidrogenase/metabolismo , Aldeído-Desidrogenase Mitocondrial , Animais , Adutos de DNA/metabolismo , Humanos , CamundongosRESUMO
Acetaldehyde and malonildialdehyde can form hybrid protein adducts, named MAA adducts that have strong immunogenic properties. The formation of MAA adducts in the liver of chronic alcohol-fed rats is associated with the development of circulating antibodies that specifically recognized these adducts. The aim of this study was to examine whether MAA adducts might participate in the immune response associated with human alcohol-induced liver disease. Circulating antibodies against MAA adducts were evaluated in 50 patients with alcohol-induced hepatitis or cirrhosis, in 40 patients with non-alcohol-induced liver disease, in 15 heavy drinkers without liver damage and in 40 healthy controls by enzyme-linked immunosorbent assays (ELISA). Immunoglobulin G (IgG) reacting with MAA-modified proteins were significantly increased in the patients with alcohol-induced cirrhosis or hepatitis. The individual levels of anti-MAA IgG in those patients were associated with the severity of liver damage. Anti-MAA antibodies were also positively correlated with the levels of IgG recognizing epitopes generated by acetaldehyde and malonildialdehyde. However, competitive inhibition experiments indicated that the anti-MAA antibodies were unrelated to those against acetaldehyde- or malonildialdehyde-derived antigens and mainly recognized a specific, cyclic MAA epitope. Some degree of immune reactivity towards MAA adducts was also observed in patients with non-alcohol-induced liver injury. However, competitive ELISA showed that the antigens recognized by these sera were not the cyclic MAA adducts. Altogether, these results showed the formation of MAA antigens during alcohol-induced liver disease and suggest their possible contribution to the development of immunologic reactions associated with alcohol-related liver damage.
Assuntos
Acetaldeído/imunologia , Anticorpos/sangue , Hepatopatias Alcoólicas/imunologia , Malondialdeído/imunologia , Adulto , Idoso , Especificidade de Anticorpos , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Feminino , Hepatite Alcoólica/imunologia , Humanos , Imunoglobulina G/sangue , Cirrose Hepática Alcoólica/imunologia , Masculino , Pessoa de Meia-IdadeRESUMO
OBJECTIVE: Rheumatoid arthritis (RA) causes substantial morbidity and mortality, and current treatments are suboptimal. Recent studies have demonstrated the short-term efficacy of minocycline in the treatment of patients with early RA. This study was undertaken to compare patients treated with conventional therapy in the early phase of their RA and those treated with minocycline, after 4 years of followup. METHODS: Forty-six patients with seropositive RA of <1 year's duration had been enrolled in a double-blind study of minocycline (100 mg twice daily) versus placebo. After the blinded portion of the study (3-6 months, depending upon response), all patients were treated with conventional therapy. This report compares those patients randomized to receive placebo for 3 months and then conventional therapy for the duration of 4 years versus those originally randomized to receive minocycline. RESULTS: Twenty of the 23 original minocycline-treated patients and 18 of the 23 original placebo-treated patients were available for followup (mean 4 years). At followup, RA was in remission (American College of Rheumatology criteria) without disease-modifying antirheumatic drug (DMARD) or steroid therapy in 8 of the patients originally treated with minocycline compared with 1 patient in the placebo group (P = 0.02). Ten patients in the minocycline group versus 16 in the original placebo group currently require DMARD therapy (P = 0.02). CONCLUSION: Among patients with seropositive RA, remissions are more frequent and the need for DMARD therapy is less in those treated early in the disease course with minocycline compared with those treated with conventional therapy delayed by an average of only 3 months. Minocycline appears to be an effective therapy for early RA; further investigation into its mechanism of action is needed.
Assuntos
Antibacterianos/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Minociclina/uso terapêutico , Antibacterianos/toxicidade , Artrite Reumatoide/sangue , Método Duplo-Cego , Feminino , Seguimentos , Hemorragia Gastrointestinal/induzido quimicamente , Humanos , Masculino , Placebos , Fatores de TempoAssuntos
Antirreumáticos/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Ciclosporina/administração & dosagem , Quimioterapia Combinada , Seguimentos , Humanos , Hidroxicloroquina/administração & dosagem , Metotrexato/administração & dosagem , Sulfassalazina/administração & dosagem , Resultado do TratamentoRESUMO
Many investigators have suggested that an immune reaction to acetaldehyde-protein adducts may be involved in the development and/or progression of alcohol liver disease. The most often reported acetaldehyde adduct is the reduced adduct prepared in vitro in the presence of strong reducing agents. However, the production of this adduct in vivo has been difficult to prove. Nevertheless, the detection of serum antibodies to this reduced adduct following alcohol exposure in animals and humans has been used to support the formation of this adduct in vivo. We have recently observed that when acetaldehyde-protein adducts prepared under nonreducing conditions are used to immunize animals, antibody to the reduced protein adduct is detected. Therefore, it was the purpose of this study to demonstrate that nonreduced (NR) adduct epitopes can be modified by intact cells to express reduced (R) adduct epitopes. This was accomplished using the monoclonal antibody RT1.1 that has been previously characterized by this laboratory and has been shown to recognize only R and not NR acetaldehyde adducts. In these studies, Balb/c mice were injected intraperitoneally (500 microg/animal) with either keyhole limpet hemocyanin (KLH)-NR or KLH-R adducted proteins. Immunization with KLH-NR produced significant amounts of antibodies that recognized both NR and R epitopes. In contrast, immunization with KLH-R produced antibodies to only R and not NR epitopes. Isolated peritoneal macrophages from nonimmunized mice were incubated in vitro with either KLH-NR, KLH-R, or unmodified KLH proteins, and the cell surface expression of the reduced epitope (RT1.1) and the activated macrophage marker (MAC-3) determined by double immunofluorescent staining. Activated macrophages incubated with KLH-NR expressed the R adduct on 11.5% of the cells, compared with 3.8% following incubation with unmodified KLH, and 19.4% following incubation with KLH-R. These data suggest that the NR adduct and/or the carrier protein are modified by peritoneal macrophages in vivo and present an epitope that is detected as a reduced adduct (RT1.1 positive). These observations may explain the presence of circulating antibodies to the reduced adduct that has been reported in human and animal studies.
Assuntos
Acetaldeído/imunologia , Células Apresentadoras de Antígenos/imunologia , Epitopos/imunologia , Fenótipo , Acetaldeído/química , Animais , Anticorpos Monoclonais , Formação de Anticorpos/imunologia , Apresentação de Antígeno/imunologia , Células Apresentadoras de Antígenos/fisiologia , Epitopos/química , Humanos , Hepatopatias Alcoólicas/imunologia , Macrófagos Peritoneais/imunologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Receptor-mediated endocytosis (RME) by a scavenger receptor on sinusoidal liver endothelial cells (LECs) for formaldehyde-treated bovine serum albumin (f-Alb) has previously been shown to be impaired following chronic ethanol consumption. These studies were initially performed by in situ perfusion, making it difficult to determine the point in the process at which RME is affected. Therefore, it was the purpose of this study to use isolated LECs to begin elucidating at what point in the process chronic ethanol consumption affects RME. Initial studies showed that degradation at the single-cell level were similarly decreased at levels that had been observed for in situ studies, suggesting that the ethanol effects can be repeated using isolated LECs, making them useful for in vitro studies. Binding studies with 125I-formaldehyde-treated bovine serum albumin (125I-f-Alb) demonstrated there was a slight, but significantly different, decrease in binding by LECs from ethanol-fed rats when compared with pair-fed or chow-fed rats. However, the affinity of these receptors was not different between these groups. In contrast, a defect in the initial stages of receptor-ligand internalization was indicated as less surface-bound ligand was internalized and subsequently degraded in cells from the ethanol-treated animals as compared with controls. Additionally, once the data were adjusted for the amount of ligand internalized, the degradation of the internalized ligand was only slightly impaired. These results indicate that chronic ethanol feeding impairs the process of RME by the liver; the major cause of this impairment appears to be caused by a decreased ability of these cells to internalize all of the surface-bound ligand, with a minimal defect in postinternalization events.
Assuntos
Consumo de Bebidas Alcoólicas/fisiopatologia , Endocitose/fisiologia , Formaldeído/farmacologia , Fígado/fisiologia , Proteínas de Membrana , Receptores Imunológicos/fisiologia , Receptores de Lipoproteínas , Soroalbumina Bovina/efeitos dos fármacos , Soroalbumina Bovina/metabolismo , Animais , Sítios de Ligação/efeitos dos fármacos , Ligação Competitiva/efeitos dos fármacos , Bovinos , Separação Celular , Endotélio/citologia , Endotélio/fisiologia , Fígado/citologia , Masculino , Ratos , Ratos Wistar , Receptores Depuradores , Receptores Depuradores Classe B , Fatores de TempoRESUMO
Recent studies have shown that the alcohol metabolites malondialdehyde and acetaldehyde can combine to form a stable adduct (MAA) on proteins. This adduct has been detected in the livers of rats chronically consuming ethanol, and serum antibodies to MAA have been observed at significantly higher concentrations in ethanol-fed when compared with pair-fed or chow-fed control rats. More recently, preliminary studies have strongly suggested that the MAA adduct is capable of stimulating antibody responses to soluble proteins in the absence of adjuvants. The antibodies produced recognize either the MAA epitope or the carrier protein itself. Therefore, it was the purpose of this study to examine the potential immunogenicity of MAA-modified exogenous proteins in the absence of adjuvants. Balb/c mice were immunized in the presence or absence of adjuvant with different concentrations of unmodified or MAA-modified proteins. The antibody response to both the MAA epitope and unmodified protein epitopes were determined by ELISA. In the absence of adjuvant, significant antibody responses were induced to both the MAA epitope and nonmodified protein epitopes. Smaller immunizing doses of MAA-protein conjugate favored the production of antibodies to nonmodified proteins, whereas larger doses induced a strong anti-MAA response. In studies to begin determining a mechanism for the specificity of the response in the absence of adjuvants, peritoneal macrophages were found to bind and degrade MAA-adducted proteins through the use of a scavenger receptor. This indicated that MAA-adducted proteins may be specifically taken up and epitopes presented to the humoral immune system in the absence of adjuvants. Importantly, these are the first data showing that an alcohol-related metabolite can induce an antibody response in the absence of adjuvant and suggesting a mechanism by which antibody to the MAA adduct or its carrier (exogenous or endogenous) proteins may be generated in vivo.
Assuntos
Acetaldeído/farmacologia , Doenças Autoimunes/imunologia , Proteínas Sanguíneas/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos dos fármacos , Malondialdeído/farmacologia , Acetaldeído/imunologia , Animais , Autoanticorpos/sangue , Proteínas Sanguíneas/imunologia , Relação Dose-Resposta a Droga , Feminino , Peroxidação de Lipídeos/imunologia , Fígado/efeitos dos fármacos , Fígado/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Malondialdeído/imunologia , Camundongos , Camundongos Endogâmicos BALB C , RatosRESUMO
Atherosclerosis is a vascular injury characterized by elevated tissue levels of tumor necrosis factor-alpha (TNF-alpha), increased expression of endothelial cell adhesion molecules, and vascular wall inflammatory cell infiltration. Foam cells are associated with atherosclerotic plaque material, and low density lipoprotein (LDL) is a lipid component of foam cells. Malondialdehyde (MDA) is an oxidative product of unsaturated fatty acids and is also present in atherosclerotic lesions. MDA-modified (adducted) proteins, including MDA-modified LDL, are present in atherosclerotic human vascular tissue. Acetaldehyde (AA) is the major metabolic product of ethanol oxidation. Both MDA and AA are highly reactive aldehydes and will combine with proteins to produce an antigenically distinct protein adduct, termed the MAA adduct. This study demonstrates that proteins modified in the presence of high concentrations of MDA can produce MAA-modified proteins in vitro. In addition, MAA adducted proteins are capable of inducing rat heart endothelial cell cultures (rHEC) to produce and release TNF-alpha, and cause rHEC upregulation of endothelial adhesion molecule expression, including ICAM-1. These adhesion molecules are required for circulating inflammatory cells to adhere to endothelium which allows inflammatory cell tissue infiltration. Additionally, MAA modified proteins were defected in human atherosclerotic aortic vascular tissue but not in normal aortic tissue. Since atherosclerosis is associated with an inflammatory vascular injury characterized by elevated tissue TNF-alpha concentrations and inflammatory cell infiltration, these data suggest that MAA-adducted proteins may be formed in atherosclerotic plaque material and may be involved in the inflammatory reaction that occurs in atherosclerosis. These data further suggest that previous studies demonstrating MDA modified protein in atherosclerotic plaque may in fact have MAA modified proteins associated with them.
Assuntos
Acetaldeído/metabolismo , Aorta/metabolismo , Arteriosclerose/metabolismo , Malondialdeído/metabolismo , Acetaldeído/farmacologia , Animais , Aorta/patologia , Arteriosclerose/patologia , Moléculas de Adesão Celular/metabolismo , Morte Celular , Células Cultivadas , Endotélio Vascular/metabolismo , Ensaio de Imunoadsorção Enzimática , Humanos , Imuno-Histoquímica , Inflamação , Masculino , Malondialdeído/farmacologia , Proteínas/metabolismo , Ratos , Ratos Wistar , Soroalbumina Bovina , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Studies have investigated the hypothesis that metabolically derived acetaldehyde (AA) is capable of complexing with liver cell proteins to form AA-protein adducts that are capable of acting as antigens and inducing an immune response, as detected by the formation of unique antibodies. In an effort to better characterize and describe these adducts, mouse monoclonal and rabbit polyclonal antibodies specific for antigens prepared with AA under non-reducing (physiologic) and reducing (presence of sodium cyanoborohydride) conditions have been prepared. Two monoclonal antibodies were developed. The first antibody was RT1.1, which is specific to N-ethyl lysine (NEL); it is of the IgG2b isotype and recognizes all proteins modified with AA under reducing conditions. The other monoclonal antibody, NR-1, was of the IgG3 isotype; it recognizes proteins modified with AA under non-reducing conditions and cannot be inhibited by NEL. Affinity-purified and/or absorbed polyclonal antibodies were also produced to these epitopes. Using this panel of monoclonal and affinity-purified polyclonal antibodies, unique antigen-antibody binding occurred that: (1) detected only NEL; (2) reacted with the alpha-amino group on proteins prepared under reducing conditions; and (3) detected adducts on proteins prepared under non-reducing conditions. However, the only antibodies that recognized antigen(s) from alcohol-fed rat livers were those that were not specific to NEL or the alpha-amino group modified under reducing conditions. These data indicate that the relevant adduct in alcohol-fed rat livers is not NEL, and that it presumably is related to proteins modified with AA under non-reducing conditions.
Assuntos
Acetaldeído/metabolismo , Fígado/metabolismo , Proteínas/metabolismo , Acetaldeído/análise , Alcoolismo/metabolismo , Animais , Anticorpos , Anticorpos Monoclonais , Especificidade de Anticorpos , Antígenos/análise , Citosol/metabolismo , Lisina , Camundongos , Camundongos Endogâmicos BALB C , Proteínas/imunologia , Coelhos , Ratos , Ratos Sprague-DawleyRESUMO
OBJECTIVE: To determine the predictive value of shared epitope alleles for response to treatment in patients with rheumatoid arthritis. METHODS: Patients from our previously published triple DMARD study were tested for the presence of shared epitope alleles (DRB1 *0401, 0404/0408, 0405, 0101, 1001, and 1402). Patients who were shared epitope positive were then compared with those who were negative to see if there was a differential effect on therapeutic response. RESULTS: Shared epitope positive patients were much more likely to achieve a 50% response if treated with methotrexate-sulphasalazine-hydroxychloroquine compared with methotrexate alone (94% responders versus 32%, p < 0.0001). In contrast shared epitope negative patients did equally well regardless of treatment (88% responders for methotrexate-sulphasalazine-hydroxychloroquine versus 83% for methotrexate). Additionally, a trend toward an inverse relation of the gene dose was seen for response to methotrexate treatment (p = 0.05). CONCLUSIONS: These data suggest that determining shared epitope status may provide clinical information useful in selecting among treatment options.
Assuntos
Antirreumáticos/uso terapêutico , Artrite Reumatoide/imunologia , Antígenos HLA-DR , Adulto , Idoso , Alelos , Artrite Reumatoide/tratamento farmacológico , Esquema de Medicação , Quimioterapia Combinada , Epitopos , Feminino , Antígenos HLA-DR/genética , Cadeias HLA-DRB1 , Teste de Histocompatibilidade , Humanos , Hidroxicloroquina/administração & dosagem , Masculino , Metotrexato/administração & dosagem , Pessoa de Meia-Idade , Seleção de Pacientes , Sulfassalazina/administração & dosagem , Resultado do TratamentoRESUMO
BACKGROUND & AIMS: Malondialdehyde and acetaldehyde react together with proteins and form hybrid protein conjugates designated as MAA adducts, which have been detected in livers of ethanol-fed rats. The aim of this study was to examine the immune response to MAA adducts and other aldehyde adducts during long-term ethanol exposure. METHODS: Rats were pair-fed for 7 months with a liquid diet containing either ethanol or isocaloric carbohydrate. Circulating antibody titers against MAA adducts and acetaldehyde adducts were measured and characterized in these animals. RESULTS: A significant increase in antibody titers against MAA-adducted proteins was observed in the ethanol-fed animals. Competitive inhibitions of antibody binding indicated that the circulating antibodies against MAA-modified proteins in the ethanol-fed rats recognized mainly a specific, chemically defined MAA epitope. Antibody titers to reduced and nonreduced acetaldehyde adducts were very low, and no significant differences were observed between ethanol-fed and control animals. Significant plasma immunoreactivity to not only MAA-adducted but also unmodified rat liver proteins (cytosol, microsomes, and especially plasma membrane) were also observed in the ethanol-fed rats. CONCLUSIONS: Long-term ethanol feeding generates circulating antibodies not only against MAA epitopes but possibly also against unmodified, native (self) protein epitopes, suggesting that MAA adducts could trigger harmful autoimmune responses.
Assuntos
Acetaldeído/imunologia , Alcoolismo/imunologia , Anticorpos/sangue , Malondialdeído/imunologia , Proteínas/imunologia , Alcoolismo/sangue , Animais , Formação de Anticorpos , Especificidade de Anticorpos , Ensaio de Imunoadsorção Enzimática , Masculino , Ratos , Ratos Wistar , Valores de ReferênciaRESUMO
Malondialdehyde (MDA) and acetaldehyde react together with proteins in a synergistic manner and form hybrid protein adducts, designated as MAA adducts. In a previous study, a polyclonal antibody specific for MAA-protein adducts was used in an immunoassay to detect the presence of MAA adducts in livers of ethanol-fed rats. In the present study, the specific epitope recognized by the antibody was defined and the chemistry of MAA adduct formation was further characterized. When several synthetic analogs were tested for their ability to inhibit antibody binding in a competitive ELISA, the results indicated that the major determinant of antibody binding was a highly fluorescent cyclic adduct composed of two molecules of MDA and one of acetaldehyde. The structure of this adduct was shown to be a 4-methyl-1,4-dihydropyridine-3,5-dicarbaldehyde derivative of an amino group of a protein. Examination of MAA adduct formation with a variety of proteins indicated that in addition to this specific fluorescent adduct, MAA adducts were also comprised of other nonfluorescent products. The amount of fluorescent epitopes present on a given protein was the major determinant of antibody binding as assessed in a competitive ELISA, although the efficiency of inhibition of antibody binding by these fluorescent epitopes on MAA-adducted proteins varied depending upon the particular protein. However, when these MAA-adducted proteins were hydrolyzed with Pronase, the concentration of these modified proteins necessary to achieve 50% inhibition of antibody binding in a competitive ELISA fell into a much narrower range of values, indicating that protein hydrolysis equalized the accessibility of the antibody to bind the epitope on these various derivatized proteins. In summary, a cyclic fluorescent adduct of defined structure has been identified as the epitope recognized by our MAA adduct antibody. In addition to this specific adduct, MAA adducts are also comprised of other nonfluorescent products.