RESUMO
Studies in rodents have shown that dietary modifications as mammary glands (MG) develop, regulates susceptibility to mammary tumor initiation. However, the effects of dietary PUFA composition on MGs in adult life, remains poorly understood. This study investigated morphological alterations and inflammatory microenvironments in the MGs of adult mice fed isocaloric and isolipidic liquid diets with varying compositions of omega (ω)-6 and long-chain (Lc)-ω3FA that were pair-fed. Despite similar consumption levels of the diets, mice fed the ω-3 diet had significantly lower body-weight gains, and abdominal-fat and mammary fat pad (MFP) weights. Fatty acid analysis showed significantly higher levels of Lc-ω-3FAs in the MFPs of mice on the ω-3 diet, while in the MFPs from the ω-6 group, Lc-ω-3FAs were undetectable. Our study revealed that MGs from ω-3 group had a significantly lower ductal end-point density, branching density, an absence of ductal sprouts, a thinner ductal stroma, fewer proliferating epithelial cells and a lower transcription levels of estrogen receptor 1 and amphiregulin. An analysis of the MFP and abdominal-fat showed significantly smaller adipocytes in the ω-3 group, which was accompanied by lower transcription levels of leptin, IGF1, and IGF1R. Further, MFPs from the ω-3 group had significantly decreased numbers and sizes of crown-like-structures (CLS), F4/80+ macrophages and decreased expression of proinflammatory mediators including Ptgs2, IL6, CCL2, TNFα, NFκB, and IFNγ. Together, these results support dietary Lc-ω-3FA regulation of MG structure and density and adipose tissue inflammation with the potential for dietary Lc-ω-3FA to decrease the risk of mammary gland tumor formation.
Assuntos
Ácidos Graxos Ômega-3/metabolismo , Inflamação/metabolismo , Glândulas Mamárias Animais/metabolismo , Adipócitos/metabolismo , Tecido Adiposo/metabolismo , Animais , Dieta/métodos , Feminino , Mediadores da Inflamação/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Precision-cut liver slices (PCLSs) provide a novel model for studies of alcoholic liver disease (ALD). This is relevant, as in vivo ethanol exposure does not appear to generate significant liver damage in ethanol-fed mice, except in the National Institute on Alcohol Abuse and Alcoholism binge model of ALD. Previous studies have shown that the two metabolites of ethanol consumption, malondialdhyde (MDA) and acetaldehyde (AA), combine to form MDA-AA (MAA) adducts, which have been correlated with the development and progression of ALD. In this study, murine PCLSs were incubated with ethanol and examined for the production of MAA adducts. PCLSs were homogenized, and homogenates were injected into C57BL/6 mice. PCLSs from control-, pair-, and ethanol-fed animals served as targets in in situ cytotoxic assays using primed T cells from mice hyperimmunized with control or ethanol-exposed PCLS homogenates. A CD45.1/CD45.2 passive-transfer model was used to determine whether T cells from the spleens of mice hyperimmunized with PCLS ethanol-exposed homogenates trafficked to the liver. PCLSs incubated with ethanol generated MAA-modified proteins in situ. Cytotoxic (CD8+) T cells from immunized mice killed naïve PCLSs from control- and pair-fed mice in vitro, a response that was blunted in PCLSs from ethanol-fed mice. Furthermore, CD45.1 CD8+ T cells from hyperimmunized mice trafficked to the liver but did not initiate liver damage. This study demonstrates that exposure to liver tissue damaged by ethanol mediates robust immune responses to well-characterized alcohol metabolites and native liver proteins in vitro. Moreover, although these proinflammatory T cells traffic to the liver, these responses appear to be dampened in vivo by locally acting pathways. NEW & NOTEWORTHY This study shows that the metabolites of ethanol and lipid breakdown produce malondialdehyde-acetaldehyde adducts in the precision-cut liver slice model system. Additionally, precision-cut liver slices exposed to ethanol and harboring malondialdehyde-acetaldehyde adducts generate liver-specific antibody and T cell responses in the spleens of naïve mice that could traffic to the liver.
Assuntos
Acetaldeído/imunologia , Autoimunidade , Fígado Gorduroso Alcoólico/imunologia , Hepatopatias Alcoólicas/imunologia , Fígado/imunologia , Malondialdeído/imunologia , Linfócitos T Citotóxicos/imunologia , Acetaldeído/metabolismo , Transferência Adotiva , Animais , Movimento Celular , Células Cultivadas , Modelos Animais de Doenças , Fígado Gorduroso Alcoólico/metabolismo , Feminino , Humanos , Técnicas In Vitro , Interleucina-6/imunologia , Interleucina-6/metabolismo , Antígenos Comuns de Leucócito/imunologia , Antígenos Comuns de Leucócito/metabolismo , Fígado/metabolismo , Hepatopatias Alcoólicas/metabolismo , Ativação Linfocitária , Malondialdeído/metabolismo , Camundongos Endogâmicos C57BL , Fenótipo , Baço/imunologia , Baço/metabolismo , Linfócitos T Citotóxicos/metabolismo , Linfócitos T Citotóxicos/transplanteRESUMO
Doxycycline (DOX), a derivative of tetracycline, is a broad-spectrum antibiotic that exhibits a number of therapeutic activities in addition to its antibacterial properties. For example, DOX has been used in the management of a number of diseases characterized by chronic inflammation. One potential mechanism by which DOX inhibits the progression of these diseases is by reducing oxidative stress, thereby inhibiting subsequent lipid peroxidation and inflammatory responses. Herein, we tested the hypothesis that DOX directly scavenges reactive oxygen species (ROS) and inhibits the formation of redox-mediated malondialdehyde-acetaldehyde (MAA) protein adducts. Using a cell-free system, we demonstrated that DOX scavenged reactive oxygen species (ROS) produced during the formation of MAA-adducts and inhibits the formation of MAA-protein adducts. To determine whether DOX scavenges specific ROS, we examined the ability of DOX to directly scavenge superoxide and hydrogen peroxide. Using electron paramagnetic resonance (EPR) spectroscopy, we found that DOX directly scavenged superoxide, but not hydrogen peroxide. Additionally, we found that DOX inhibits MAA-induced activation of Nrf2, a redox-sensitive transcription factor. Together, these findings demonstrate the under-recognized direct antioxidant property of DOX that may help to explain its therapeutic potential in the treatment of conditions characterized by chronic inflammation and increased oxidative stress.
Assuntos
Doxiciclina/química , Sequestradores de Radicais Livres/química , Sistema Livre de Células , Doxiciclina/farmacologia , Sequestradores de Radicais Livres/farmacologia , Células HEK293 , Humanos , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/metabolismo , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Malondialdeído/química , Malondialdeído/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Superóxidos/química , Superóxidos/metabolismoRESUMO
Objective: To characterize the expression of malondialdehdye-acetaldehyde (MAA) adducts and anti-MAA antibody in articular tissues and serum of patients with RA. Methods: Paired sera and SF were examined from 29 RA and 13 OA patients. Anti-MAA antibody, RF, ACPA and total immunoglobulin were quantified. SF-serum measures were compared within and between disease groups. The presence and co-localization of MAA, citrulline and select leukocyte antigens in RA and OA synovial tissues were examined using immunohistochemistry. Results: Circulating and SF anti-MAA antibody concentrations were higher in RA vs OA by 1.5- to 5-fold. IgG (P < 0.001), IgM (P = 0.006) and IgA (P = 0.036) anti-MAA antibodies were higher in paired RA SF than serum, differences not observed for total immunoglobulin, RF or ACPA. In RA synovial tissues, co-localization of MAA with citrulline and CD19+ or CD27+ B cells was demonstrated and was much higher in magnitude than MAA or citrulline co-localization with T cells, monocytes, macrophages or dendritic cells (P < 0.01). Conclusion: Anti-MAA antibodies are present in higher concentrations in the RA joint compared with sera, a finding not observed for other disease-related autoantibodies. Co-localization of MAA and citrulline with mature B cells, coupled with the local enrichment of anti-MAA immune responses, implicates MAA-adduct formation in local autoantibody production.
Assuntos
Acetaldeído/imunologia , Artrite Reumatoide/imunologia , Autoanticorpos/análise , Articulações/imunologia , Malondialdeído/imunologia , Idoso , Artrite Reumatoide/sangue , Estudos de Casos e Controles , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Osteoartrite/sangue , Osteoartrite/imunologia , Fator Reumatoide/sangue , Líquido Sinovial/imunologiaRESUMO
BACKGROUND: Malondialdehyde (MDA) and acetaldehyde (AA) exist following ethanol metabolism and tobacco pyrolysis. As such, lungs of individuals with alcohol use disorders (AUDs) are a target for the effects of combined alcohol and cigarette smoke metabolites. MDA and AA form a stable protein adduct, malondialdehyde-acetaldehyde (MAA) adduct, known to be immunogenic, profibrotic, and proinflammatory. MAA adduct is the dominant epitope in anti-MAA antibody formation. We hypothesized that MAA-adducted protein forms in lungs of those who both abuse alcohol and smoke cigarettes, and that this would be associated with systemically elevated anti-MAA antibodies. METHODS: Four groups were established: AUD subjects who smoked cigarettes (+AUD/+smoke), smokers without AUD (-AUD/+smoke), AUD without smoke (+AUD/-smoke), and non-AUD/nonsmokers (-AUD/-smoke). RESULTS: We observed a significant increase in MAA adducts in lung cells of +AUD/+smoke versus -AUD/-smoke. No significant increase in MAA adducts was observed in -AUD/+smoke or in +AUD/-smoke compared to -AUD/-smoke. Serum from +AUD/+smoke had significantly increased levels of circulating anti-MAA IgA antibodies. After 1 week of alcohol that MAA-adducted protein is formed in the lungs of those who smoke cigarettes and abuse alcohol, leading to a subsequent increase in serum IgA antibodies. CONCLUSIONS: MAA-adducted proteins could play a role in pneumonia and other diseases of the lung in the setting of AUD and smoking.
Assuntos
Acetaldeído/metabolismo , Alcoolismo/metabolismo , Autoanticorpos/sangue , Pulmão/metabolismo , Malondialdeído/metabolismo , Proteínas/metabolismo , Fumantes , Fumar/metabolismo , Acetaldeído/química , Adulto , Alcoolismo/complicações , Feminino , Humanos , Masculino , Malondialdeído/química , Ligação Proteica , Proteínas/química , Adulto JovemRESUMO
Agricultural workers have high rates of airway and skeletal health disease. Studies recently demonstrated that inhaled agricultural organic dust extract (ODE)-induced airway injury is associated with bone deterioration in an animal model. However, the effect of age in governing these responses to organic dusts is unclear, but might be important in future approaches. Young (7-9 wk) and older (12-14,o) male C57BL/6 mice received intranasal (i.n.) inhalation exposure to ODE from swine confinement facilities once or daily for 3 wk. Acute ODE-induced neutrophil influx and cytokine and chemokine (tumor necrosis factor [TNF]-α, interleukin [IL]-6, keratinocyte chemoattractant [CXCL1], macrophage inflammatory protein-2 [CXCL2]) airway production were reduced in older compared to young mice. Repetitive ODE treatment, however, increased lymphocyte recruitment and alveolar compartment histopathologic inflammatory changes in older mice. Whole lung cell infiltrate analysis revealed that young, but not older, mice repetitively treated with ODE demonstrated an elevated CD4:CD8 lymphocyte response. Acute inhalant ODE exposure resulted in a 4-fold and 1.5-fold rise in blood neutrophils in young and older mice, respectively. Serum IL-6 and CXCL1 levels were elevated in young and older mice i.n. exposed once to ODE, with increased CXCL1 levels in younger compared to older mice. Although older mice displayed reduced bone measurements compared to younger mice, younger rodents demonstrated ODE-induced decrease in bone mineral density, bone volume, and bone microarchitecture quality as determined by computed tomography (CT) analysis. Collectively, age impacts the airway injury and systemic inflammatory and bone loss response to inhalant ODE, suggesting an altered and enhanced immunologic response in younger as compared to older counterparts.
Assuntos
Osso e Ossos/efeitos dos fármacos , Poeira , Exposição por Inalação/efeitos adversos , Pneumonia/induzido quimicamente , Administração Intranasal , Fatores Etários , Animais , Densidade Óssea/efeitos dos fármacos , Quimiocina CXCL1/sangue , Quimiocina CXCL2/sangue , Interleucina-6/sangue , Ativação Linfocitária/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fator de Necrose Tumoral alfa/sangueRESUMO
Oxidative stress from fat accumulation in the liver has many deleterious effects. Many believe that there is a second hit that causes relatively benign fat accumulation to transform into liver failure. Therefore, we evaluated the effects of ethanol on ex vivo precision-cut liver slice cultures (PCLS) from rats fed a high-fat diet resulting in fatty liver. Age-matched male Sprague-Dawley rats were fed either high-fat (obese) (45% calories from fat, 4.73 kcal/g) or control diet for 13 mo. PCLS were prepared, incubated with 25 mM ethanol for 24, 48, and 72 h, harvested, and evaluated for ethanol metabolism, triglyceride production, oxidative stress, and cytokine expression. Ethanol metabolism and acetaldehyde production decreased in PCLS from obese rats compared with age-matched controls (AMC). Increased triglyceride and smooth muscle actin production was observed in PCLS from obese rats compared with AMC, which further increased following ethanol incubation. Lipid peroxidation, measured by thiobarbituric acid reactive substances assay, increased in response to ethanol, whereas GSH and heme oxygenase I levels were decreased. TNF-α and IL-6 levels were increased in the PCLS from obese rats and increased further with ethanol incubation. Diet-induced fatty liver increases the susceptibility of the liver to toxins such as ethanol, possibly by the increased oxidative stress and cytokine production. These findings support the concept that the development of fatty liver sensitizes the liver to the effects of ethanol and leads to the start of liver failure, necrosis, and eventually cirrhosis.
Assuntos
Etanol/farmacologia , Ácidos Graxos/biossíntese , Fígado/efeitos dos fármacos , Obesidade/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Animais , Dieta Hiperlipídica , Etanol/metabolismo , Fígado Gorduroso/metabolismo , Interleucina-6/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Peroxidação de Lipídeos/fisiologia , Fígado/metabolismo , Masculino , Modelos Animais , Ratos , Ratos Sprague-Dawley , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Skeletal health consequences associated with chronic inflammatory respiratory disease, and particularly chronic obstructive pulmonary disease (COPD), contribute to overall disease morbidity. Agricultural environmental exposures induce significant airway diseases, including COPD. However, animal models to understand inhalant exposure-induced lung injury and bone disease have not been described. Using micro-computed tomography (micro-CT) imaging technology and histology, bone quantity and quality measurements were investigated in mice after repetitive intranasal inhalation exposures to complex organic dust extracts (ODEs) from swine confinement facilities. Comparison experiments with LPS and peptidoglycan (PGN) alone were also performed. After 3 weeks of repetitive ODE inhalation exposure, significant loss of bone mineral density and trabecular bone volume fraction was evident, with altered morphological microarchitecture changes in the trabecular bone, compared with saline-treated control animals. Torsional resistance was also significantly reduced. Compared with saline treatment, ODE-treated mice demonstrated decreased collagen and proteoglycan content in their articular cartilage, according to histopathology. Significant bone deterioration was also evident after repetitive intranasal inhalant treatment with LPS and PGN. These findings were not secondary to animal distress, and not entirely dependent on the degree of induced lung parenchymal inflammation. Repetitive LPS treatment demonstrated the most pronounced changes in bone parameters, and PGN treatment resulted in the greatest lung parenchymal inflammatory changes. Collectively, repetitive inhalation exposures to noninfectious inflammatory agents such as complex organic dust, LPS, and PGN resulted in bone loss. This animal model may contribute to efforts toward understanding the mechanisms and evaluating the therapeutics associated with adverse skeletal health consequences after subchronic airway injury.
Assuntos
Doenças Ósseas Metabólicas/induzido quimicamente , Osso e Ossos/efeitos dos fármacos , Poeira , Exposição por Inalação/efeitos adversos , Lipopolissacarídeos/toxicidade , Compostos Orgânicos/toxicidade , Peptidoglicano/toxicidade , Animais , Densidade Óssea , Doenças Ósseas Metabólicas/diagnóstico por imagem , Doenças Ósseas Metabólicas/metabolismo , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/metabolismo , Cartilagem Articular/diagnóstico por imagem , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/metabolismo , Colágeno/metabolismo , Abrigo para Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pneumonia/induzido quimicamente , Pneumonia/diagnóstico por imagem , Pneumonia/metabolismo , Proteoglicanas/metabolismo , Medição de Risco , Fatores de Risco , Suínos , Fatores de Tempo , Microtomografia por Raio-XRESUMO
Biomarkers such as interleukin-6 (IL-6), soluble interleukin-6 receptor (sIL-6R), and high sensitive C-reactive protein (hsCRP) have been reported to be elevated in acute myocardial infarction (AMI). The aim of this study is to determine the relationship between these markers during AMI, as well as their relationship to clinical parameters in an effort to discern their predictive potential in cardiac events. Serum was collected from 73 patients with; AMI, stable coronary artery disease (CAD), and controls during cardiac catheterization. Biomarker levels were determined and correlated with clinical data. IL-6 (11.75pg/ml, P<0.05) and sIL-6R (41,340pg/ml, P=0.05) were elevated in AMI compared with CAD and controls. At presentation, hsCRP was elevated in AMI patients (4.69mg/L) compared to controls (2.69mg/L, P<0.05); however, there was a significant decrease in hsCRP between AMI (4.69mg/L) and CAD patients (7.4mg/L, P<0.05). After 24h post-AMI hsCRP levels were increased compared to stable CAD (60.46mg/L, P<0.05) and were preceded by increased IL-6 at presentation. Soluble Gp130 (sGp130) showed no significant change between AMI, CAD, and control patients. However, sGp130 positively correlated with peak troponin in AMI (R=0.587, P<0.01), and negatively correlated with previous AMI (R=-0.382, P<0.05). Circulating monocyte mRNA expression isolated from selected AMI patients showed an increase in IL-6 mRNA (5.28-fold, P<0.01) and a decrease in both IL-6R (0.374-fold, P<0.01) and sGp130 mRNA (0.38-fold, P<0.01) as compared to CAD and controls. Results demonstrate that IL-6 and sIL-6R are associated with AMI and cardiac injury. These data support the hypothesis that trans-IL-6 receptor binding may alter intracellular signaling, and blocking of IL-6 receptor binding may be pathogenic in AMI. These data may be predictive of mechanism(s) by which plaques become unstable and rupture.
Assuntos
Doença da Artéria Coronariana/sangue , Doença da Artéria Coronariana/genética , Interleucina-6/sangue , Infarto do Miocárdio/sangue , Infarto do Miocárdio/genética , Receptores de Interleucina-6/sangue , Proteína C-Reativa/metabolismo , Estudos de Casos e Controles , Receptor gp130 de Citocina/sangue , Demografia , Ensaio de Imunoadsorção Enzimática , Feminino , Regulação da Expressão Gênica , Humanos , Interleucina-6/genética , Masculino , Pessoa de Meia-Idade , Monócitos/metabolismo , Receptores de Interleucina-6/genética , Análise de RegressãoRESUMO
Soman forms a stable, covalent bond with tyrosine 411 of human albumin, with tyrosines 257 and 593 in human transferrin, and with tyrosine in many other proteins. The pinacolyl group of soman is retained, suggesting that pinacolyl methylphosphonate bound to tyrosine could generate specific antibodies. Tyrosine in the pentapeptide RYGRK was covalently modified with soman simply by adding soman to the peptide. The phosphonylated-peptide was linked to keyhole limpet hemocyanin, and the conjugate was injected into rabbits. The polyclonal antiserum recognized soman-labeled human albumin, soman-mouse albumin, and soman human transferrin but not nonphosphonylated control proteins. The soman-labeled tyrosines in these proteins are surrounded by different amino acid sequences, suggesting that the polyclonal recognizes soman-tyrosine independent of the amino acid sequence. Antiserum obtained after 4 antigen injections over a period of 18 weeks was tested in a competition ELISA where it had an IC50 of 10(-11) M. The limit of detection on Western blots was 0.01 µg (15 picomoles) of soman-labeled albumin. In conclusion, a high-affinity, polyclonal antibody that specifically recognizes soman adducts on tyrosine in a variety of proteins has been produced. Such an antibody could be useful for identifying secondary targets of soman toxicity.
Assuntos
Anticorpos/imunologia , Antígenos/imunologia , Soman/imunologia , Tirosina/imunologia , Animais , Antígenos/química , Antígenos/metabolismo , Ensaio de Imunoadsorção Enzimática , Hemocianinas/química , Hemocianinas/imunologia , Humanos , Camundongos , Oligopeptídeos/química , Oligopeptídeos/imunologia , Coelhos , Soman/química , Soman/metabolismo , Tirosina/química , Tirosina/metabolismoRESUMO
We have shown previously that perfluorocarbon-exposed sonicated dextrose albumin (PESDA) microbubbles bind to injured vascular tissue and can be detected with ultrasound imaging techniques. Prior studies have shown that scavenger receptors (SRs) are regulators of innate and adaptive immune responses and are involved in the progression of vascular disease such as atherosclerosis. In this study, we sought to determine the molecular mechanism of PESDA binding to balloon-injured vasculature. RT-PCR analysis of angioplastied aortas demonstrated a significantly (p ≤ 0.01) increased expression of SRs. Binding to SRs was confirmed using SR-expressing CHO cells, and this binding was blocked by competitive inhibition with the SR-binding ligands oxidized LDL and malondialdehyde-acetaldehyde-modified LDL. Confocal imaging confirmed the co-localization of PESDA microbubbles to CD36, SRB-1, and Toll-like receptor 4, but not to monocytes/macrophages. This study demonstrates that PESDA binds to SRs and that this binding is in major part dependent upon the oxidized nature of PESDA microbubble shell proteins. The extent of SR mRNA expression was increased with injury and associated with microbubble retention as defined by scanning electron microscopy and immunohistochemistry. These findings clarify the mechanisms of how albumin-based microbubbles bind to injured and inflamed vasculature and further support the potential of this imaging technique to detect early vascular innate inflammatory pathophysiologic processes.
Assuntos
Aorta , Meios de Contraste/farmacologia , Fluorocarbonos/farmacologia , Glucose/farmacologia , Microbolhas , Receptores Depuradores/biossíntese , Albumina Sérica/farmacologia , Regulação para Cima/efeitos dos fármacos , Animais , Aorta/lesões , Aorta/metabolismo , Aorta/ultraestrutura , Células CHO , Bovinos , Cricetinae , Cricetulus , Humanos , Inflamação/metabolismo , Inflamação/patologia , Masculino , Ratos , Ratos Sprague-Dawley , Albumina Sérica HumanaRESUMO
Ethanol metabolism in the liver induces oxidative stress and altered cytokine production preceding myofibroblast activation and fibrogenic responses. The purpose of this study was to determine how ethanol affects the fibrogenic response in precision-cut liver slices (PCLS). PCLS were obtained from chow-fed male Wistar rats (200-300 g) and were cultured up to 96 h in medium, 25 mM ethanol, or 25 mM ethanol and 0.5 mM 4-methylpyrazole (4-MP), an inhibitor of ethanol metabolism. Slices from every time point (24, 48, 72, and 96 h) were examined for glutathione (GSH) levels, lipid peroxidation [thiobarbituric acid-reactive substance (TBARS) assay], cytokine production (ELISA and RT-PCR), and myofibroblast activation [immunoblotting and immunohistochemistry for smooth muscle actin (SMA) and collagen]. Treatment of PCLS with 25 mM ethanol induced significant oxidative stress within 24 h, including depletion of cellular GSH and increased lipid peroxidation compared with controls (P < 0.05). Ethanol treatment also elicited a significant and sustained increase in interleukin-6 (IL-6) production (P < 0.05). Importantly, ethanol treatment accelerates a fibrogenic response after 48 h, represented by significant increases in SMA and collagen 1alpha(I) production (P < 0.05). These ethanol-induced effects were prevented by the addition of 4-MP. Ethanol metabolism induces oxidative stress (GSH depletion and increased lipid peroxidation) and sustained IL-6 expression in rat PCLS. These phenomena precede and coincide with myofibroblast activation, which occurs within 48 h of treatment. These results indicate the PCLS can be used as in vitro model for studying multicellular interactions during the early stages of ethanol-induced liver injury and fibrogenesis.
Assuntos
Etanol/toxicidade , Fibroblastos/efeitos dos fármacos , Cirrose Hepática/induzido quimicamente , Fígado/efeitos dos fármacos , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Interleucina-6/genética , Interleucina-6/metabolismo , Peroxidação de Lipídeos , Fígado/citologia , Cirrose Hepática/patologia , Masculino , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
BACKGROUND AND AIMS: Aldehydes that are produced following the breakdown of ethanol (acetaldehyde) and lipid peroxidation of membranes (malondialdehyde) have been shown to bind (adduct) proteins. Additionally, these two aldehydes can combine (MAA) on nonsyngeneic and syngeneic proteins to initiate numerous immune responses to the unmodified part of the protein in the absence of an adjuvant. Therefore, these studies provide a potential mechanism for the development of antigen-specific immune responses resulting in liver damage should syngeneic liver proteins be adducted with MAA. METHODS: This study sought to test whether MAA-modified syngeneic liver cytosolic proteins administered daily in the absence of adjuvant into C57BL/6 mice abrogates tolerance to initiate a MAA-induced autoimmune-like hepatitis. RESULTS: In mice immunized with MAA-modified cytosols, there was an increase in liver damage as assessed by aspartate aminotransferase/alanine aminotransferase levels that correlated with liver pathology scores and the presence of the pro-fibrotic factors, smooth muscle actin, TGF-ß, and collagen. IgG antibodies and T-cell proliferative responses specific for cytosolic proteins were also detected. Pro-inflammatory cytokines were produced in the livers of animals exposed to MAA-modified cytosols. Finally, transfer of immunized T cells to naïve animals caused biochemical and histological evidence of liver damage. CONCLUSIONS: These data demonstrate that a disease with an autoimmune-like pathophysiology can be generated in this animal model using soluble MAA-modified syngeneic liver cytosols as the immunogen. These studies provide insight into potential mechanism(s) that the metabolites of alcohol may play in contributing to the onset of an autoimmune-like disease in patients with alcoholic liver disease.
Assuntos
Acetaldeído/efeitos adversos , Etanol/efeitos adversos , Hepatite Autoimune/metabolismo , Fígado/efeitos dos fármacos , Malondialdeído/efeitos adversos , Proteínas/efeitos adversos , Acetaldeído/metabolismo , Animais , Biotransformação/efeitos dos fármacos , Citosol/metabolismo , Modelos Animais de Doenças , Etanol/metabolismo , Feminino , Hepatite Autoimune/imunologia , Hepatite Autoimune/patologia , Fígado/imunologia , Fígado/metabolismo , Fígado/patologia , Malondialdeído/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas/química , Proteínas/metabolismo , Proteínas S100/síntese química , Proteínas S100/imunologiaRESUMO
Many medications exhibit clinical benefits that are unrelated to their primary therapeutic uses. In many cases, the mechanisms underpinning these pleotropic effects are unknown. Two commonly prescribed medications that exhibit pleotropic benefits in cardiovascular disease and other diseases associated with chronic inflammation are methotrexate (MTX) and doxycycline (DOX). The vast majority of cardiovascular disease is associated with atherosclerosis. Because atherosclerosis is a chronic inflammatory disease, possible mechanisms by which MTX and DOX reduce inflammation have been investigated. Interestingly, the primary structure of both of these medications contain aromatic phenolic rings, which resemble polyphenols that are known to possess antioxidant activity. Inflammation and oxidative stress are intimately related. Inflammation promotes oxidative stress, which in turn leads to further inflammation; in this way, oxidative stress and inflammation can establish a self-perpetuating cycle. It has been shown that MTX and DOX act as antioxidants and are capable of scavenging free radicals and the reactive oxygen species (ROS) superoxide (O2-). Furthermore, both MTX and DOX inhibit the formation of malondialdehyde acetaldehyde (MAA) adducts, products of oxidative stress and lipid peroxidation. Importantly, MAA-adducts are highly immunogenic and initiate inflammatory responses; thereby, fueling the cycle of inflammation and oxidative stress that results in chronic inflammation. Thus, reducing the formation of MAA-adducts may ameliorate inflammation that leads to ROS production and in this way, break the self-sustaining cycle of oxidative stress and inflammation. It is possible that the under-recognized antioxidant properties of these medications may be a mechanism by which they and other medications provide pleotropic benefit in the treatment of chronic inflammatory disease.
Assuntos
Antioxidantes/farmacologia , Doxiciclina/farmacologia , Metotrexato/farmacologia , Animais , Aterosclerose/tratamento farmacológico , Aterosclerose/fisiopatologia , Doenças Cardiovasculares/tratamento farmacológico , Doenças Cardiovasculares/fisiopatologia , Humanos , Inflamação/tratamento farmacológico , Inflamação/fisiopatologia , Peroxidação de Lipídeos/efeitos dos fármacos , Malondialdeído/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismoRESUMO
BACKGROUND/OBJECTIVE: Malondialdehyde-acetaldehyde adducts (MAA) act as potent immune adjuvants and co-localize with citrullinated antigens in tissues effected by rheumatoid arthritis (RA). We sought to examine the role of MAA-adducts in promoting RA-related autoimmunity and inflammation. METHODS: DBA/J1 mice were immunized with human serum albumin (HSA), HSA-MAA, citrullinated HSA (HSA-Cit), or HSA-MAA-Cit with subsequent measurement of serum anti-citrullinated protein antibody (ACPA) and anti-Cit T cell responses. Cellular binding of the same antigens was examined using THP-1 monocytes and Chinese Hamster Ovary (CHO) cells transfected with specific scavenger receptors (SRs: TLR4, SR-B2, SREC-1). The effects of these antigens on THP-1 activation were then examined by quantifying plate adherence, pro-inflammatory (TNFα, IL-1ß, IL-10) cytokine release, and SR (CD14, SR-B2)/co-stimulatory molecule (CD80, HLA-DR) expression. Comparisons were completed using one-way ANOVA with Tukey's post-hoc test. RESULTS: Mice immunized with co-modified HSA produced significantly higher ACPA concentrations than all other groups whereas T cell responses to citrullinated proteins were highest following immunization with HSA-MAA. Both transfected CHO and THP-1 cells demonstrated significantly higher binding of HSA-MAA-Cit vs. HSA or HSA-Cit. THP-1 cells exposed to HSA-MAA-Cit expressed significantly higher concentrations of TNFα, IL-1ß, and IL-10 vs. all other groups. Furthermore, THP-1 cells demonstrated significantly increased plate adherence and higher expression of CD14, SR-B2, and HLA-DR following incubation with HSA-MAA-Cit vs. HSA or HSA-Cit. CONCLUSION: These studies demonstrate that MAA-adduction of citrullinated antigen greatly enhances immune and cellular responses, potentially acting as a key co-factor in RA pathogenesis.
Assuntos
Acetaldeído/imunologia , Anticorpos Antiproteína Citrulinada/sangue , Citrulinação/imunologia , Malondialdeído/imunologia , Acetaldeído/química , Adjuvantes Imunológicos/química , Animais , Anticorpos Antiproteína Citrulinada/imunologia , Artrite Reumatoide/imunologia , Células CHO , Cricetulus , Citocinas/metabolismo , Humanos , Imunogenicidade da Vacina , Inflamação/metabolismo , Masculino , Malondialdeído/química , Camundongos Endogâmicos DBA , Monócitos/metabolismo , Receptores Depuradores/metabolismo , Albumina Sérica Humana/química , Albumina Sérica Humana/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Células THP-1RESUMO
Rheumatoid arthritis (RA) is characterized by extra-articular involvement including lung disease, yet the mechanisms linking the two conditions are poorly understood. The collagen-induced arthritis (CIA) model was combined with the organic dust extract (ODE) airway inflammatory model to assess bone/joint-lung inflammatory outcomes. DBA/1J mice were intranasally treated with saline or ODE daily for 5 weeks. CIA was induced on days 1 and 21. Treatment groups included sham (saline injection/saline inhalation), CIA (CIA/saline), ODE (saline/ODE), and CIA + ODE (CIA/ODE). Arthritis inflammatory scores, bones, bronchoalveolar lavage fluid, lung tissues, and serum were assessed. In DBA/1J male mice, arthritis was increased in CIA + ODE > CIA > ODE versus sham. Micro-computed tomography (µCT) demonstrated that loss of BMD and volume and deterioration of bone microarchitecture was greatest in CIA + ODE. However, ODE-induced airway neutrophil influx and inflammatory cytokine/chemokine levels in lavage fluids were increased in ODE > CIA + ODE versus sham. Activated lung CD11c+ CD11b+ macrophages were increased in ODE > CIA + ODE > CIA pattern, whereas lung hyaluronan, fibronectin, and amphiregulin levels were greatest in CIA + ODE. Serum autoantibody and inflammatory marker concentrations varied among experimental groups. Compared with male mice, female mice showed less articular and pulmonary disease. The interaction of inhalation-induced airway inflammation and arthritis induction resulted in compartmentalized responses with the greatest degree of arthritis and bone loss in male mice with combined exposures. Data also support suppression of the lung inflammatory response, but increases in extracellular matrix protein deposition/interstitial disease in the setting of arthritis. This coexposure model could be exploited to better understand and treat RA-lung disease. © 2019 American Society for Bone and Mineral Research.
Assuntos
Artrite Experimental/complicações , Artrite Reumatoide/complicações , Poeira , Inflamação/complicações , Pneumopatias/etiologia , Pulmão/patologia , Animais , Artrite Experimental/sangue , Artrite Experimental/patologia , Artrite Reumatoide/sangue , Artrite Reumatoide/patologia , Autoanticorpos/sangue , Biomarcadores/sangue , Osso Esponjoso/patologia , Colágeno , Proteínas da Matriz Extracelular/metabolismo , Feminino , Inflamação/sangue , Inflamação/patologia , Articulações/patologia , Pneumopatias/sangue , Pneumopatias/patologia , Masculino , Camundongos , Coloração e RotulagemRESUMO
OBJECTIVE: To compare serum anti-malondialdehyde-acetaldehyde (anti-MAA) antibody levels and MAA expression in lung tissue from patients with rheumatoid arthritis-associated interstitial lung disease (RA-ILD) to those found in controls. METHODS: Anti-MAA antibody (IgA, IgM, IgG) concentrations were measured in patients with RA-ILD and compared to those of RA patients with chronic obstructive pulmonary disease (COPD) and RA patients without lung disease. Associations between anti-MAA antibody with RA-ILD were assessed using multivariable logistic regression. Lung tissue from patients with RA-ILD, other ILD, or emphysema, and from controls (n = 3 per group) were stained for MAA, citrulline, macrophages (CD68), T cells (CD3), B cells (CD19/CD27), and extracellular matrix proteins (type II collagen, fibronectin, vimentin). Tissue expression and colocalization with MAA were quantified and compared. RESULTS: Among 1,823 RA patients, 90 had prevalent RA-ILD. Serum IgA and IgM anti-MAA antibody concentrations were higher in RA-ILD than in RA with COPD or RA alone (P = 0.005). After adjustment for covariates, the highest quartiles of IgA anti-MAA antibody concentration (odds ratio 2.09 [95% confidence interval 1.11-3.90]) and IgM (odds ratio 2.23 [95% confidence interval 1.19-4.15]) were significantly associated with the presence of RA-ILD. MAA expression in RA-ILD lung tissue was greater than in tissue from all other groups (P < 0.001), and it colocalized with citrulline (r = 0.79), CD19+ B cells (r = 0.78), and extracellular matrix proteins (type II collagen [r = 0.72] and vimentin [r = 0.77]) to the greatest degree in RA-ILD. CONCLUSION: Serum IgA and IgM anti-MAA antibody is associated with ILD among RA patients. MAA is highly expressed in RA-ILD lung tissue, where it colocalizes with other RA autoantigens, autoreactive B cells, and extracellular matrix proteins, highlighting its potential role in the pathogenesis of RA-ILD.
Assuntos
Acetaldeído/imunologia , Artrite Reumatoide/imunologia , Autoanticorpos/sangue , Doenças Pulmonares Intersticiais/imunologia , Malondialdeído/imunologia , Idoso , Formação de Anticorpos/imunologia , Artrite Reumatoide/sangue , Autoanticorpos/imunologia , Autoantígenos/sangue , Autoantígenos/imunologia , Estudos de Casos e Controles , Feminino , Humanos , Pulmão/imunologia , Masculino , Pessoa de Meia-IdadeRESUMO
Aldehyde modified proteins have been associated with the development and/or progression of alcoholic liver disease (ALD). These protein adducts are capable of initiating many immunological responses that are harmful to the normal homeostasis of organism function. Previous studies have shown that malondialdehyde (MDA) and acetaldehyde (AA) synergistically form a unique adduct (MAA) with soluble proteins, which are capable of inducing cytokine release, T-cell proliferation, and antibody production. The purpose of this study was to determine whether MAA adduction can elicit similar responses to cells using a well-defined tumor model. The mouse mastocytoma P815 tumor cell line was modified with MAA (P815-MAA) or left unmodified (P815) and 10(6) irradiated cells were injected into DBA/2 mice once a week for 5 weeks. Serum was collected and tested for antibody responses to P815 cells and the MAA epitope. Immunization of MAA adducted P815 cells into syngeneic DBA/2 mice induced a strong antibody response to the MAA epitope as determined by ELISA on Alb and MAA-Alb (508 microg/ml and 1092 microg/ml, respectively). In addition, antibody to unmodified P815 cells was detected by fluorescent technique. Mice immunized with P815 cells or PBS showed little or no reactivity to the MAA epitope or P815 cells. Studies to assess IL-12 stimulation showed that peritoneal macrophages from P815 and PBS immunized animals produced modest amounts of IL-12 (20 and 35 pg/ml) when stimulated with Alb or MAA-Alb. However, macrophage from P815-MAA immunized mice responded to soluble MAA adduct (142 pg/ml). Finally, in tumor survival studies the mean survival was 14.25 days in PBS treated mice; 15.75 days with P815 immunized mice and 18.25 days with P815-MAA immunized mice. Therefore, these data strongly suggest that antibody responses are induced by P815 cells modified with MAA adducts. This may be a possible tool to begin looking at how alcohol metabolites potentially modify cells and/or cellular components making them recognizable to the immune system as foreign. It is thought that these studies define a model system that will be useful in assessing antibody and potentially T-cell responses to cells that are modified by MAA.
Assuntos
Acetaldeído/metabolismo , Anticorpos Antineoplásicos/sangue , Interleucina-12/metabolismo , Macrófagos Peritoneais/imunologia , Malondialdeído/metabolismo , Mastocitoma/imunologia , Acetaldeído/imunologia , Animais , Anticorpos Antineoplásicos/imunologia , Linhagem Celular Tumoral , Imunização , Interleucina-12/imunologia , Macrófagos Peritoneais/metabolismo , Malondialdeído/imunologia , Mastocitoma/metabolismo , Mastocitoma/mortalidade , Camundongos , Camundongos Endogâmicos DBA , Transplante de NeoplasiasRESUMO
Most ingested ethanol is eliminated from the body through oxidative metabolism in the liver. Alcohol dehydrogenase is the enzyme that is most important in the oxidation of ethanol to acetaldehyde. However, it has also been demonstrated that cytochrome P4502E1 also can contribute to this process. However, this is not the only aldehyde that is produced after chronic ethanol consumption because oxidative stress and lipid peroxidation can be induced in the liver, which results in the production of malondialdehyde and 4-hydroxy-2-nonenal. These aldehydes are highly reactive and have the ability to react with (adduct) many macromolecules to alter their structure and play a major role in the derangements of hepatic function. Therefore, the formation of these types of adducts in the liver has been proposed as key events leading to the development and/or progression of alcoholic liver disease. In this chapter, methods for the production and detection of these modified proteins will be discussed.
Assuntos
Consumo de Bebidas Alcoólicas/metabolismo , Aldeídos/metabolismo , Etanol/metabolismo , Técnicas Imunológicas , Hepatopatias Alcoólicas/metabolismo , Fígado/metabolismo , Estresse Oxidativo , Proteínas/metabolismo , Acetaldeído/imunologia , Acetaldeído/metabolismo , Aldeídos/imunologia , Animais , Anticorpos , Anticorpos Monoclonais , Especificidade de Anticorpos , Biomarcadores/metabolismo , Western Blotting , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Etanol/administração & dosagem , Humanos , Imunoprecipitação , Fígado/efeitos dos fármacos , Malondialdeído/imunologia , Malondialdeído/metabolismo , Camundongos , Modelos Animais , Estresse Oxidativo/efeitos dos fármacos , Proteínas/imunologia , Coelhos , Linfócitos T/metabolismoRESUMO
Epidemiological studies show a reduced risk of breast cancer (BC) in women consuming high levels of long-chain (LC) omega-3 (ω-3) fatty acids (FAs) compared with women who consumed low levels. However, the regulatory and mechanistic roles of dietary ω-6 and LC-ω-3 FAs on tumor progression, metastasis and survival are poorly understood. Female BALB/c mice (10-week old) were pair-fed with a diet containing ω-3 or an isocaloric, isolipidic ω-6 diet for 16 weeks prior to the orthotopic implantation of 4T1 mammary tumor cells. Major outcomes studied included: mammary tumor growth, survival analysis, and metastases analyses in multiple organs including pulmonary, hepatic, bone, cardiac, renal, ovarian, and contralateral MG (CMG). The dietary regulation of the tumor microenvironment was evaluated in mice autopsied on day-35 post tumor injection. In mice fed the ω-3 containing diet, there was a significant delay in tumor initiation and prolonged survival relative to the ω-6 diet-fed group. The tumor size on day 35 post tumor injection in the ω-3 group was 50% smaller and the frequencies of pulmonary and bone metastases were significantly lower relative to the ω-6 group. Similarly, the incidence/frequencies and/or size of cardiac, renal, ovarian metastases were significantly lower in mice fed the ω-3 diet. The analyses of the tumor microenvironment showed that tumors in the ω-3 group had significantly lower numbers of proliferating tumor cells (Ki67+)/high power field (HPF), and higher numbers of apoptotic tumor cells (TUNEL+)/HPF, lower neo-vascularization (CD31+ vessels/HPF), infiltration by neutrophil elastase+ cells, and macrophages (F4/80+) relative to the tumors from the ω-6 group. Further, in tumors from the ω-3 diet-fed mice, T-cell infiltration was 102% higher resulting in a neutrophil to T-lymphocyte ratio (NLR) that was 76% lower (p < 0.05). Direct correlations were observed between NLR with tumor size and T-cell infiltration with the number of apoptotic tumor cells. qRT-PCR analysis revealed that tumor IL10 mRNA levels were significantly higher (six-fold) in the tumors from mice fed the ω-3 diet and inversely correlated with the tumor size. Our data suggest that dietary LC-ω-3FAs modulates the mammary tumor microenvironment slowing tumor growth, and reducing metastases to both common and less preferential organs resulting in prolonged survival. The surrogate analyses undertaken support a mechanism of action by dietary LC-ω-3FAs that includes, but is not limited to decreased infiltration by myeloid cells (neutrophils and macrophages), an increase in CD3+ lymphocyte infiltration and IL10 associated anti-inflammatory activity.