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Background and aims In focal areas of advanced human atherosclerotic lesions, the intimal fluid is acidic. An acidic medium impairs the ABCA1-mediated cholesterol efflux from macrophages, so tending to increase their content of free cholesterol, which is then available for esterification by the macrophage enzyme ACAT1. Here we investigated whether low extracellular pH would affect the activity of ACAT1. Methods – Human monocyte-derived macrophages were first incubated with acetyl-LDL at neutral and acidic conditions (pH 7.5, 6.5, and 5.5) to generate foam cells, and then the foam cells were incubated with [3H]oleate-BSA complexes, and the formation of [3H]oleate-labeled cholesteryl esters was measured. ACAT1 activity was also measured in cell-free macrophage extracts. Results – In acidic media, ACAT1-dependent cholesteryl [3H]oleate generation became compromised in the developing foam cells and their content of free cholesterol increased. In line with this finding, ACAT1 activity in the soluble cell-free fraction derived from macrophage foam cells peaked at pH 7, and gradually decreased under acidic pH with a rapid drop below pH 6.5. Incubation of macrophages under progressively more acidic conditions (until pH 5.5) lowered the cytosolic pH of macrophages (down to pH 6.0). Such intracellular acidification did not affect macrophage gene expression of ACAT1 or the neutral CEH. Conclusions Exposure of human macrophage foam cells to acidic conditions lowers their intracellular pH with simultaneous decrease in ACAT1 activity. This reduces cholesterol esterification and thus leads to accumulation of potentially toxic levels of free cholesterol, a contributing factor to macrophage foam cell death.

Atherosclerotic arterial plaques and malignant solid tumors contain macrophages, which participate in anaerobic metabolism, acidosis, and inflammatory processes inherent in the development of either disease. The tissue-resident macrophage populations originate from precursor cells derived from the yolk sac and from circulating bone marrow-derived monocytes. In the tissues, they differentiate into varying functional phenotypes in response to local microenvironmental stimulation. Broadly categorized, the macrophages are activated to polarize into proinflammatory M1 and anti-inflammatory M2 phenotypes; yet, noticeable plasticity allows them to dynamically shift between several distinct functional subtypes. In atherosclerosis, low-density lipoprotein (LDL)-derived cholesterol accumulates within macrophages as cytoplasmic lipid droplets thereby generating macrophage foam cells, which are involved in all steps of atherosclerosis. The conversion of macrophages into foam cells may suppress the expression of given proinflammatory genes and thereby initiate their transcriptional reprogramming toward an anti-inflammatory phenotype. In this particular sense, foam cell formation can be considered anti-atherogenic. The tumor-associated macrophages (TAMs) may become polarized into anti-tumoral M1 and pro-tumoral M2 phenotypes. Mechanistically, the TAMs can regulate the survival and proliferation of the surrounding cancer cells and participate in various aspects of tumor formation, progression, and metastasis. The TAMs may accumulate lipids, but their type and their specific roles in tumorigenesis are still poorly understood. Here, we discuss how the phenotypic and functional plasticity of macrophages allows their multifunctional response to the distinct microenvironments in developing atherosclerotic lesions and in developing malignant tumors. We also discuss how the inflammatory reactions of the macrophages may influence the development of atherosclerotic plaques and malignant tumors, and highlight the potential therapeutic effects of targeting lipid-laden macrophages in either disease.

In atherosclerotic lesions, blood-derived monocytes differentiate into distinct macrophage subpopulations, and further into cholesterol-filled foam cells under a complex milieu of cytokines, which also contains macrophage-colony stimulating factor (M-CSF) and granulocyte–macrophage-colony stimulating factor (GM-CSF). Here we generated human macrophages in the presence of either M-CSF or GM-CSF to obtain M-MØ and GM-MØ, respectively. The macrophages were converted into cholesterol-loaded foam cells by incubating them with acetyl-LDL, and their atheroinflammatory gene expression profiles were then assessed. Compared with GM-MØ, the M-MØ expressed higher levels of CD36, SRA1, and ACAT1, and also exhibited a greater ability to take up acetyl-LDL, esterify cholesterol, and become converted to foam cells. M-MØ foam cells expressed higher levels of ABCA1 and ABCG1, and, correspondingly, exhibited higher rates of cholesterol efflux to apoA-I and HDL2. Cholesterol loading of M-MØ strongly suppressed the high baseline expression of CCL2, whereas in GM-MØ the low baseline expression CCL2 remained unchanged during cholesterol loading. The expression of TNFA, IL1B, and CXCL8 were reduced in LPS-activated macrophage foam cells of either subtype. In summary, cholesterol loading converged the CSF-dependent expression of key genes related to intracellular cholesterol balance and inflammation. These findings suggest that transformation of CSF-polarized macrophages into foam cells may reduce their atheroinflammatory potential in atherogenesis.

There are clear benefits of physical activity and especially exercise training on performance, including adaptation of muscle mass and strength. Maintenance of skeletal muscle mass is determined by a fine balance between protein synthesis and degradation. Loss of muscle mass ensues when degradation exceeds protein synthesis, which is observed in numerous conditions, including immobilization and age-related sarcopenia. Heat shock proteins (HSPs) are a family of highly conserved proteins that contribute to cellular protection, protein homeostasis, and survival against a variety of environmental and metabolic stresses. Some HSPs have been suggested for roles controlling skeletal muscle mass, and they may play a fundamental role in skeletal muscle function. Conversely, inability to induce individual HSPs could have deleterious effects on skeletal muscle protection and tolerance to external stressors. Both acute and chronic exercise induces HSP expression. As a physiological tool, physical exercise can be considered the safest strategy to enhance HSP levels in skeletal muscle. Studies on the modulation of skeletal muscle HSP levels using antioxidant and dietary supplementation strategies have provided contradictory results and warrant further investigation.

Background In atherosclerotic lesions, cholesterol-laden macrophage foam cells are formed and exposed to M1- and M2-polarizing factors. However, the effects of the polarizing factors on the proinflammatory and the anti-inflammatory potential of foam cells are not known. Objective To investigate the effects of M1- and M2-polarizing factors on the expression of pro- and anti-inflammatory genes in cultured human macrophage foam cells. Methods and Results Human monocytes were differentiated into macrophages in the presence of M-CSF, and then converted into cholesterol-loaded foam cells by incubation with acetylated LDL. The generated macrophages and foam cells were polarized into the M1 phenotype by classical activation with LPS and IFN-γ, or into the M2 phenotype by alternative activation with IL-4. When subjected to the M1-polarizing factors, the macrophages responded by typical upregulation of several key proinflammatory genes (TNFA , IL1B , CXCL8 , CCL19 , and COX2) , while the anti-inflammatory genes ( MRC1 , CCL17 , and IL10 ) displayed variable responses. The foam cells, again, showed a weaker response to the M1-polarizing factors, as indicated by reduced upregulation of the proinflammatory genes, reduced secretion of TNF-α, and a trend towards lower NF-κB activity. When subjected to alternative M2 polarization, both macrophages and foam cells responded by a typical upregulation of the anti-inflammatory genes, which was of equal magnitude in both cell types. Conclusions Conversion of cultured human macrophages into foam cells suppresses their proinflammatory responses to M1-polarizing factors. Thus, in M1-polarizing microenvironments of atherosclerotic lesions, foam cell formation may locally weaken the macrophage-dependent inflammatory component of atherogenesis.

Supplemental Digital Content is available in the text., Objective— Apolipoprotein A-I (apoA-I) has been shown to possess several atheroprotective functions, including inhibition of inflammation. Protease-secreting activated mast cells reside in human atherosclerotic lesions. Here we investigated the effects of the neutral proteases released by activated mast cells on the anti-inflammatory properties of apoA-I. Approach and Results— Activation of human mast cells triggered the release of granule-associated proteases chymase, tryptase, cathepsin G, carboxypeptidase A, and granzyme B. Among them, chymase cleaved apoA-I with the greatest efficiency and generated C-terminally truncated apoA-I, which failed to bind with high affinity to human coronary artery endothelial cells. In tumor necrosis factor-α–activated human coronary artery endothelial cells, the chymase-cleaved apoA-I was unable to suppress nuclear factor-κB–dependent upregulation of vascular cell adhesion molecule-1 (VCAM-1) and to block THP-1 cells from adhering to and transmigrating across the human coronary artery endothelial cells. Chymase-cleaved apoA-I also had an impaired ability to downregulate the expression of tumor necrosis factor-α, interleukin-1β, interleukin-6, and interleukin-8 in lipopolysaccharide-activated GM-CSF (granulocyte-macrophage colony-stimulating factor)– and M-CSF (macrophage colony-stimulating factor)–differentiated human macrophage foam cells and to inhibit reactive oxygen species formation in PMA (phorbol 12-myristate 13-acetate)–activated human neutrophils. Importantly, chymase-cleaved apoA-I showed reduced ability to inhibit lipopolysaccharide-induced inflammation in vivo in mice. Treatment with chymase blocked the ability of the apoA-I mimetic peptide L-4F, but not of the protease-resistant D-4F, to inhibit proinflammatory gene expression in activated human coronary artery endothelial cells and macrophage foam cells and to prevent reactive oxygen species formation in activated neutrophils. Conclusions— The findings identify C-terminal cleavage of apoA-I by human mast cell chymase as a novel mechanism leading to loss of its anti-inflammatory functions. When targeting inflamed protease-rich atherosclerotic lesions with apoA-I, infusions of protease-resistant apoA-I might be the appropriate approach.

Background— The HLA-DRB1*01 allele of the human leukocyte antigen has been associated with acute coronary syndrome. Genome-wide association studies have revealed associations with human leukocyte antigen and non–human leukocyte antigen genes of 3 major histocompatibility complex gene classes but not at allelic level. Methods and Results— We conducted a large-scale genetic analysis on a case–control cohort comprising 5376 acute coronary syndrome cases and 4852 unrelated controls from 4 populations of 2 European countries. We analyzed the risk candidate allele of HLA-DRB1*01 by genomic real-time polymerase chain reaction together with high-density single nucleotide polymorphisms of the major histocompatibility complex to precisely identify risk loci for acute coronary syndrome with effective clinical implications. We found a risk haplotype for the disease containing single nucleotide polymorphisms from BTNL2 and HLA-DRA genes and the HLA-DRB1*01 allele. The association of the haplotype appeared in 3 of the 4 populations, and the direction of the effect was consistent in the fourth. Coronary samples from subjects homozygous for the disease-associated haplotype showed higher BTNL2 mRNA levels ( r =0.760; P + FOXP3 + regulatory T cell proliferation significantly (blocking versus nonblocking; P Conclusions— In cases with the risk haplotype for acute coronary syndrome, these results suggest involvement of enhanced immune reactions. BTNL2 may have an inhibitory effect on FOXP3 + T cell proliferation, especially in patients homozygous for the risk alleles. Clinical Trial Registration— https://www.clinicaltrials.gov ; Unique Identifier: NCT00417534.

Impaired expression of heat shock proteins (HSPs) and increased oxidative stress may contribute to the pathophysiology of diabetes by disrupted tissue protection. Acute exercise induces oxidative stress, whereas exercise training up-regulates endogenous antioxidant defenses and HSP expression. Although diabetic nephropathy is a major contributor to diabetic morbidity, information regarding the effect of HSPs on kidney protection is limited. This study evaluated the effects of eight-week exercise training on kidney HSP expression and markers of oxidative stress at rest and after acute exercise in rats with or without streptozotocin-induced diabetes. Induction of diabetes increased DNA-binding activity of heat shock factor-1, but decreased the expression of HSP72, HSP60, and HSP90. The inflammatory markers IL-6 and TNF-alpha were increased in the kidney tissue of diabetic animals. Both exercise training and acute exercise increased HSP72 and HSP90 protein levels only in non-diabetic rats. On the other hand, exercise training appeared to reverse the diabetes-induced histological changes together with decreased expression of TGF-beta as a key inducer of glomerulosclerosis, and decreased levels of IL-6 and TNF-alpha. Notably, HSP72 and TGF-beta were negatively correlated. In conclusion, impaired HSP defense seems to contribute to kidney injury vulnerability in diabetes and exercise training does not up-regulate kidney HSP expression despite the improvements in histopathological and inflammatory markers.

Summary Mast cells have been implicated in the first line of defence against parasites and bacteria, but less is known about their role in anti-viral responses. Allergic diseases often exacerbate during viral infection, suggesting an increased activation of mast cells in the process. In this study we investigated human mast cell response to double-stranded RNA and viral infection. Cultured human mast cells were incubated with poly(I:C), a synthetic RNA analogue and live Sendai virus as a model of RNA parainfluenza virus infection, and analysed for their anti-viral response. Mast cells responded to intracellular poly(I:C) by inducing type 1 and type 3 interferons and TNF-α. In contrast, extracellular Toll-like receptor 3 (TLR)-3-activating poly(I:C) failed to induce such response. Infection of mast cells with live Sendai virus induced an anti-viral response similar to that of intracellular poly(I:C). Type 1, but not type 3 interferons, up-regulated the expression of melanoma differentiation–associated gene 5 (MDA-5) and retinoic acid-inducible gene-1 (RIG-1), and TLR-3, demonstrating that human mast cells do not express functional receptors for type 3 interferons. Furthermore, virus infection induced the anti-viral proteins MxA and IFIT3 in human mast cells. In conclusion, our results support the notion that mast cells can recognize an invading virus through intracellular virus sensors and produce high amounts of type 1 and type 3 interferons and the anti-viral proteins human myxovirus resistance gene A (MxA) and interferon-induced protein with tetratricopeptide repeats 3 (IFIT3) in response to the virus infection.

Objective To investigate mechanisms of lymphangiogenesis in aortic valve stenosis (AS). Methods Lymphatic vessels were visualized with LYVE-1 staining in 20 control, 5 sclerotic, and 40 stenotic human aortic valves. Vascular endothelial growth factors (VEGFs) VEGF-C and VEGF-D, and their lymphangiogenic receptor VEGFR-3, and the angiogenic VEGFR-2 were analysed by quantitative real-time PCR and immunohistochemistry. Cultured myofibroblasts derived from human stenotic aortic valves, and cultured human mast cells were used to study VEGF-C regulation, and VEGF-C and VEGF-A were quantified from cell culture media by enzyme immunoassays. Results Lymphatic vessels, VEGF-C, VEGF-D, VEGFR-3 and VEGFR-2 all were present in the aortic valves. In AS, the number of lymphatic vessels and the expression of VEGF-D, VEGFR-3, and VEGFR-2 were increased. Moreover, the numbers of lymphatic vessels correlated positively with those of neovessels ( r =0.525, p =0.001) and mast cells ( r =0.374, p =0.017). Cultured valvular myofibroblasts produced VEGF-C, and addition of tumour necrosis factor alpha (TNF-α) to the cells augmented its secretion. In contrast, proteases released by activated human mast cells degraded VEGF-C. Conclusion These results show that lymphangiogenesis is induced in advancing AS. Furthermore, valvular myofibroblasts and activated mast cells were identified as novel regulators of lymphangiogenesis in aortic valves.

Lipid droplets, also called lipid bodies (LB) in inflammatory cells, are important cytoplasmic organelles. However, little is known about the molecular characteristics and functions of LBs in human mast cells (MC). Here, we have analyzed the genesis and components of LBs during differentiation of human peripheral blood-derived CD34(+) progenitors into connective tissue-type MCs. In our serum-free culture system, the maturing MCs, derived from 18 different donors, invariably developed triacylglycerol (TG)-rich LBs. Not known heretofore, the MCs transcribe the genes for perilipins (PLIN)1-4, but not PLIN5, and PLIN2 and PLIN3 display different degrees of LB association. Upon MC activation and ensuing degranulation, the LBs were not cosecreted with the cytoplasmic secretory granules. Exogenous arachidonic acid (AA) enhanced LB genesis in Triacsin C-sensitive fashion, and it was found to be preferentially incorporated into the TGs of LBs. The large TG-associated pool of AA in LBs likely is a major precursor for eicosanoid production by MCs. In summary, we demonstrate that cultured human MCs derived from CD34(+) progenitors in peripheral blood provide a new tool to study regulatory mechanisms involving LB functions, with particular emphasis on AA metabolism, eicosanoid biosynthesis, and subsequent release of proinflammatory lipid mediators from these cells.

Objective Human atherosclerotic lesions contain mast cells and immunoglobulin G immune complexes containing oxidized low-density lipoproteins (oxLDL-IgG ICs). Here we studied whether such oxLDL-IgG ICs can activate human mast cells and induce them to express and secrete pro-inflammatory cytokines that are potentially capable of inducing and amplifying atherogenic processes. Methods and results Incubation of cultured human mast cells in the presence of oxLDL-IgG ICs led to a significant dose-dependent upregulation of the expression and secretion of tumor necrosis factor-alpha (TNF-a) and interleukin-8 (IL-8), and the chemotactic cytokine monocyte chemoattractant protein-1 (MCP-1). The secretory responses were dose-dependent and associated with moderate release of histamine and tryptase, which are preformed mast cell mediators contained in the cytoplasmic secretory granules of the cells. Also native LDL-IgG ICs induced similar pro-inflammatory cytokine response, suggesting that ICs per se are important for the IgG IC-induced mast cell activation. Conclusion Mast cells in atherosclerotic lesions which also contain oxLDL-IgG ICs may become activated by the ICs and secrete many pro-inflammatory cytokines. Our results suggest that intimal mast cells act as a cellular link between oxLDL-IgG ICs and the inflammatory response in atherosclerosis.

To cite this article: Ribbing C, Engblom C, Lappalainen J, Lindstedt K, Kovanen PT, Karlsson MA, Lundeberg L, Johansson C, Nilsson G, Lunderius-Andersson C, Scheynius A. Mast cells generated from patients with atopic eczema have enhanced levels of granule mediators and an impaired Dectin-1 expression. Allergy 2011; 66: 110–119. Abstract Background: The disrupted skin barrier of patients with atopic eczema (AE) might facilitate contact between mast cells (MCs) in the skin and environmental triggers of the disease. One such trigger is the skin-colonizing yeast Malassezia sympodialis (M. sympodialis). In this study, we investigated the interaction of MC with M. sympodialis. Methods: Mast cells were generated from peripheral blood CD34+ progenitor cells of healthy controls (HC) and M. sympodialis-sensitized AE patients. Biopsy specimens were taken from HC and lesional AE skin for immunohistological stainings. Results: The progenitor-derived MCs expressed the macrophage-inducible C-type lectin receptor Mincle, and exposure of these cells to M. sympodialis induced up-regulation of the mRNA expression of Mincle. Furthermore, we demonstrate that, when compared to HC, the progenitor-derived MCs from AE patients (i) contain more intrinsic granule mediators such as histamine, (ii) exhibit enhanced IL-6 release in response to M. sympodialis exposure, and (iii) have an impaired up-regulation of the fungal recognition receptor Dectin-1. In addition, analysis of skin sections from HC and AE patients revealed MCs as the predominant Dectin-1-expressing cell type in the skin. Conclusion: Our data indicate that progenitor-derived MCs from AE patients differ from those from HC. Further investigations with skin-derived MCs are necessary to confirm the observed differences which could provide new insights into the pathogenic mechanisms underlying AE.

Heat shock proteins (HSPs) are molecular chaperones which may act protective in cerebrovascular insults and peripheral diabetic neuropathy. We hypothesized that alpha-lipoic acid (LA), a natural thiol antioxidant, may enhance brain HSP response in diabetes. Rats with or without streptozotocin-induced diabetes were treated with LA or saline for 8 weeks. Half of the rats were subjected to exhaustive exercise to investigate HSP induction, and the brain tissue was analyzed. Diabetes increased constitutive HSC70 mRNA, and decreased HSP90 and glucose-regulated protein 75 (GRP75) mRNA without affecting protein levels. Exercise increased HSP90 protein and mRNA, and also GRP75 and heme oxygenase-1 (HO-1) mRNA only in non-diabetic animals. LA had no significant effect on brain HSPs, although LA increased HSC70 and HO-1 mRNA in diabetic animals and decreased HSC70 mRNA in non-diabetic animals. Eukaryotic translation elongation factor-2, essential for protein synthesis, was decreased by diabetes and suggesting a mechanism for the impaired HSP response related to translocation of the nascent chain during protein synthesis. LA supplementation does not offset the adverse effects of diabetes on brain HSP mRNA expression. Diabetes may impair HSP translation through elongation factors related to nascent chain translocation and subsequent responses to acute stress.

Objective— In the deep microenvironments of advanced human atherosclerotic lesions, the intimal fluid becomes acidic. We examined the effect of an acidic extracellular pH on cholesterol removal (efflux) from primary human macrophages. Methods and Results— When cholesterol efflux from acetyl-low-density lipoprotein-loaded macrophages to various cholesterol acceptors was evaluated at pH 7.5, 6.5, or 5.5, the lower the pH the more was cholesterol efflux reduced. The reduction of efflux to lipid-free apolipoprotein A-I was stronger than to high-density lipoprotein 2 or to plasma. Cholesterol efflux to every acceptor was severely compromised also at neutral pH when the macrophages had been loaded with cholesterol at acidic pH, or when both loading and efflux were carried out at acidic pH. Compatible with these observations, the typical upregulation of ABCA1 and ABCG1 mRNA levels in macrophages loaded with cholesterol at neutral pH was rapidly attenuated in acidic medium. The secondary structure of apolipoprotein A-I did not changed over the pH range studied, supporting the notion that the inhibitory effect of acidic pH on cholesterol efflux rather impaired the ability of the foam cells to facilitate ABCA1-mediated cholesterol release. Secretion of apolipoprotein E from the foam cells was fully inhibited when the pH was 5.5, which further reduced cholesterol efflux. Conclusion— An acidic pH reduces cholesterol efflux via different pathways and particularly impairs the function of the ABCA1 transporter. The pH-sensitive function of human macrophage foam cells in releasing cholesterol may accelerate lipid accumulation in deep areas of advanced atherosclerotic plaques where the intimal fluid is acidic.

Leptin regulates body weight, metabolism, and tissue adaptations to environmental stressors. We examined the association of single nucleotide polymorphism (SNP) of leptin promoter G-2548A (rs7799039) and leptin receptor Gln223Arg (rs1137101) with body composition, plasma leptin levels, and peak oxygen uptake (VO(2)peak) in response to 8 weeks of physical training in 48 male military conscripts. AA homozygotes of leptin promoter SNP-2548 showed higher body fat and BMI values than G allele carriers. Acute exercise decreased leptin levels in G allele carriers, but increased in AA homozygotes. Physical training significantly decreased BMI values and also a tendency for decreased plasma leptin levels was observed in all subjects. In G allele carriers, BMI loss was mainly due to decreased fat mass, whereas in AA homozygotes due to loss of fat-free mass. Training increased VO(2)peak in all subjects with most prominent effects in G allele carriers. Regarding leptin receptor SNP, there were no statistically significant differences in BMI values between the genotype groups at baseline or after physical training. Our results suggest that physical training-induced alterations in body composition and plasma leptin may be influenced by a genetic variation of leptin promoter but not of leptin receptor.

Thioredoxin (TRX) is a protein disulfide reductase that plays an important role in many thiol-dependent cellular reductive processes, antioxidant protection, and signal transduction. Moreover, TRX reduces and maintains the function of many proteins during oxidative stress, which is increased in diabetes. The authors recently reported that diabetes impairs brain redox status and TRX response to exercise training. As a continuation of their studies, they hypothesized that alpha-lipoic acid, a natural thiol antioxidant, has a favorable effect on the brain TRX and glutathione (GSH) system in diabetes. Streptozotocin-induced diabetes was used as a chronic model and exhaustive exercise as an acute model for disrupted redox balance. Half the diabetic and nondiabetic animals were subjected to a bout of exhaustive exercise after 8 wk with or without lipoic acid and analyzed for key thiol antioxidants. Lipoic acid neither altered diabetes-induced oxidative stress as assessed by the increased ratio of oxidized to total GSH nor had any impact on the antioxidant protein response to exercise. However, lipoic acid increased mRNA of TRX-interacting protein, an inhibitor of TRX-1, and glutaredoxin-1 in diabetes. Exercise increased TRX-1 mRNA in both diabetic and nondiabetic animals but had no effect on TRX-1 protein. Cytosolic superoxide dismutase mRNA was only increased in diabetes, whereas exercise increased the protein levels in nondiabetic animals. The findings suggest that exhaustive exercise induces mRNA of TRX-1 in the brain and that lipoic acid cannot prevent diabetes-induced disturbances in GSH homeostasis. Because lipoic acid increased TRX-interacting protein transcription in diabetes, high doses may impair TRX-1 homeostasis.

Objective— To examine the proangiogenic potential of myofibroblasts and mast cells, 2 types of cells present in human aortic valves. Methods and Results— Aortic valve stenosis is an active atheroinflammatory disease, characterized by the accumulation of inflammatory cells and the neovascularization of the valves. A total of 85 stenotic valves and 20 control valves were obtained during valve replacement surgery. The results of immunohistochemistry analysis revealed stenotic aortic valves that contained 3 types of neovessels: small microvessels, medium microvessels, and organized arterioles. The distribution density of the neovessels was significantly higher in stenotic valves than in control valves ( P r =0.26, P =0.02) and mast cell density ( r =0.38, P Conclusion— Mast cells and myofibroblasts may accelerate the progression of aortic valve stenosis by altering the balance between angiogenic and antiangiogenic factors in the valves, thus promoting valvular neovascularization.

Circadian rhythms are known to influence muscular performance and regulate leptin hormone secretion, which assists in the regulation of body weight and energy homeostasis. In this study we examined the time-of-day effect on skeletal muscle eccentric contractile properties and the response of serum leptin to acute eccentric exhaustive isokinetic exercise. Male subjects (n = 26) performed a bout of exhaustive eccentric exercise on an isokinetic dynamometer in the morning or late afternoon time on different days. The test was repeated after 7-10 days at different time-of-day. Blood samples were collected at baseline and immediately after exercise for determination of serum leptin. Leg extensor muscle torque and work parameters were recorded, and compared with the time-of-day. Maximal eccentric torque values were not significantly affected by time-of-day. The maximal work performed during single repetition as well as total work values were significantly higher in the afternoon time. Leptin levels significantly decreased after exercise, but no time-of-day effect on serum leptin could be observed. Diurnal variations during short-term isokinetic exhaustive eccentric exercise seem to reflect muscle work capacity, with higher contractile efficiency towards the evening time. Although short-term exhaustive eccentric exercise decreased serum leptin, the levels did not to have a significant diurnal variation.

Intercellular adhesion molecules (ICAMs) bind to leukocyte β2 integrins, which, among other functions, provide costimulatory signals for T-cell activation. ICAM-5 (telencephalin) is expressed in the somadendritic region of neurons of the mammalian brain. The receptor for ICAM-5 is the integrin LFA-1, a major leukocyte integ-rin expressed in lymphocytes and microglia. In conditions of brain ischemia, epilepsy, and encephalitis, the soluble form of ICAM-5 (sICAM-5) has been detected in physiologic fluids. Here, we report that sICAM-5 attenuates the T-cell receptor-mediated activation of T cells as demonstrated by the decreased expression of the activation markers CD69, CD40L, and CD25 (IL-2R). This effect is most clearly seen in CD45ROLow (naive), and not in CD45ROHigh (memory) T cells, and is most effective early in priming, but not in the presence of strong costimulatory signals. Furthermore, sICAM-5 promotes the mRNA expression of the cytokines TGF-β1 and IFN-γ, but not TNF. The formation of sICAM-5 is promoted by activated T cells through the cleavage of ICAM-5 from neurons. This suggests that ICAM-5 is involved in immune privilege of the brain and acts as an anti-inflammatory agent.

A broad variety of microbes are present in atherosclerotic plaques and chronic bacterial infection increases the risk of atherosclerosis by mechanisms that have remained vague. One possible mechanism is that bacteria or bacterial products activate plaque mast cells that are known to participate in the pathogenesis of atherosclerosis. Here, we show by real-time PCR analysis and ELISA that Chlamydia pneumoniae (Cpn) and a periodontal pathogen, Aggregatibacter actinomycetemcomitans (Aa), both induce a time and concentration-dependent expression and secretion of interleukin 8 (IL-8), tumour necrosis factor-alpha (TNF-alpha) and monocyte chemoattractant protein-1 (MCP-1) by cultured human peripheral blood-derived mast cells, but not anti-inflammatory molecules, such as IL-10 or transforming growth factor beta 1 (TGF-beta 1). The IL-8 and MCP-1 responses were immediate, whereas the onset of TNF-alpha secretion was delayed. The Cpn-mediated pro-inflammatory effect was attenuated when the bacteria were inactivated by UV-treatment. Human monocyte-derived macrophages that were pre-infected with Cpn also induced a significant pro-inflammatory response in human mast cells, both in cocultures and when preconditioned media from Cpn-infected macrophages were used. Intranasal and intravenous administration of live Cpn and Aa, respectively induced an accumulation of activated mast cells in the aortic sinus of apolipoprotein E-deficient mice, however, with varying responses in the systemic levels of lipopolysaccharide (LPS) and TNF-alpha. Pro-atherogenic Cpn and Aa induce a pro-inflammatory response in cultured human connective tissue-type mast cells and activation of mouse aortic mast cells in vivo.

Summary Background Mast cells (MCs) are multi-functional effector cells with an essential role in innate immunity and host defence, and under several pathological conditions, such as allergy. Here, we aimed at defining the culture conditions that would allow efficient generation of mature and functional human MCs from their progenitor cells. Methods Human peripheral blood-derived CD34+ progenitor cells were cultured in vitro under serum-free conditions with human stem cell factor for 9 weeks. Growth and differentiation of the cells into MCs were optimized by selected cytokines and a combination of hypoxic and normoxic conditions. MCs were phenotypically characterized by immunocytochemistry, their preformed mediators were quantified, and their functional ability to degranulate and release histamine was tested. Results On average, 20 × 106 mature MCs were generated from 0.5 × 106 progenitor cells during 9 weeks of culture, i.e. at least a 40-fold increase in cell number was achieved. The mature MCs had oval-shaped non-lobular nuclei, contained histamine, heparin, tryptase, chymase, and cathepsin G in their secretory granules, and strongly expressed c-kit (CD117) and Fc epsilon receptor I on their surface. Histamine release from the cells could be brought about by IgE–anti-IgE cross-linkage, compound 48/80, substance P, and anaphylatoxin C3a. The MCs remained functional for several weeks after their maturation. Conclusion This study describes an efficient protocol for generating mature MCs from human peripheral blood with a functional phenotype of connective tissue-type MCs. Use of these cultured human MCs will increase our knowledge and understanding about human MC development and biology in human disease.

To study the consequences of mast cell activation in human synovial tissue.Synovial tissue was obtained from 18 RA patients and mast cells was selectively activated in synovial tissue explant cultures. Expression of TNF-alpha, IL-1beta and IL-1Ra were determined and tissue distribution of IL-1beta was studied.Compared to untreated synovia, selective activation of synovial mast cells increased significantly the production of TNF-alpha (0.49 +/- 0.88 vs. 4.56 +/- 3.18 pg/mg wet tissue, p0.001) and IL-1beta (0.058 +/- 0.032 vs. 2.55 +/- 1.98 pg/mg wet tissue, p = 0.013). The expression of TNF-alpha and IL-1beta mRNA increased significantly (19-fold (p = 0.009) and 13-fold (p = 0.031), respectively). Mast cell activation induced IL-1beta expression in particular in nearby CD68 positive synovial macrophages. Secretion of IL-1Ra was also increased but to a lesser degree than that of IL-1beta.Synovial mast cells produce proinflammmatory cytokines and may thus contribute to the inflammation in RA.

Increased oxidative stress and impaired heat shock protein (HSP) synthesis may contribute to diabetic nephropathy. The question of whether 8-week thiol antioxidant alpha-lipoic acid (LA) supplementation modulates HSP response and oxidative stress was studied in the kidney of streptozotocin-induced diabetic (SID) and nondiabetic rats. SID caused a histological mesangial expansion, tubular dilatation, and increased levels of transforming growth factor-beta (TGF-beta), a mediator of glomerulosclerosis. SID increased 4-hydroxynonenal (4-HNE) protein adduct formation, a marker of lipid peroxidation, and heme oxygenase-1 (HO-1), also a marker of oxidative stress. Moreover, SID increased the DNA-binding activity of heat shock factor-1 (HSF-1) and expression of heat shock protein 60 (HSP60). In contrast, LA supplementation partially reversed histological findings of glomerulosclerosis and decreased TGF-beta. LA also increased HSF-1 and decreased HO-1 protein expression, without affecting 4-HNE protein adduct levels. At the mRNA level, LA increased expression of HSF-1, HSP90, and glucose-regulated protein (GRP75) in both control and diabetic animals and HSP72 in SID rats. However, LA supplementation did not affect these HSPs at the protein level. These findings suggest that in addition to its antiglomerulosclerotic effects, LA can induce cytoprotective response in SID.

Since her shipwreck in 1771, the wooden vessel Vrouw Maria has been lying on the seabed at 41. m depth on the border between the Archipelago Sea and the northern Baltic proper, off the southern coast of Finland. The wreck lies in an upward position in a right angle to the dominating bottom current, which is potentially affecting seabed topography, sediment characteristics and zoobenthic communities both upstream (NE) and downstream (SW) of the wreck. This multidisciplinary study attempts to clarify abiotic and biotic patterns and processes in the vicinity of the wreck by combining field investigations with physical simulation studies in the field and in the laboratory. Multibeam echo-sounder techniques were utilised to generate a map of the wreck area and sediment grab samples were taken to characterize the sediment type and its zoobenthic community. A medium-sized field experiment generated data on the accumulation of sediment organic matter in the presence and absence of a current and/or a barrier on the seafloor and a small-scaled laboratory study was conducted to simulate scour forming processes. The results showed that a deeper basin, scour area, with the dimensions 150×300m, was present downstream of the wreck and there was also a smaller scour area upstream of the wreck. Similar traces on the bottom were simulated in the laboratory. The organic content (recent mud) and the proportion of finer sediment were more pronounced in areas closer to both sides of the wreck. These results were in turn imitated in a field experiment, where the accumulation of organic matter in the sediment increased significantly downstream, when a current was interrupted by a barrier. Regarding zoobenthos in the wreck area, 11 taxa and a mean total abundance of 773 individuals per m2 were registered. The dominant species were Macoma balthica and Marenzelleria sp., which together made up >80% of the total number of individuals. Multivariate data analyses showed significant differences in community structure between upstream and downstream sides of the wreck and these differences were basically expressed as more individuals upstream of the wreck of the seven most common species. Univariate data analyses also showed significantly more species and higher total abundance in the scour area upstream of the wreck compared to the downstream scour area. The weakened communities downstream of the wreck were possibly due to accumulation of organic matter and more frequent occurrence of hypoxia. The results are useful for describing bottom areas around long-term physical barriers/artificial reefs, as background measurements ahead of possibly increased archaeological diving activities, sediment excavations or measures taken to raise the wreck to the surface, as well as for any attempts to preserve the wreck in situ.

The pathophysiology of diabetes includes oxidative stress and impaired heat shock protein (HSP) expression. We studied the effects of α-lipoic acid (LA) supplementation for 8 weeks and acute exercise on HSP60 expression and the oxidative stress marker 4-hydroxynonenal adducts (4-HNE) in streptozotocin-induced diabetic (SID) and nondiabetic control rats. Diabetes was associated with decreased HSP60 in the heart and increased levels of HSP60 and 4-HNE in the liver. LA increased HSP60 in the liver of control and diabetic rats and decreased 4-HNE in the liver and heart. Acute exercise increased liver 4-HNE, which was offset by LA. In conclusion, diabetes induced oxidative stress and impaired myocardial HSP60 expression, while LA partially offsets these alterations in a tissue-specific manner.

OBJECTIVE—The aim of this study was to examine how the major components of the metabolic syndrome relate to each other and to the development of diabetes using factor analysis. RESEARCH DESIGN AND METHODS—The screening survey for type 2 diabetes was conducted in 1994, and a follow-up study of nondiabetic individuals at baseline was carried out in 1999 in the Beijing area. Among 934 nondiabetic and 305 diabetic subjects at baseline, factor analysis was performed using the principle components analysis with varimax orthogonal rotation of continuously distributed variables considered to represent the components of the metabolic syndrome. Fasting insulin was used as a marker for insulin resistance. Of the 559 subjects without diabetes at baseline, 129 developed diabetes during the 5-year follow-up. Factors identified at baseline were used as independent variables in univariate and multivariate logistic regression models to determine risk factor clusters predicting the development of diabetes. RESULTS—Four factors were identified in nondiabetic and diabetic subjects. Fasting insulin levels, BMI, and waist-to-hip ratio were associated with one factor. Systolic and diastolic blood pressures were associated with the second factor. Two-hour postload plasma glucose (2-h PG) and serum insulin and fasting plasma glucose were associated with the third factor. Serum total cholesterol and triglycerides were associated with the fourth factor. The first and the third factors predicted the development of diabetes. In diabetic patients at baseline, the combination of systolic and diastolic blood pressure was the most important factor, and urinary albumin excretion rate clustered with fasting and 2-h PG levels. CONCLUSIONS—Insulin resistance alone does not underlie all features of the metabolic syndrome. Different physiological processes associated with various components of the metabolic syndrome contain unique information about diabetes risk. Microalbunuria is more likely to be a complication of type 2 diabetes or hypertension than a marker for the metabolic syndrome.

Strenuous exercise induces oxidative stress and modification of intracellular proteins. Exercise training, however, upregulates endogenous antioxidant defenses and heat shock protein (HSP) expression. In diabetes, perturbations in the endogenous antioxidant and HSP protection have been reported. The aim of this study was to examine the effect of 8 wk of endurance training on HSP expression and oxidative stress markers in the skeletal muscle, heart, and liver of streptozotocin-induced diabetic (SID) and nondiabetic control rats. Induction of diabetes decreased HSP72 expression in heart, liver, and vastus lateralis muscles. SID increased heme oxygenase-1, an oxidative stress-inducible HSP, in liver, red gastrocnemius muscle, and vastus lateralis muscle and glucose-regulated protein 75 in liver. SID increased HSP90 levels in the heart, but levels decreased in the liver. Diabetes induced oxidative stress marker protein carbonyl levels and tissue inflammation. Although endurance training increased the expression of HSP72 in all of the tissues examined, this induction was less pronounced in diabetic rats than in nondiabetic controls. Furthermore, endurance training induced the activation and expression of transcriptional regulator heat shock factor-1 only in nondiabetic control animals. In summary, diabetes may increase susceptibility to oxidative damage and impair HSP protection, but endurance training may offset some of the adverse effects of diabetes by upregulating tissue HSP expression. Our results suggest that diabetes impairs HSP protection, possibly via transcriptionally mediated mechanisms.

Acute exercise induces oxidative stress and heat shock protein (HSP) expression. Information on the protection of stress proteins against oxidant insult and muscle damage during moderate exercise is scanty. We aimed to show how a single bout of moderate exercise affects the markers of oxidative stress and heat shock factor-1 (HSF1; the transcriptional regulator of HSP synthesis), and HSP70, HSP90 and glucose-regulated protein (GRP75) expression in horses. Eight clinically normal and regularly trained standardbred trotters were treadmill-exercised for 45 min at moderate intensity. Blood samples were collected prior to and immediately after exercise and at 4 and 24 h of recovery. Muscle biopsy samples from the middle gluteal muscle were taken before exercise and after 4 h of recovery. Acute exercise did not activate HSF1 or induce expression of HSP70, HSP90 or GRP75 in skeletal muscle. One bout of acute exercise increased protein oxidation, which was measured by protein carbonyls in plasma and muscle, but it did not effect 4-hydroxynonenal protein adducts, which are markers of lipid peroxidation. Furthermore, mild muscle damage was observed 4 h after exercise. Our results showed that horses are susceptible to oxidative stress. One bout of exercise at moderate intensity and duration did not induce HSP responses despite the increased protein oxidation and tissue inflammation in equine muscle.

Objective Activated mast cells in atherosclerotic lesions degranulate and release bioactive compounds capable of regulating atherogenesis. Here we examined the ability of activated human primary mast cells to regulate the expression of the major scavenger receptors in cultured human primary monocyte-derived macrophages (HMDMs). Results Components released by immunologically activated human primary mast cells induced a transient expression of lectin-like oxidized LDL receptor (LOX-1) mRNA in HMDMs, while the expression of two other scavenger receptors, MSR1 and CD36, remained unaffected. The LOX-1-inducing secretory components were identified as histamine, tumor necrosis factor alpha (TNF-α), and transforming growth factor beta (TGF-β1), which exhibited a synergistic effect on LOX-1 mRNA expression. Histamine induced a transient expression of LOX-1 protein. Mast cell -induced increase in LOX-1 expression was not associated with increased uptake of oxidized LDL by the macrophages. Conclusions Mast cell-derived histamine, TNF-α, and TGF-β1 act in concert to induce a transient increase in LOX-1 expression in human primary monocyte-derived macrophages. The LOX-1-inducing activity potentially endows mast cells a hitherto unrecognized role in the regulation of innate immune reactions in atherogenesis.

Kokkola, Tarja/0000-0002-3303-3912; Kaarniranta, Kai/0000-0003-2600-8679 WOS: 000350812500004 PubMed: 25462062 Hear shock proteins (HSPs), originally identified as heat-inducible gene products, are a family of highly conserved proteins that respond to a wide variety of stress including oxidative stress. Although both acute and chronic oxidative stress have been well demonstrated to induce HSP responses, Rae evidence is available whether increased HSP levels provide enhanced protection against oxidative stress under elevated yet sublethal temperatures. We studied relationships between oxidative stress and HSPs in a physiological model by using Garra rufa (doctor fish), a fish species naturally acclimatized to different thermal conditions. We compared fish naturally living in a hot spring with relatively high water temperature (34.4 +/- 0.6 degrees C) to those living in normal river water temperature (25.4 +/- 4.7 degrees C), and found that levels of all the studied HSPs (HSP70, RSPB, HSP90, HSC70 and GRP75) were higher in fish living in elevated water temperature compared with normal river water temperature. In contrast, indicators of oxidative stress, including protein carbonyls and lipid hydroperoxides, were decreased in fish living in the elevated temperature, indicating that HSP levels are inversely associated with oxidative stress. The present results provide evidence that physiologically increased HSP levels provide protection against oxidative stress and enhance cyLoprotection. (C) 2014 The Authors. Published by Elsevier B.V. Finnish Ministry of Education and Culture, Juho Vainio; Yrjo Jahnsson Foundations, Helsinki, Finland, High technology Foundation of eastern Finland This work has been supported by the grants from the Finnish Ministry of Education and Culture, Juho Vainio and Yrjo Jahnsson Foundations, Helsinki, Finland, High technology Foundation of eastern Finland. University of Hacettepe. Unit of Scientific Researches (0202601006) and COST actions CM1001 "Chemistry of non-enzymatic protein modification - modulation of protein structure and function" and TD1304 "Zinc-Net". We thank Taina Vihavainen and Taija Hukkanen for the skillful technical assistance

The current understanding of the pathogenesis of type 2 diabetes is mainly based on a large number of cross-sectional and prospective studies (1– 4) in which nondiabetic individuals were followed for several years to determine incident cases of diabetes without repeating assessment of other metabolic characteristics except for assay of plasma glucose during follow-up. In fact, only the studies in the Pima Indian population (5–7) have examined the changes in anthropometric characteristics, insulin secretion, and insulin action during the progression from normal glucose tolerance (NGT) to impaired glucose tolerance (IGT) to diabetes. Thus far, no study has directly addressed the effects of changes in the components of the metabolic syndrome on the transition to diabetes in other populations. The purpose of this study was to investigate the changes in the features of the metabolic syndrome during the transition from one state of glucose homeostasis to another in a cohort of Chinese people and to understand the relative contributions of these changes to the development of impaired glucose regulation (IGR) or type 2 diabetes. RESEARCH DESIGN AND METHODS — A total of 627 subjects without diabetes at baseline who participated in the National Diabetes Survey in 1994 and the follow-up survey in 1999 in the Beijing area (8) were included in this study. All of the subjects had measurements of BMI, blood pressure, fasting serum total cholesterol, triglecerides, insulin, plasma fasting glucose, and 2-h postload glucose (2-hPG) at baseline and follow-up separately. Past medical history, family history of diabetes, history of pharmacological treatment, smoking status, education, and occupation were determined with a standardized questionnaire. Obesity was defined as BMI 25.0 kg/m 2 , according to the recommendations for Asians (9). Hy

A high-fat diet disturbs the composition and function of the gut microbiota and generates local gut-associated and also systemic responses. Intestinal mast cells, for their part, secrete mediators which play a role in the orchestration of physiological and immunological functions of the intestine. Probiotic bacteria, again, help to maintain the homeostasis of the gut microbiota by protecting the gut epithelium and regulating the local immune system. In the present study, we explored the effects of two probiotic bacteria, Lactobacillus rhamnosus GG (GG) and Propionibacterium freudenreichii spp. shermanii JS (PJS), on high fat-fed ApoE*3Leiden mice by estimating the mast cell numbers and the immunoreactivity of TNF-α and IL-10 in the intestine, as well as plasma levels of several markers of inflammation and parameters of lipid metabolism. We found that mice that received GG and PJS exhibited significantly lower numbers of intestinal mast cells compared with control mice. PJS lowered intestinal immunoreactivity of TNF-α, while GG increased intestinal IL-10. PJS was also observed to lower the plasma levels of markers of inflammation including vascular cell adhesion molecule 1, and also the amount of gonadal adipose tissue. GG lowered alanine aminotransferase, a marker of hepatocellular activation. Collectively, these data demonstrate that probiotic GG and PJS tend to down-regulate both intestinal and systemic pro-inflammatory changes induced by a high-fat diet in this humanised mouse model.

Background Human tissue mast cells (MCs) have the potential to express several neutral granule proteases, which are the most precise markers of the phenotypic heterogeneity of MCs. However, the full extent of such heterogeneity is limited by the fact that MCs containing either tryptase only or tryptase and chymase have long been considered to be the sole MC phenotypes. Moreover, the potential developmental relationship between human MCs of different protease phenotypes has remained a matter of dispute. Objective We attempted to define how human MCs with different protease phenotypes relate to their circulating progenitors. Methods MCs were generated from human peripheral blood–derived CD34 + progenitors in the presence of kit ligand (KITLG) and the cytokines IL-3, IL-9, and IL-6 under serum-free conditions, or by KITLG alone in the presence or absence of serum. The expression of chymase, carboxypeptidase A3, cathepsin G, granzyme B, and the tryptases derived from the TPSAB1 , TPSB2 , TPSD1 , and TPSG1/PRSS31 genes were determined weekly at the mRNA and/or protein levels. Results Incubation of CD34 + progenitors in the presence of KITLG and the cytokines IL-3, IL-9, and IL-6 promoted the development of a single population of MCs with a uniform tryptase + , chymase + , CPA3 + , cathepsin G + , and granzyme B + phenotype. Interestingly, the presence of KITLG alone was sufficient to induce the expression of all the above proteases. Conclusion All circulating human MC progenitors have the potential to differentiate into MCs expressing the complete panel of neutral granule proteases, implying that human MCs originate from a common MC-committed progenitor.

Most, if not all, disk galaxies have a thin (classical) disk and a thick disk. In most models thick disks are thought to be a necessary consequence of the disk formation and/or evolution of the galaxy. We present the results of a study of the thick disk properties in a sample of carefully selected edge-on galaxies with types ranging from T=3 to T=8. We fitted one-dimensional luminosity profiles with physically motivated functions - the solutions of two stellar and one gaseous isothermal coupled disks in equilibrium - which are likely to yield more accurate results than other functions used in previous studies. The images used for the fits come from the Spitzer Survey of Stellar Structure in Galaxies (S4G). We found that thick disks are on average more massive than previously reported, mostly due to the selected fitting function. Typically, the thin and the thick disk have similar masses. We also found that thick disks do not flare significantly within the observed range in galactocentric radii and that the ratio of thick to thin disk scaleheights is higher for galaxies of earlier types. Our results tend to favor an in situ origin for most of the stars in the thick disk. In addition the thick disk may contain a significant amount of stars coming from satellites accreted after the initial build-up of the galaxy and an extra fraction of stars coming from the secular heating of the thin disk by its own overdensities. Assigning thick disk light to the thin disk component may lead to an underestimate of the overall stellar mass in galaxies, because of different mass to light ratios in the two disk components. On the basis of our new results, we estimate that disk stellar masses are between 10% and 50% higher than previously thought and we suggest that thick disks are a reservoir of "local missing baryons"., Accepted for publication in ApJ

Serum amyloid A (SAA) is an acute-phase protein, the serum levels of which can increase up to 1000-fold during inflammation. SAA has a pathogenic role in amyloid A-type amyloidosis, and increased serum levels of SAA correlate with the risk for cardiovascular diseases. IL-1β is a key proinflammatory cytokine, and its secretion is strictly controlled by the inflammasomes. We studied the role of SAA in the regulation of IL-1β production and activation of the inflammasome cascade in human and mouse macrophages, as well as in THP-1 cells. SAA could provide a signal for the induction of pro–IL-1β expression and for inflammasome activation, resulting in secretion of mature IL-1β. Blocking TLR2 and TLR4 attenuated SAA-induced expression of IL1B, whereas inhibition of caspase-1 and the ATP receptor P2X7 abrogated the release of mature IL-1β. NLRP3 inflammasome consists of the NLRP3 receptor and the adaptor protein apoptosis-associated speck-like protein containing CARD (a caspase-recruitment domain) (ASC). SAA-mediated IL-1β secretion was markedly reduced in ASC−/− macrophages, and silencing NLRP3 decreased IL-1β secretion, confirming NLRP3 as the SAA-responsive inflammasome. Inflammasome activation was dependent on cathepsin B activity, but it was not associated with lysosomal destabilization. SAA also induced secretion of cathepsin B and ASC. In conclusion, SAA can induce the expression of pro–IL-1β and activation of the NLRP3 inflammasome via P2X7 receptor and a cathepsin B-sensitive pathway. Thus, during systemic inflammation, SAA may promote the production of IL-1β in tissues. Furthermore, the SAA-induced secretion of active cathepsin B may lead to extracellular processing of SAA and, thus, potentially to the development of amyloid A amyloidosis.

Background Chronic inflammation of the arterial wall is a key element in the pathogenesis of atherosclerosis, yet the factors that trigger and sustain the inflammation remain elusive. Inflammasomes are cytoplasmic caspase-1-activating protein complexes that promote maturation and secretion of the proinflammatory cytokines interleukin(IL)-1beta and IL-18. The most intensively studied inflammasome, NLRP3 inflammasome, is activated by diverse substances, including crystalline and particulate materials. As cholesterol crystals are abundant in atherosclerotic lesions, and IL-1beta has been linked to atherogenesis, we explored the possibility that cholesterol crystals promote inflammation by activating the inflammasome pathway. Principal findings Here we show that human macrophages avidly phagocytose cholesterol crystals and store the ingested cholesterol as cholesteryl esters. Importantly, cholesterol crystals induced dose-dependent secretion of mature IL-1beta from human monocytes and macrophages. The cholesterol crystal-induced secretion of IL-1beta was caspase-1-dependent, suggesting the involvement of an inflammasome-mediated pathway. Silencing of the NLRP3 receptor, the crucial component in NLRP3 inflammasome, completely abolished crystal-induced IL-1beta secretion, thus identifying NLRP3 inflammasome as the cholesterol crystal-responsive element in macrophages. The crystals were shown to induce leakage of the lysosomal protease cathepsin B into the cytoplasm and inhibition of this enzyme reduced cholesterol crystal-induced IL-1beta secretion, suggesting that NLRP3 inflammasome activation occurred via lysosomal destabilization. Conclusions The cholesterol crystal-induced inflammasome activation in macrophages may represent an important link between cholesterol metabolism and inflammation in atherosclerotic lesions.

Background Upon IgE-mediated activation, mast cells (MC) exocytose their cytoplasmic secretory granules and release a variety of bioactive substances that trigger inflammatory responses. Polyamines mediate numerous cellular and physiological functions. We report here that MCs express antizyme inhibitor 2 (AZIN2), an activator of polyamine biosynthesis, previously reported to be exclusively expressed in the brain and testis. We have investigated the intracellular localization of AZIN2 both in resting and activated MCs. In addition, we have examined the functional role of polyamines, downstream effectors of AZIN2, as potential regulators of MC activity. Methodology/Principal Findings Immunostainings show that AZIN2 is expressed in primary and neoplastic human and rodent MCs. We demonstrate that AZIN2 localizes in the Vamp-8 positive, serotonin-containing subset of MC granules, but not in tryptase-containing granules, as revealed by double immunofluorescence stainings. Furthermore, activation of MCs induces rapid upregulation of AZIN2 expression and its redistribution, suggesting a role for AZIN2 in secretory granule exocytosis. We also demonstrate that release of serotonin from activated MCs is polyamine-dependent whereas release of histamine and β-hexosaminidase is not, indicating a granule subtype-specific function for polyamines. Conclusions/Significance The study reports for the first time the expression of AZIN2 outside the brain and testis, and demonstrates the intracellular localization of endogenous AZIN2 in MCs. The granule subtype-specific expression and its induction after MC activation suggest a role for AZIN2 as a local, in situ regulator of polyamine biosynthesis in association with serotonin-containing granules of MCs. Furthermore, our data indicates a novel function for polyamines as selective regulators of serotonin release from MCs.

The heat shock proteins (HSPs), originally identified as heat-inducible gene products, are a highly conserved family of proteins that respond to a wide variety of stress. Although HSPs are among the most abundant intracellular proteins, they are expressed at low levels under normal physiological conditions, and show marked induction in response to various stressors. HSPs function primarily as molecular chaperones, facilitating the folding of other cellular proteins, preventing protein aggregation, or targeting improperly folded proteins to specific pathways for degradation. By modulating inflammation, wound debris clearance, cell proliferation, migration and collagen synthesis, HSPs are essential for normal wound healing of the skin. In this review, our goal is to discuss the role and clinical implications of HSP with respect to skin wound healing and diabetes. The numerous defects in the function of HSPs associated with diabetes could contribute to the commonly observed complications and delayed wound healing in diabetics. Several physical, pharmacological and genetic approaches may be considered to address HSP-directed therapies both in the laboratory and in the clinics.

Regular exercise plays an important preventive and therapeutic role in oxidative stress-associated diseases such as diabetes and its complications. Thiol antioxidants including thioredoxin (TRX) and glutathione (GSH) have a crucial role in controlling cellular redox status. In this study, the effects of 8 wk of exercise training on brain TRX and GSH systems, and antioxidant enzymes were tested in rats with or without streptozotocin-induced diabetes. We found that in untrained animals, the levels of TRX-1 (TRX1) protein and activity, and thioredoxin-interacting protein (TXNip) were similar in diabetic and nondiabetic animals. Exercise training, however, increased TRX1 protein in nondiabetic animals without affecting TXNip levels, whereas diabetes inhibited the effect of training on TRX1 protein and also increased TXNip mRNA. In addition, the proportion of oxidized glutathione (GSSG) to total GSH was increased in animals with diabetes, indicating altered redox status and possibly increased oxidative stress. Glutathione peroxidase-1 (GPX1) levels were not affected by diabetes or exercise training, although diabetes increased total GPX activity. Both diabetes and exercise training decreased glutathione reductase (GRD) activity and cytosolic superoxide dismutase (Cu,Zn-SOD) levels. Nevertheless, diabetes or training had no effect on Cu,Zn-SOD mRNA, Mn-SOD protein, total SOD activity, or catalase mRNA, protein, or activity. Our findings suggest that exercise training increases TRX1 levels in brain without a concomitant rise in TXNip, and that experimental diabetes is associated with an incomplete TRX response to training. Increased oxidative stress may be both a cause and a consequence of perturbed antioxidant defenses in the diabetic brain.