Halothane potentiates the alcohol-adduct induced TNF-alpha release in heart endothelial cells

BACKGROUND: The possibility exists for major complications to occur when individuals are intoxicated with alcohol prior to anesthetization. Halothane is an anesthetic that can be metabolized by the liver into a highly reactive product, trifluoroacetyl chloride, which reacts with endogenous proteins to form a trifluoroacetyl-adduct (TFA-adduct). The MAA-adduct which is formed by acetaldehyde (AA) and malondialdehyde reacting with endogenous proteins, has been found in both patients and animals chronically consuming alcohol. These TFA and MAA-adducts have been shown to cause the release of inflammatory products by various cell types. If both adducts share a similar mechanism of cell activation, receiving halothane anesthesia while intoxicated with alcohol could exacerbate the inflammatory response and lead to cardiovascular injury. METHODS: We have recently demonstrated that the MAA-adduct induces tumor necrosis factor-α (TNF-α) release by heart endothelial cells (HECs). In this study, pair and alcohol-fed rats were randomized to receive halothane pretreatments intra peritoneal. Following the pretreatments, the intact heart was removed, HECs were isolated and stimulated with unmodified bovine serum albumin (Alb), MAA-modified Alb (MAA-Alb), Hexyl-MAA, or lipopolysaccharide (LPS), and supernatant concentrations of TNF-α were measured by ELISA. RESULTS: Halothane pre-treated rat HECs released significantly greater TNF-α concentration following MAA-adduct and LPS stimulation than the non-halothane pre-treated in both pair and alcohol-fed rats, but was significantly greater in the alcohol-fed rats. CONCLUSION: These results demonstrate that halothane and MAA-adduct pre-treatment increases the inflammatory response (TNF-α release). Also, these results suggest that halothane exposure may increase the risk of alcohol-induced heart injury, since halothane pre-treatment potentiates the HEC TNF-α release measured following both MAA-Alb and LPS stimulation

Increased immunogenicity to P815 cells modified with malondialdehyde and acetaldehyde

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 106 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 µg/ml and 1092 µg/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

Toll-Like Receptor 4 Signaling Pathway Mediates Inhalant Organic Dust-Induced Bone Loss.

Agriculture workers have increased rates of airway and skeletal disease. Inhalant exposure to agricultural organic dust extract (ODE) induces bone deterioration in mice; yet, mechanisms underlying lung-bone crosstalk remain unclear. Because Toll-like receptor 2 (TLR2) and TLR4 are important in mediating the airway consequences of ODE, this study investigated their role in regulating bone responses. First, swine facility ODE stimulated wild-type (WT) bone marrow macrophages to form osteoclasts, and this finding was inhibited in TLR4 knock-out (KO), but not TLR2 KO cells. Next, using an established intranasal inhalation exposure model, WT, TLR2 KO and TLR4 KO mice were treated daily with ODE or saline for 3 weeks. ODE-induced airway neutrophil influx and cytokine/chemokine release were similarly reduced in TLR2 and TLR4 KO animals as compared to WT mice. Utilizing micro-computed tomography (CT), analysis of tibia showed loss of bone mineral density, volume and deterioration of bone micro-architecture and mechanical strength induced by ODE in WT mice were significantly reduced in TLR4 but not TLR2 KO animals. Bone marrow osteoclast precursor cell populations were analyzed by flow cytometry from exposed animals. In WT animals, exposure to inhalant ODE increased osteoclast precursor cell populations as compared to saline, an effect that was reduced in TLR4 but not TLR2 KO mice. These results show that TLR2 and TLR4 pathways mediate ODE-induced airway inflammation, but bone deterioration consequences following inhalant ODE treatment is strongly dependent upon TLR4. Thus, the TLR4 signaling pathway appears critical in regulating the lung-bone inflammatory axis to microbial component-enriched organic dust exposures

Direct antioxidant properties of methotrexate: Inhibition of malondialdehyde-acetaldehyde-protein adduct formation and superoxide scavenging.

Methotrexate (MTX) is an immunosuppressant commonly used for the treatment of autoimmune diseases. Recent observations have shown that patients treated with MTX also exhibit a reduced risk for the development of cardiovascular disease (CVD). Although MTX reduces systemic inflammation and tissue damage, the mechanisms by which MTX exerts these beneficial effects are not entirely known. We have previously demonstrated that protein adducts formed by the interaction of malondialdehyde (MDA) and acetaldehyde (AA), known as MAA-protein adducts, are present in diseased tissues of individuals with rheumatoid arthritis (RA) or CVD. In previously reported studies, MAA-adducts were shown to be highly immunogenic, supporting the concept that MAA-adducts not only serve as markers of oxidative stress but may have a direct role in the pathogenesis of inflammatory diseases. Because MAA-adducts are commonly detected in diseased tissues and are proposed to mitigate disease progression in both RA and CVD, we tested the hypothesis that MTX inhibits the generation of MAA-protein adducts by scavenging reactive oxygen species. Using a cell free system, we found that MTX reduces MAA-adduct formation by approximately 6-fold, and scavenges free radicals produced during MAA-adduct formation. Further investigation revealed that MTX directly scavenges superoxide, but not hydrogen peroxide. Additionally, using the Nrf2/ARE luciferase reporter cell line, which responds to intracellular redox changes, we observed that MTX inhibits the activation of Nrf2 in cells treated with MDA and AA. These studies define previously unrecognized mechanisms by which MTX can reduce inflammation and subsequent tissue damage, namely, scavenging free radicals, reducing oxidative stress, and inhibiting MAA-adduct formation

Autoimmune Hepatitis Induced by Syngeneic Liver Cytosolic Proteins Biotransformed by Alcohol Metabolites

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 non-syngeneic 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

The Induction of Autoimmune Arthritis and Sex differences in Mice Impact the Lung Inflammatory Response to Repetitive Inhalant Organic Dust Extract Exposures

Asthma, chronic bronchitis and COPD are common adverse respiratory health effects among persons exposed to agriculture organic dust work environments. Occupational inhalant exposures have been increasingly associated with the risk of rheumatoid arthritis (RA) disease development, particularly among males. Agriculture workers have increased risk of RA and generalized bone disease. Chronic lung disease is associated with production of characteristic autoantibodies associated with RA (e.g.anti-citrullinated antibodies), even in absence of RA disease. The mechanistic link between pulmonary inflammation and arthritis (and vice versa) remains poorly understood. Animal models are lacking.https://digitalcommons.unmc.edu/emet_posters/1004/thumbnail.jp

Novel Antioxidant Properties of Doxycycline

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