Oxidative deamination of lysine residues by polyphenols generates an equilibrium of aldehyde and 2-piperidinol products.

Polyphenols, especially catechol-type polyphenols, exhibit lysyl oxidase-like activity and mediate oxidative deamination of lysine residues in proteins. Previous studies have shown that polyphenol-mediated oxidative deamination of lysine residues can be associated with altered electrical properties of proteins and increased crossreactivity with natural immunoglobulin M antibodies. This interaction suggested that oxidized proteins could act as innate antigens and elicit an innate immune response. However, the structural basis for oxidatively deaminated lysine residues remains unclear. In the present study, to establish the chemistry of lysine oxidation, we characterized oxidation products obtained via incubation of the lysine analog N-biotinyl-5-aminopentylamine with eggshell membranes containing lysyl oxidase and identified a unique six-membered ring 2-piperidinol derivative equilibrated with a ring-open product (aldehyde) as the major product. By monitoring these aldehyde-2-piperidinol products, we evaluated the lysyl oxidase-like activity of polyphenols. We also observed that this reaction was mediated by some polyphenols, especially o-diphenolic-type polyphenols, in the presence of copper ions. Interestingly, the natural immunoglobulin M monoclonal antibody recognized these aldehyde-2-piperidinol products as an innate epitope. These findings establish the existence of a dynamic equilibrium of oxidized lysine and provide important insights into the chemopreventive function of dietary polyphenols for chronic diseases.

Occupational contact sensitization in female geriatric nurses: Data of the Information Network of Departments of Dermatology (IVDK) 2005-2014.

BACKGROUND: Geriatric nurses (GN) have a high risk of occupational contact dermatitis (OCD), with chronic irritant contact dermatitis predominating. However, allergic contact dermatitis is an important issue as well. Little is known whether the relevant occupational allergen spectrum reported in the 1990s, including fragrances, preservatives, rubber chemicals and ingredients of surface disinfectants to be the most common sensitizers in GN, is still valid. OBJECTIVES: To monitor the current allergen spectrum in GN with OCD and verify the validity of the patch test recommendations (baseline-, preservative-, ointment base-, rubber-, disinfectant, series and fragrances) in GN with suspected OCD given by the German Contact Dermatitis Research Group (DKG). METHODS: Retrospective analysis of IVDK data (2005-2014) of 743 female GN with OCD, in comparison to 695 GN without OCD. RESULTS: GN with OCD reacted significantly more frequently to both fragrance mixes, hydroxyisohexyl 3-cyclohexene carboxaldehyde (HICC), thiuram mix, zinc diethyldithiocarbamate and mercaptobenzothiazole than GN without OCD. Reactions to MDBGN, methylchloroisothiazolinone/methylisothiazolinone and oil of turpentine occurred substantially, but not significantly more frequently among GN with OCD. The latter may be due to former use of a special alcoholic liniment in geriatric care. Among material from the patients' workplaces, tetrazepam was a frequent allergen, due to dust exposure from pill crushing. Furthermore, occupationally used protective gloves, body care products as well as surface disinfectants were often tested positively. CONCLUSIONS: The general allergen spectrum in GN with OCD is unchanged, so the DKG patch test recommendations are still valid. Prevention of occupational sensitization should focus on fragrance-free hygiene and body care products, usage of accelerator-free protective gloves and avoidance of drug dust exposure.

Metastasis-associated protein 1 (MTA1) signaling in rheumatoid synovium: Regulation of inflammatory response and cytokine-mediated production of prostaglandin E2 (PGE2).

Abnormal perpetual inflammatory response and sequential cytokine-induced prostaglandin E2 (PGE2) play important roles in the pathogenesis of rheumatoid arthritis (RA). The underlying regulatory mechanism, however, remain largely unknown. Here, we discovered that expression level of Metastasis associated protein 1 (MTA1), an important chromatin modifier that plays a critical role in transcriptional regulation by modifying DNA accessibility for cofactors, was upregulated in human rheumatoid synovial tissues. Furthermore, a knockdown of MTA1 by siRNA in the human fibroblast-like synovial cell line MH7A was found to impair the 4-hydroxynonenal (4-HNE)-induced transcriptional expression levels of certain proinflammatory cytokines including IL-1ß, TNF-α and IL-6. Moreover, endogenous MTA1 was required for the cytokines-induced PGE2 synthesis by rheumatoid synoviocytes. Collectively, the coordinated existence of MTA1 inside distinct cascade loops points to its indispensable role in the modulation of the integrated cytokine network along the pathogenesis of RA. Further exploration of the functional details of this master transcriptional regulator should be an attractive strategy to identify novel therapeutic target for RA and warrants execution.

Increased 4-hydroxynonenal protein adducts in male GSTA4-4/PPAR-α double knockout mice enhance injury during early stages of alcoholic liver disease.

To test the significance of lipid peroxidation in the development of alcoholic liver injury, an ethanol (EtOH) liquid diet was fed to male 129/SvJ mice (wild-type, WT) and glutathione S-transferase A4-4-null (GSTA4-/-) mice for 40 days. GSTA4-/- mice were crossed with peroxisome proliferator-activated receptor-α-null mice (PPAR-α-/-), and the effects of EtOH in the resulting double knockout (dKO) mice were compared with the other strains. EtOH increased lipid peroxidation in all except WT mice (P < 0.05). Increased steatosis and mRNA expression of the inflammatory markers CXCL2, tumor necrosis factor-α (TNF-α), and α-smooth muscle actin (α-SMA) were observed in EtOH GSTA4-/- compared with EtOH WT mice (P < 0.05). EtOH PPAR-α-/- mice had increased steatosis, serum alanine aminotransferase (ALT), and hepatic CD3+ T cell populations and elevated mRNA encoding CD14, CXCL2, TNF-α, IL-6, CD138, transforming growth factor-ß, platelet-derived growth factor receptor-ß (PDGFR-ß), matrix metalloproteinase (MMP)-9, MMP-13, α-SMA, and collagen type 1 compared with EtOH WT mice. EtOH-fed dKO mice displayed elevation of periportal hepatic 4-hydroxynonenal adducts and serum antibodies against malondialdehyde adducts compared with EtOH feeding of GSTA4-/-, PPAR-α-/-, and WT mice (P < 0.05). ALT was higher in EtOH dKO mice compared with all other groups (P < 0.001). EtOH-fed dKO mice displayed elevated mRNAs for TNF-α and CD14, histological evidence of fibrosis, and increased PDGFR, MMP-9, and MMP-13 mRNAs compared with the EtOH GSTA4-/- or EtOH PPAR-α-/- genotype (P < 0.05). These findings demonstrate the central role lipid peroxidation plays in mediating progression of alcohol-induced necroinflammatory liver injury, stellate cell activation, matrix remodeling, and fibrosis.

4-Hydroxy-2-nonenal modified histone-H2A: a possible antigenic stimulus for systemic lupus erythematosus autoantibodies.

Protein modifications by 4-hydroxy-2-nonenals (HNE) are involved in various diseases. Histones are DNA protective nucleoprotein, which adopt different structures under oxidative stress. This study was undertaken to test the role of HNE-modified-histone-H2A (HNE-H2A) in systemic lupus erythematosus (SLE). Our data revealed that HNE-mediated-lipid peroxidation in histone-H2A caused alteration in histidine, lysine and cystein residues. In addition, protein carbonyl contents were also high in HNE-H2A. HNE-specific quencher, L-carnosine further reiterates HNE-modifications. Specificity of autoantibodies from SLE patients (n=48) were analyzed towards HNE-H2A and their results were compared with sex- and age-matched controls (n=36). SLE autoantibodies show preferential binding to HNE-H2A in comparison with histone-H2A (p<0.0001). Furthermore, HNE-H2A was also detected in SLE peripheral blood mononuclear cells. In conclusion, this is the first study to demonstrate the role of HNE-modified-histone in SLE. Preferential binding of HNE-H2A by affinity purified SLE-IgG pointed out the likely role of HNE-H2A in the initiation/progression of SLE.

Effects of regular exercise on neutrophil functions, oxidative stress parameters and antibody responses against 4-hydroxy-2-nonenal adducts in middle aged humans.

Regular exercise has recognized health benefits, partly because it reportedly lowers the levels of the oxidation products of proteins and DNA at rest, in contrast with the effect of acute exercise. However, when we compared oxidative response markers in active middle-aged subjects with those in sedentary ones, the level of urinary 8-OHdG was higher in active subjects. Because neutrophils are the first line of defense against a variety of infectious diseases, we then compared the cell density, functions and apoptosis of neutrophils in active subjects with those in sedentary ones. The cell density of neutrophils and phagocytosis of opsonized zymosan by neutrophils were higher in active subjects, being similar with the reported effects of acute exercise. To determine any beneficial effects of oxidative stress in active subjects, we then compared the levels of antibodies against 4-hydroxy-2-nonenal adducts in active subjects with those in sedentary ones, because 4-hydroxy-2-nonenal is one of the most common bioactive aldehyde products of oxidative stress, and because the IgM class of antibodies against oxidized low-density lipoprotein is associated with atheroprotective properties. The level of the IgM but not the IgG class of antibodies against 4-hydroxy-2-nonenal adducts was higher in active subjects. Overall, this study revealed that our active middle-aged subjects showed both oxidative responses and a higher IgM response to reactive carbonyl derivatives, possibly providing a basis for a health benefit by exercise in our active subjects.

Reactive carbonyls are a major Th2-inducing damage-associated molecular pattern generated by oxidative stress.

Oxidative stress is widespread and entwined with pathological processes, yet its linkage to adaptive immunity remains elusive. Reactive carbonyl (RC) adduction, a common feature of oxidative stress, has been shown to target proteins to the adaptive immune system. Because aldehydes are important mediators of carbonylation, we explored the immunomodulatory properties of model Ags modified by common bioactive aldehyde by-products of oxidative stress: 4-hydroxy-2-nonenal, malondialdehyde, and glycolaldehyde. Ag modification with all three aldehydes resulted in Ag-specific IgG1-dominated responses in adjuvant-free murine immunizations in an RC-dependent manner. The central role of RCs was confirmed, as their reduction into nonreactive groups abrogated all adaptive responses, despite the presence of other well-known aldehyde-driven adducts such as N(ε)-carboxymethyllysine and glycolaldehyde-pyridine. Moreover, Ag-specific Ab responses robustly correlated with the extent of RC adduction, regardless of the means of their generation. T cell responses mirrored the Th2-biased Ab isotypes by Ag-specific splenocyte production of IL-4, IL-5, and IL-13, but not IFN-γ. The RC-induced Th2 response was in sharp contrast to that induced by Th1/Th2 balanced or Th1-biasing adjuvants and was maintained in a range of mouse strains. In vitro studies revealed that RC adduction enhanced Ag presentation with Th2 polarization in the absence of conventional dendritic cell activation. Taken together, these data implicate commonly occurring RC as an important oxidation-derived Th2 immunomodulatory damage-associated molecular pattern with potentially important roles in health and disease.

Elevated neutrophil elastase and acrolein-protein adducts are associated with W256 regression.

The involvement of granulocytes in immune response against cancer is not well understood. Depending on the cytokine milieu in which they act and on their oxidative burst, granulocytes may play either an inhibitory or stimulatory role in tumour growth. Unsaturated fatty acids, essential components of cellular membranes and storage lipids, are susceptible to granulocyte-derived reactive oxygen species (ROS). ROS can induce lipid peroxidation (LPO) resulting in the destruction of biomembranes. Thus, murine W256 tumour progressing and tumour regressing animal models were used to study the involvement of plasma inflammatory mediators and oxidative burst of circulating granulocytes in malignant destruction and detrimental tumour growth. The involvement of LPO-derived aldehydes (i.e. acrolein, 4-hydroxy-2-nonenal and malondialdehyde) and myeloperoxidase (MPO) appearance in the granulocyte anti-cancer response were further evaluated. The results obtained revealed a significant increase in neutrophil elastase in animals with regressing tumour. Furthermore, the presence of MPO in tumour microenvironment was accompanied by the formation of acrolein only 5 h after tumour transplantation and its presence increased during tumour regression. Later, at an early stage of tumour regression, the presence of other LPO-derived aldehydes were also observed. The results obtained suggest that elevated neutrophil elastase and initiation of LPO may play an important role in the tumour development leading to tumour regression.

Induction of anti-Ro60/anti-La by immunisation with spectrin and induction of anti-spectrin by immunisation with Ro60 and 4-hydroxy-2-nonenal-modified Ro60 immunisation.

OBJECTIVES: The Ro ribonucleoprotein particle, targeted in systemic lupus erythematosus (SLE) and Sjögren's syndrome (SS), includes Ro60 (SSA) and La (SSA) autoantigens. Anti-Ro60 occurs in SLE and SS. The importance of α-fodrin and spectrin as well as anti-Ro and anti-fodrin/spectrin antibodies in SS and SLE, led us to hypothesise that rabbit immunisation with Ro60 or 4-hydroxy-2-nonenal-modified Ro60 would induce anti-spectrin. In addition, we hypothesised that antibodies to Ro60 and La will develop in animals immunised with spectrin. METHODS: Two NZW rabbits each were immunised with 4-hydroxy-2-nonenal-modified Ro60 or unmodified Ro60. Methods used included ELISA, including an inside-out RBC membrane ELISA, and Crithidia lucilae assays. RESULTS: Commercial anti-spectrin sera bound significantly to Ro60 (OD 2.6 ± 0.1), Ro60 multiple antigenic peptides (MAPs) (3 out of 21 Ro60 MAPs), La (OD 4.4±0.5), and La fragments as well as to double stranded DNA but not to BSA (OD 0.6±0.1). Anti-spectrin binding to purified spectrin could be inhibited by spectrin (>95%), and Ro60 or La (70%). When the binding of anti-spectrin was tested against a nested set of La fragments we found that a N4 fragment representing the C-terminal 250 aa (aa 159 to 408) bound the strongest (OD=4.12) followed by a N9 fragment (the C-terminal 36aa; aa373 to 408 (OD=1.36). Also, significant anti-spectrin antibody levels were induced by Ro60 and HNE-modified Ro60 immunisation. CONCLUSIONS: We found intermolecular epitope spreading from Ro60/La to spectrin and vice versa, and this may have pathological significance in these animal models of autoimmunity.

Serum levels of 4-hydroxynonenal adducts and responding autoantibodies correlate with the pathogenesis from hyperglycemia to Alzheimer's disease.

OBJECTIVE: Hyperglycemia leads to lipid peroxidation, producing 4-hydroxynonenal (HNE) adducts which correlate with the production of amyloid-beta (Aß), one of the hallmarks of Alzheimer's disease (AD). This study is to investigate the interactions of Aß, HNE adducts and responding autoantibodies during the pathogenesis from hyperglycemia to AD. METHODS: A total of 239 Taiwanese serum samples from a healthy control group and patients with hyperglycemia, and AD with and without hyperglycemia were analyzed. Aß was immunoprecipitated from randomly pooled serum in each group and immunoblotted. Synthetic Aß1-16 and Aß17-28 peptides were modified with HNE in vitro and verified with LC-MS/MS. The levels of Aß, HNE adducts, and autoantibody isotypes IgG and IgM against either native or HNE-modified Aß were determined with ELISA. The diagnostic power of potential biomarkers was evaluated. RESULTS: Increased fasting glucose and decreased high-density-lipoprotein cholesterol in AD groups indicated abnormal metabolism in the pathogenesis progression from hyperglycemia to AD. Indeed, serum Aß, HNE adducts and most of the autoantibodies recognizing either native or HNE-modified Aß were increased in the diseased groups. However, HNE adducts had better diagnostic performances than Aß for both hyperglycemia and AD. Additionally, HNE-Aß peptide levels were increased, and the responding autoantibodies (most notably IgM) were decreased in hyperglycemic AD group compared to the hyperglycemia only group, suggesting an immunity disturbance in the pathogenesis progression from hyperglycemia to AD. CONCLUSION: Hyperglycemia increases the level of HNE adducts which may be neutralized by responding autoantibodies. Depletion of these autoantibodies promotes AD-like pathogenesis. Thus, levels of a patient's HNE adducts and associated responding autoantibodies are potential biomarkers for AD with diabetes.

Lipid peroxidation generates body odor component trans-2-nonenal covalently bound to protein in vivo.

trans-2-Nonenal is an unsaturated aldehyde with an unpleasant greasy and grassy odor endogenously generated during the peroxidation of polyunsaturated fatty acids. 2-Nonenal covalently modified human serum albumin through a reaction in which the aldehyde preferentially reacted with the lysine residues. Modified proteins were immunogenic, and a specific monoclonal antibody (mAb) 27Q4 that cross-reacted with the protein covalently modified with 2-nonenal was raised from mouse. To verify the presence of the protein-bound 2-nonenal in vivo, the mAb 27Q4 against the 2-nonenal-modified keyhole limpet hemocyanin was raised. It was found that a novel 2-nonenal-lysine adduct, cis- and trans-N(epsilon)-3-[(hept-1-enyl)-4-hexylpyridinium]lysine (HHP-lysine), constitutes an epitope of the antibody. The immunoreactive materials with mAb 27Q4 were detected in the kidney of rats exposed to ferric nitrilotriacetate, an iron chelate that induces free radical-mediated oxidative tissue damage. Using high performance liquid chromatography with on-line electrospray ionization tandem mass spectrometry, we also established a highly sensitive method for detection of the cis- and trans-HHP-lysine and confirmed that the 2-nonenal-lysine adducts were indeed formed during the lipid peroxidation-mediated modification of protein in vitro and in vivo. Furthermore, we examined the involvement of the scavenger receptor lectin-like oxidized low density lipoprotein receptor-1 in the recognition of 2-nonenal-modified proteins and established that the receptor recognized the HHP-lysine adducts as a ligand.

Enhanced sensitization and elicitation responses caused by mixtures of common fragrance allergens.

BACKGROUND: Perfumes are complex mixtures composed of many fragrance ingredients, many of which are known to be only weak allergens when tested individually. It is therefore surprising that fragrance contact allergy is one of the most common forms of contact allergy. OBJECTIVES: To investigate whether mixing different fragrance allergens leads to increased sensitization potency, and to examine the difference in the challenge response to one chemical in mice sensitized either with the mixture of allergens or with only the relevant allergen. METHODS: CBA mice were sensitized with three different concentrations of three fragrance allergens alone or as a mixture. The sensitization and elicitation responses were measured by ear thickness plus infiltration of B and T cells and T cell proliferation in the draining lymph nodes. RESULTS: We found a dose-dependent sensitization response for each of the allergens. An increased response was seen when the allergens were mixed. A stronger challenge response to cinnamal was seen in mice sensitized with the allergen mixture than in mice sensitized with cinnamal alone. CONCLUSIONS: Our findings suggest that mixtures of allergens increase the primary response that potentiates the generation of memory T cells in response to the specific allergen. Thus, allergen mixtures enhance both induction and elicitation of contact allergy.

[Current contact allergens]. / Aktuelle Kontaktallergene.

Ever-changing exposure to contact allergens, partly due to statutory directives (e.g. nickel, chromate, methyldibromo glutaronitrile) or recommendations from industrial associations (e.g. hydroxyisohexyl 3-cyclohexene carboxaldehyde), requires on-going epidemiologic surveillance of contact allergy. In this paper, the current state with special focus in fragrances and preservatives is described on the basis of data of the Information Network of Departments of Dermatology (IVDK) of the year 2010. In 2010, 12,574 patients were patch tested in the dermatology departments belonging to the IVDK. Nickel is still the most frequent contact allergen. However the continuously improved EU nickel directive already has some beneficial effect; sensitization frequency in young women is dropping. In Germany, chromate-reduced cement has been in use now for several years, leading to a decline in chromate sensitization in brick-layers. Two fragrance mixes are part of the German baseline series; they are still relevant. The most important fragrances in these mixes still are oak moss absolute and hydroxyisohexyl 3-cyclohexene carboxaldehyde. However, in relation to these leading allergens, sensitization frequency to other fragrances contained in the mixes seems to be increasing. Among the preservatives, MCI/MI has not lost its importance as contact allergen, in contrast to MDBGN. Sources of MCI/MI sensitization obviously are increasingly found in occupational context. Methylisothiazolinone is a significant allergen in occupational settings, and less frequently in body care products.

Chlorinated lipid species in activated human neutrophils: lipid metabolites of 2-chlorohexadecanal.

Neutrophils are important in the host response against invading pathogens. One chemical defense mechanism employed by neutrophils involves the production of myeloperoxidase (MPO)-derived HOCl. 2-Chlorohexadecanal (2-ClHDA) is a naturally occurring lipid product of HOCl targeting the vinyl ether bond of plasmalogens. Previous studies have shown that exogenously-added 2-ClHDA is oxidized to 2-chlorohexadecanoic acid (2-ClHA) and reduced to 2-chlorohexadecanol (2-ClHOH) by endothelial cells. These studies show that both 2-ClHA and 2-ClHOH are produced in activated neutrophils in an MPO- and time-dependent manner and are released by neutrophils into media. 2-ClHDA levels peak following 30 min of phorbol 12-myristate-13-acetate stimulation. In contrast, 2-ClHA and 2-ClHOH levels steadily increased over 60 min, suggesting a precursor-product relationship between 2-ClHDA and both 2-ClHA and 2-ClHOH. Additional experiments using wild-type CHO.K1 and CHO.K1 cells deficient in fatty aldehyde dehydrogenase (FALDH), FAA.K1A, demonstrated that 2-ClHDA oxidation to 2-ClHA is dependent on FALDH activity. Furthermore, mice exposed to intranasal Sendai virus displayed lung neutrophil recruitment, as well as elevated 2-ClHA levels in plasma and bronchoalveolar lavage compared with control-treated mice. Taken together, these data demonstrate, for the first time, that metabolites of 2-ClHDA are produced both in vivo as well as in isolated human neutrophils.

Natural IgM antibodies against oxidation-specific epitopes.

INTRODUCTION: Natural antibodies (NAbs) are mostly antibodies of the IgM isotype with germline or close to germline encoded variable regions that provide them with specificity for both microbial and altered self antigens. Thus, natural IgM possess an important function in the first line defense against invading pathogens and in tissue homeostasis by regulating the clearance of cellular debris. RESULTS: Oxidation-specific epitopes represent prominent examples of stress-induced altered self, as they are generated ubiquitously as a consequence of lipid peroxidation during many physiological and pathological situations, and are found on the membranes of apoptotic cells. Importantly, oxidation-specific epitopes are dominant targets of natural IgM antibodies, indicating an important role for natural IgM in their clearance and the neutralization of their pro-inflammatory properties. SUMMARY AND CONCLUSION: These findings and the insights they provide into the ability of NAbs in mediating host homeostasis in health and diseases, including atherosclerosis and other chronic inflammatory diseases, will be discussed.

Antibodies raised against aldehyde-fixed antigens improve sensitivity for postembedding electron microscopy.

BACKGROUND: Antibodies are one of the most important tools in biological research. High specificity and sensitivity of antibodies are crucial to obtain reliable results. Tissue fixed with glutaraldehyde (GA) is commonly used in electron microscopical investigations. The fixation and embedding routine in preparation of tissue for post-embedding electron microscopy (EM) will mask and structurally alter epitopes, making antibody-antigen interaction inefficient, with low labeling intensities. One of the main factors in this regard is the use of GA as fixative. NEW METHOD: To alleviate these technical challenges, we immunized rabbits with antigen pre-fixed with GA. We hypothesized that the resulting antibodies would have stronger affinity to antigens that have been conformationally changed and denatured by GA, the way they are in fixed tissue. COMPARISON WITH EXISTING METHOD AND RESULTS: An initial screening with western blotting (WB) showed results consistent with our hypothesis. In-house antibodies raised against GA-fixed SNARE proteins SNAP-25 and VAMP2, binds more strongly to fixed proteins compared to non-fixed proteins, while the pattern is opposite with the commercially available antibodies raised against non-fixed antigens (standard antibodies). Quantitative post-embedding EM of hippocampal synapses gave higher labeling intensities with anti-GA-SNAP-25 and anti-GA-VAMP2 compared to standard antibodies. Importantly, light microscopy (LM) and EM with our antibodies revealed stronger labeling of GA-fixed than formaldehyde (FH) treated brains. CONCLUSION: Our results highlight the experimental potential of raising antibodies against GA-treated antigen to improve sensitivity of the antibodies for postembedding immunogold EM.

Extracellular vesicle-mediated macrophage activation: An insight into the mechanism of thioredoxin-mediated immune activation.

Extracellular vesicles (EVs) generated from redox active anticancer drugs are released into the extracellular environment. These EVs contain oxidized molecules and trigger inflammatory responses by macrophages. Using a mouse model of doxorubicin (DOX)-induced tissue injury, we previously found that the major sources of circulating EVs are from heart and liver, organs that are differentially affected by DOX. Here, we investigated the effects of EVs from cardiomyocytes and those from hepatocytes on macrophage activation. EVs from H9c2 rat cardiomyocytes (H9c2 EVs) and EVs from FL83b mouse hepatocytes (FL83 b EVs) have different levels of protein-bound 4-hydroxynonenal and thus different immunostimulatory effects on mouse RAW264.7 macrophages. H9c2 EVs but not FL83 b EVs induced both pro-inflammatory and anti-inflammatory macrophage activation, mediated by NFκB and Nrf-2 pathways, respectively. DOX enhanced the effects of H9c2 EVs but not FL83 b EVs. While EVs from DOX-treated H9c2 cells (H9c2 DOXEVs) suppressed mitochondrial respiration and increased glycolysis of macrophages, EVs from DOX-treated FL83b cells (FL83b DOXEVs) enhanced mitochondrial reserve capacity. Mechanistically, the different immunostimulatory functions of H9c2 EVs and FL83 b EVs are regulated, in part, by the redox status of the cytoplasmic thioredoxin 1 (Trx1) of macrophages. H9c2 DOXEVs lowered the level of reduced Trx1 in cytoplasm while FL83b DOXEVs did the opposite. Trx1 overexpression alleviated the effect of H9c2 DOXEVs on NFκB and Nrf-2 activation and prevented the upregulation of their target genes. Our findings identify EVs as a novel Trx1-mediated redox mediator of immune response, which greatly enhances our understanding of innate immune responses during cancer therapy.

LncRNA NEAT1 alleviates sepsis-induced myocardial injury by regulating the TLR2/NF-κB signaling pathway.

OBJECTIVE: To investigate the effect of long non-coding ribonucleic acid nuclear paraspeckle assembly transcript 1 (lncRNA NEAT1) on lipopolysaccharide (LPS)-induced myocardial injury in mice and the underlying mechanism. This study aims to provide some references for the prevention and treatment of sepsis-induced myocardial injury. MATERIALS AND METHODS: According to the random number table, 60 male C57 mice were divided into the Sham group (n=20), LPS group (n=20) and LPS + NEAT1 small interfering ribonucleic acid (siRNA) group (n=20). Sepsis-induced myocardial injury model in mice was established by intraperitoneal injection of LPS (10 mg/kg), and the NEAT1 knockout model was established by tail vein injection of NEAT1 siRNAs. After 12 h, the cardiac function of mice in each group was detected via the two-dimensional ultrasound; ejection fraction [EF (%)] and fraction shortening [FS (%)] were recorded. Hematoxylin and eosin (H&E) staining was conducted to evaluate the pathological changes in the heart tissues in each group. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was used to detect the apoptotic levels of myocardial cells and fibroblasts in each group. In addition, the expression level of the oxidative stress marker 4-hydroxynonena (4-HNE) and the positive proportions of cluster of differentiation 45 (CD45) and CD68 in the mouse heart of three groups were detected via immunohistochemical staining. Moreover, the messenger RNA (mRNA) expression levels of inflammatory indicators [interleukin-1 (IL-1), IL-6, monocyte chemotactic protein 1 (MCP-1) and tumor necrosis factor-alpha (TNF-α)] in mouse serum of the three groups were examined by enzyme-linked immunosorbent assay (ELISA). Finally, the effects of NEAT1 siRNAs on the Toll-like receptor 2 (TLR2)/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway were detected by Western blotting. RESULTS: ENEAT1 knockdown could significantly improve ischemia/reperfusion (I/R)-induced cardiac insufficiency in rats, and increase EF (%) and FS (%) (p<0.05). Besides, NEAT1 knockdown remarkably inhibited the LPS-induced myocardial injury. Compared with the LPS group, LPS + NEAT 1 siRNA group has more orderly arranged cardiac myofilament, a lower degree of degradation and necrosis, and significantly reduced cell edema. TUNEL staining showed that NEAT1 knockdown markedly reduced LPS-induced apoptosis of cardiac cells (p<0.05). Immunohistochemical results revealed that NEAT1 knockdown could remarkably reverse LPS-induced elevation of the myocardial 4-HNE expression and decrease the oxidative stress in the heart (p<0.05). At the same time, CD45+ and CD68+ cells were reduced after NEAT1 knockdown in myocardial tissues (p<0.05). Reverse Transcription-Polymerase Chain Reaction (RT-PCR) showed that the mRNA levels of inflammatory indicators in LPS + NEAT1 siRNA group were lower than that in the LPS group (p<0.05). According to Western blotting results, NEAT1 siRNAs could significantly downregulate the protein expressions of TLR2 and p-p65. CONCLUSIONS: NEAT1 knockdown can improve LPS-induced myocardial injury in mice by inhibiting the TLR2/NF-κB signaling pathway. LncRNA NEAT1 is expected to be a potential target for clinical treatment of the sepsis-induced myocardial injury.

Induction of allergic contact dermatitis by astigmatid mite-derived monoterpene, alpha-acaridial.

alpha-Acaridial [2(E)-(4-methyl-3-pentenyl)butenedial] is a novel monoterpene secreted from the house dust mites. Because of its molecular nature of a highly reactive, small lipidic compound, we addressed whether alpha-acaridial might function as a haptenic allergen that induced allergic contact dermatitis. Mice sensitized with alpha-acaridial were challenged by the same antigen on the ear skin. After 2 days, significant ear swelling with a prominent infiltration of CD4(+) T lymphocytes was observed. In vitro, alpha-acaridial exhibited an outstanding ability to quickly interact with and chemically modify a reference protein. Virtually all cysteine residues and a sizable fraction of lysine residues were found to be selectively modified, suggesting that alpha-acaridial could potentially interact with any proteins. Previously, numerous mite-derived proteinaceous allergens have been associated with contact dermatitis. Our study now emphasizes that small lipidic compounds released from mites comprise a new class of mite allergens, and therefore, is of significant medical implications.