BBA - Molecular Basis of Disease (v.1812, #9)

Roles of platelets and macrophages in the protective effects of lipopolysaccharide against concanavalin A-induced murine hepatitis by Zhiqian Yu; Hirotada Otsuka; Kouji Yamaguchi; Toshinobu Kuroishi; Takashi Sasano; Shunji Sugawara; Masanori Nakamura; Yasuo Endo (1069-1079).
Platelets are reportedly causal in hepatitis. We previously showed that in mice, lipopolysaccharide (LPS) induces a reversible and macrophage-dependent hepatic platelet accumulation (HPA), including translocation of platelets into Disse spaces and their entry into hepatocytes. Concanavalin A (ConA), which induces hepatitis in mice via both T cells and macrophages, also induces HPA. Here, we examined the relationship between HPA and ConA-hepatitis. ConA-hepatitis and HPA were evaluated by serum transaminases, hepatic 5-hydroxytryptamine, and/or electron microscopy. Unlike LPS-induced HPA, ConA-induced HPA was only moderately dependent on phagocytic macrophages. Against expectations, platelet-depletion significantly exacerbated ConA-hepatitis, and anti-P-selectin antibody and P-selectin receptor blockade reduced both ConA-induced HPA and hepatitis. Prior induction of HPA by pretreatment with low-dose LPS powerfully reduced ConA-hepatitis. Such protection by LPS-pretreatment was not effective in mice depleted of phagocytic macrophages. In platelet-depleted mice, LPS-pretreatment severely exacerbated ConA-hepatitis. In mice depleted of both macrophages and platelets, neither ConA nor LPS-pretreatment + ConA induced hepatitis. In mice deficient in IL-1α and IL-1β (but not in TNFα), ConA-induced hepatitis was mild, and a protective effect of LPS was not detected. These results suggest that (i) there are causal and protective types of HPA, (ii) the causal type involves hepatic aggregation of platelets, which may be induced by platelet stimulants leaked from injured hepatocytes, (iii) the protective type is inducible by administration of prior low-dose LPS in a manner dependent on phagocytic (or F4/80-positive) macrophages, and (iv) IL-1 is involved in both the causal and protective types.► There are causal and protective types of hepatic platelet accumulation (HPA). ► Lipopolysaccharide (LPS) induces a protective HPA depending on macrophages. ► HPA by pretreatment with LPS reduces hepatitis induced by concanavalin A (ConA). ► In platelet-depleted mice, LPS-pretreatment severely exacerbates ConA-hepatitis. ► IL-1 is involved in both the causal and protective types of HPA.
Keywords: Macrophage; Platelet; Concanavalin A; Lipopolysaccharide; Liver; Kupffer cell;

Various growth factors and cytokines are implicated in endothelial dysfunction and blood–retinal barrier (BRB) breakdown in early diabetic retinopathy (DR). However, cellular and molecular mechanisms that may underlie the pathology of DR are not fully understood yet. We therefore examined the effect of insulin-like growth factor (IGF)-1 on ECM/adhesion molecule expression, cell cycle regulation and monolayer permeability in an endothelial cell line (TR-iBRB2). We investigate whether the action of IGF-1 (1) involves glycogen synthase kinase 3beta (GSK-3β) and cAMP responsive transcription factor (CREB) and (2) alters ECM/adhesion molecule gene expression. Treatment of TR-iBRB2 cell with IGF-1 (100 ng/ml for 0–24 h) increases phosphorylation of (i) Akt Thr308, and its substrates including GSK-3β at Ser9, which inactivates its kinase function, and (ii) CREB at Ser133 (activation). These phosphorylations correlate positively with enhanced expression of CREB targets such as ECM protein fibronectin and cell cycle progression factor cyclin D1. However, stable transfection of a mutant GSK3β(S9A) or a dominant negative K-CREB in TR-iBRB2 prevents IGF-1-induced fibronectin and cyclin D1 expression. Furthermore, IGF-1 reduces the level of intercellular adherence molecule VE-cadherin and increases monolayer permeability in TR-iBRB2 cells when measured by FITC-dextran leakage. The effect of IGF-1 on VE-cadherin and membrane permeability is absent in TR-iBRB2 cells expressing the GSK-3β(S9A). Similarly, K-CREB reverses IGF-1 down-regulation of VE-cadherin and up-regulation of fibronectin. These results indicate that GSK-3β/CREB axis alters ECM/adhesion molecule expression and cell cycle progression in retinal endothelial cells, and may potentially contribute to endothelial dysfunction and BRB leakage in DR.► GSK-3β/CREB axis mediates aberrant ECM/cell adhesion molecule gene expression. ► GSK-3β/CREB axis regulates cell viability and cyclin D1 expression. ► GSK-3β mediates IGF-1-induced endothelial monolayer permeability. ► Stable expression of a constitutively active GSK3β(Ser9Ala) or a dominant negative K-CREB blocks IGF-1's effects on aberrant gene expression and monolayer permeability. ► Modulation of GSK-3β/CREB pathway may prevent microvascular dysfunction in diabetic retinopathy.
Keywords: IGF-1; GSK-3β/CREB axis; ECM/adhesion molecule expression; Cell cycle regulation; Monolayer permeability;

Unique and analogous functions of aquaporin 0 for fiber cell architecture and ocular lens transparency by S. Sindhu Kumari; Subramaniam Eswaramoorthy; Richard T. Mathias; Kulandaiappan Varadaraj (1089-1097).
Aquaporin (AQP) 1 and AQP0 water channels are expressed in lens epithelial and fiber cells, respectively, facilitating fluid circulation for nourishing the avascular lens to maintain transparency. Even though AQP0 water permeability is 40-fold less than AQP1, AQP0 is selectively expressed in the fibers. Delimited AQP0 fiber expression is attributed to a unique structural role as an adhesion protein. To validate this notion, we determined if wild type (WT) lens ultrastructure and fiber cell adhesion are different in AQP0−/−, and TgAQP1+/+/AQP0−/− mice that transgenically express AQP1 (TgAQP1) in fiber cells without AQP0 (AQP0−/−). In WT, lenses were transparent with ‘Y’ sutures. Fibers contained opposite end curvature, lateral interdigitations, hexagonal shape, and were arranged as concentric growth shells. AQP0−/− lenses were cataractous, lacked ‘Y’ sutures, ordered packing and well-defined lateral interdigitations. TgAQP1+/+/AQP0−/− lenses showed improvement in transparency and lateral interdigitations in the outer cortex while inner cortex and nuclear fibers were severely disintegrated. Transmission electron micrographs exhibited tightly packed fiber cells in WT whereas AQP0−/− and TgAQP1+/+/AQP0−/− lenses had wide extracellular spaces. Fibers were easily separable by teasing in AQP0−/− and TgAQP1+/+/AQP0−/− lenses compared to WT. Our data suggest that the increased water permeability through AQP1 does not compensate for loss of AQP0 expression in TgAQP1+/+/AQP0−/− mice. Fiber cell AQP0 expression is required to maintain their organization, which is a requisite for lens transparency. AQP0 appears necessary for cell-to-cell adhesion and thereby to minimize light scattering since in the AQP0−/− and TgAQP1+/+/AQP0−/− lenses, fiber cell disorganization was evident.Display Omitted► First report to demonstrate that lens AQP0 protein has analogous and unique functions. ► AQP0 and AQP1 have water permeability as an analogous function. ► AQP0 may have cell-to-cell adhesion as a unique function in the fiber cells. ► Loss of AQP0 leads to failure of suture formation and disorganization of the fiber cells, eventually causing lens cataract. ► AQP0 mediated cell-to-cell adhesion facilitates cell-to-cell communication.
Keywords: AQP0; Lens cataract; Cell-to-cell adhesion; Analogous function; Unique function; Lenticular architecture and transparency;

Although the importance of adipose tissue (AT) glucose transport in regulating whole-body insulin sensitivity is becoming increasingly evident and insulin resistance (IR) has been widely recognized, the underlying mechanisms of IR are still not well understood. The purpose of the present study was to determine the early pathological changes in glucose transport by characterizing the alterations in glucose transporters (GLUT) in multiple visceral and subcutaneous adipose depots in a large animal model of naturally occurring compensated IR. AT biopsies were collected from horses, which were classified as insulin-sensitive (IS) or compensated IR based on the results of an insulin-modified frequently sampled intravenous glucose tolerance test. Protein expression of GLUT4 (major isoform) and GLUT12 (one of the most recently discovered isoforms) were measured by Western blotting in multiple AT depots, as well as AS160 (a potential key player in GLUT trafficking pathway). Using a biotinylated bis-mannose photolabeled technique, active cell surface GLUT content was quantified. Omental AT had the highest total GLUT content compared to other sites during the IS state. IR was associated with a significantly reduced total GLUT4 content in omental AT, without a change in content in other visceral or subcutaneous adipose sites. In addition, active cell surface GLUT-4, but not -12, was significantly lower in AT of IR compared to IS horses, without change in AS160 phosphorylation between groups. Our data suggest that GLUT4, but not GLUT12, is a pathogenic factor in AT during naturally occurring compensated IR, despite normal AS160 activation.►Study of glucose transporters 4 & 12 in a large animal model of naturally occurring IR. ►Using an innovative methodology, we successfully quantified active cell surface GLUT-4 & -12 in AT. ►Sampling multiple AT depots elucidated key selective impairments in the glucose transport pathway. ►Novel mechanistic insights into AT glucose metabolism and early pathogenesis of IR.
Keywords: GLUT trafficking; Biotinylated photo-affinity label; Omental; GLUT12;

Cigarette smoke induces the release of CXCL-8 from human bronchial epithelial cells via TLRs and induction of the inflammasome by E. Mortaz; P.A.J. Henricks; A.D. Kraneveld; M.E. Givi; J. Garssen; G. Folkerts (1104-1110).
COPD is a chronic airway disease associated with inflammation and cigarette smoking. Airway epithelial cells are the first cells exposed to cigarette smoke (CS) and can release CXCL-8 and IL-1β. These cytokines are involved in acute and chronic inflammatory processes in COPD. The aim of this study was to investigate whether toll-like receptors (TLRs) located in/on epithelial cells were involved in cigarette smoke-induced cytokine production. Here we demonstrate that CS induces the release of CXCL-8 and IL-1β from human bronchial epithelial cells (HBE-14o). CS-induced CXCL-8 production was inhibited by an antibody against TLR4 and by inhibitory ODN suggesting the involvement of TLR4 and TLR9. In addition, exposure of HBE-14o cells to TLR4 or TLR9 ligands resulted in the release of CXCL-8 and IL1β. TLR4 and also TLR9 were present on the cell surface and the expression of both receptors decreased after CS exposure. The molecular mechanism of the CS-induced CXCL-8 production by the epithelial cells was further investigated. It was found that P2X7 receptors and reactive oxygen species were involved. Interestingly, the inflammasome activator monosodium urate crystals (MSU) induced the release of CXCL-8 and IL-1β and the caspase-1 inhibitor Z-VADDCB suppressed the CS-induced release of CXCL-8. In addition, CS, CpGODN, lipopolysaccharide and MSU all increased the expression of caspase-1 and IL-1β. In conclusion, our results demonstrate that CS releases CXCL-8 from HBE-14o cells via TLR4 and TLR9 and inflammasome activation. Therefore, inflammasome signaling in airway epithelial cells may play an important role in pathogenesis of diseases like COPD.► Cigarette smoking is a risk factor for COPD. ► Airway epithelial cells are the first cells exposed to cigarette smoke. ► The involvement of TLRs and inflammasome signaling was investigated. ► Cigarette smoke induces CXCL-8 and IL-1 release from the epithelial cells. ► This release is mediated via TLRs and inflammasome signaling.
Keywords: COPD; Inflammation; IL-1β; Caspase-1;

The dysfunction of hepatic transcriptional factors in mice with Huntington's Disease by Ming-Chang Chiang; Yijuang Chern; Chiun-Gung Juo (1111-1120).
Huntington's Disease (HD) is an autosomal dominant neurodegenerative disease caused by a CAG trinucleotide expansion in the Huntingtin (Htt) gene. The resultant mutant Htt protein (mHtt) forms aggregates in the brain and several peripheral tissues (e.g., the liver), and causes devastating widespread pathology. Since aggregates of mHtt have been found in the liver, defects in liver function might contribute to peripheral abnormalities in HD mice. We previously reported that two crucial transcription factors PPARγ (peroxisome proliferator-activated receptor-γ) and C/EBPα (CCAAT/enhancer-binding protein α) are potential therapeutic targets of HD. We herein demonstrate that the transcript level of PPARγ was markedly downregulated in the livers of a transgenic mouse model of HD (R6/2). Treatment of R6/2 mice with an agonist of PPARγ (thiazolidinedione, TZD) normalized the reduced PPARγ transcript. By reducing Htt aggregates and thereby ameliorating the recruitment of PPARγ into Htt aggregates, TZD treatment also elevated the availability of the PPARγ level and subsequently normalized the expression of its downstream genes [including PGC-1α (PPAR coactivator-1α) and several mitochondrial genes] and C/EBPα in the liver. The aforementioned protective effects appeared to be exerted by a direct activation of the PPARγ agonist (rosiglitazone) because rosiglitazone reduced mHtt aggregates and rescued energy deficiency in a hepatoma cell line (HepG2). These findings show that the impairment of PPARγ contributes to the liver dysfunction observed in HD. Treatment with PPARγ agents (TZD and rosiglitazone) enhanced the function of PPARγ, and might lead to therapeutic benefits.► PPARγ agonist (thiazolidinedione, TZD) elevated reduced hepatic transcriptional factors in the livers of R6/2 mice. ► TZD treatment reduced mHtt aggregates and ameliorated the recruitment of hepatic transcriptional factors. ► PPARγ agonist reduced mHtt aggregates, enhanced PPRE promoter activity, and ATP level in mHtt expressing HepG2 cells.
Keywords: Huntington's Disease; Thiazolidinedione; PPARγ; PGC-1α; C/EBPα; Mitochondrial function;

Niemann-Pick Type C1 deficiency in microglia does not cause neuron death in vitro by Kyle B. Peake; Robert B. Campenot; Dennis E. Vance; Jean E. Vance (1121-1129).
Niemann-Pick Type C (NPC) disease is an autosomal recessive disorder that results in accumulation of cholesterol and other lipids in late endosomes/lysosomes and leads to progressive neurodegeneration and premature death. The mechanism by which lipid accumulation causes neurodegeneration remains unclear. Inappropriate activation of microglia, the resident immune cells of the central nervous system, has been implicated in several neurodegenerative disorders including NPC disease. Immunohistochemical analysis demonstrates that NPC1 deficiency in mouse brains alters microglial morphology and increases the number of microglia. In primary cultures of microglia from Npc1 −/− mice cholesterol is sequestered intracellularly, as occurs in other NPC-deficient cells. Activated microglia secrete potentially neurotoxic molecules such as tumor necrosis factor-α (TNFα). However, NPC1 deficiency in isolated microglia did not increase TNFα mRNA or TNFα secretion in vitro. In addition, qPCR analysis shows that expression of pro-inflammatory and oxidative stress genes is the same in Npc1 +/+ and Npc1 −/− microglia, whereas the mRNA encoding the anti-inflammatory cytokine, interleukin-10 in Npc1 −/− microglia is ~ 60% lower than in Npc1 +/+ microglia. The survival of cultured neurons was not impaired by NPC1 deficiency, nor was death of Npc1 −/− and Npc1 +/+ neurons in microglia–neuron co-cultures increased by NPC1 deficiency in microglia. However, a high concentration of Npc1 −/− microglia appeared to promote neuron survival. Thus, although microglia exhibit an active morphology in NPC1-deficient brains, lack of NPC1 in microglia does not promote neuron death in vitro in microglia–neuron co-cultures, supporting the view that microglial NPC1 deficiency is not the primary cause of neuron death in NPC disease.► Microglia are activated and proliferate in NPC1-deficient mouse brains. ► Tumor necrosis factor mRNA and secretion are not increased in Npc1 −/− microglia. ► Survival of cultured neurons was not compromised by NPC1 deficiency. ► Npc1 −/− microglia do not increase neuron death in microglia–neuron co-cultures.
Keywords: Niemann-Pick Type C; Microglia; Tumor necrosis factor; Interleukin-10; Neurodegeneration;

Atorvastatin exerts its anti-atherosclerotic effects by targeting the receptor for advanced glycation end products by Bo Feng; Lei Xu; Hua Wang; Xinfeng Yan; Junli Xue; Fengjing Liu; Ji-Fan Hu (1130-1137).
Recent studies demonstrated the beneficial role of atorvastatin in reducing the risk of cardiovascular morbidity and mortality in patients with diabetes mellitus and/or metabolic syndrome. To investigate the mechanisms underlying the anti-atheroscleroic action of atorvastatin, we examined the expression of the receptor for advanced glycation end products (RAGE) and its downstream target gene, monocyte chemoattractant protein-1 (MCP-1) using real-time PCR. In in vitro studies, exposure to high glucose or AGE induced oxidative stress and activation of the AGE/RAGE system in human umbilical vein endothelial cells. Treatment of the cells with atorvastatin significantly released the oxidative stress by restoring the levels of glutathione and inhibited the RAGE upregulation. In diabetic Goto Kakisaki (GK) rats fed with a high-fat diet for 12 weeks, RAGE and MCP-1 were upregulated in the aortas, and there was a significant correlation between RAGE and MCP-1 mRNA abundance (r  = 0.482, P  = 0.031). Treatment with atorvastatin (20 mg/kg qd) significantly downregulated the expression of RAGE and MCP-1. These data thus demonstrate a novel “pleiotropic” activity of atorvastatin in reducing the risk of cardiovascular diseases by targeting RAGE expression.► High glucose or AGE activates the AGE/RAGE system in HUVECs. ► Atorvastatin releases oxidative stress by restoring the level of glutathione. ► Suppression of the activated RAGE and MCP-1 by atorvastatin in HUVECs. ► Atorvastatin downregulats RAGE and MCP-1 in diabetic Goto Kakisaki rats. ► A novel “pleiotropic” activity of atorvastatin by targeting RAGE expression.
Keywords: Atorvastatin; Receptor for advanced glycation end products; RAGE; AGE; Diabetes; Atherosclerosis;

Physical and catalytic properties of a peroxidase derived from cytochrome c by Johannes Everse; Chyong-Jy J. Liu; Penelope W. Coates (1138-1145).
Except for its redox properties, cytochrome c is an inert protein. However, dissociation of the bond between methionine-80 and the heme iron converts the cytochrome into a peroxidase. Dissociation is accomplished by subjecting the cytochrome to various conditions, including proteolysis and hydrogen peroxide (H2O2)-mediated oxidation. In affected cells of various neurological diseases, including Parkinson's disease, cytochrome c is released from the mitochondrial membrane and enters the cytosol. In the cytosol cytochrome c is exposed to cellular proteases and to H2O2 produced by dysfunctional mitochondria and activated microglial cells. These could promote the formation of the peroxidase form of cytochrome c. In this study we investigated the catalytic and cytolytic properties of the peroxidase form of cytochrome c. These properties are qualitatively similar to those of other heme-containing peroxidases. Dopamine as well as sulfhydryl group-containing metabolites, including reduced glutathione and coenzyme A, are readily oxidized in the presence of H2O2. This peroxidase also has cytolytic properties similar to myeloperoxidase, lactoperoxidase, and horseradish peroxidase. Cytolysis is inhibited by various reducing agents, including dopamine. Our data show that the peroxidase form of cytochrome c has catalytic and cytolytic properties that could account for at least some of the damage that leads to neuronal death in the parkinsonian brain.► Cytochrome c in which the methionine-iron bond is broken by proteolysis acts as a potent peroxidase. ► Proteolysis of released cytochrome c is likely to occur in the cytosol of Parkinsonian neurons. ► This peroxidase is able to oxidize metabolites including glutathione and coenzyme A. ► This peroxidase is also able to oxidize sulfhydryl groups in proteins leading to their inactivation. ► This peroxidase could account for most of the aberrant reactions occurring in Parkinsonian neurons.
Keywords: Cytochrome c; Peroxidase; Parkinson's disease; Neurodegeneration; Cytolysis; Cellular metabolites;

Candidate tumour suppressor Fau regulates apoptosis in human cells: An essential role for Bcl-G by Mark R. Pickard; Mirna Mourtada-Maarabouni; Gwyn T. Williams (1146-1153).
FAU, which encodes a ubiquitin-like protein (termed FUBI) with ribosomal protein S30 as a carboxy-terminal extension, has recently been identified as a pro-apoptotic regulatory gene. This activity may be mediated by Bcl-G (a pro-apoptotic member of the Bcl-2 family) which can be covalently modified by FUBI. FAU gene expression has been shown to be down-regulated in human breast, prostate and ovarian tumours, and this down-regulation is strongly associated with poor prognosis in breast cancer. We demonstrate here that ectopic FAU expression increases basal apoptosis in human T-cell lines and 293T/17 cells, whereas it has only a transient stimulatory effect on ultraviolet-C (UVC)-induced apoptosis. Conversely, siRNA-mediated silencing of FAU gene expression has no effect on basal apoptosis, but attenuates UV-induced apoptosis. Importantly, prior knockdown of Bcl-G expression ablates the stimulation of basal apoptosis by FAU, consistent with an essential downstream role for Bcl-G, itself a candidate tumour suppressor, in mediating the apoptosis regulatory role of FAU. In 293T/17 cells, Bcl-G knockdown also attenuates UV-induced apoptosis, so that Bcl-G may constitute a common factor in the pathways by which both FAU and UV-irradiation induce apoptosis. UV irradiation increases Bcl-G mRNA levels, providing an explanation for the transient nature of the effect of ectopic FAU expression on UV-induced apoptosis. Since failure of apoptosis is fundamental to the development of many cancers, the pro-apoptotic activity of the Fau/Bcl-G pathway offers an attractive explanation for the putative tumour suppressor role of FAU.► Bcl-G acts downstream of FAU in apoptosis control. ► FAU pro-apoptotic action may explain its tumour suppressor activity. ► Knock-down of FAU gene expression attenuates UV-induced apoptosis. ► Enhanced FAU gene expression induces apoptosis. ► Prior knockdown of Bcl-G expression ablates pro-apoptotic activity of FAU.
Keywords: Apoptosis; Bcl-2 family; BCL-L14; FUBI; Oncogenesis; Tumour suppressor;

Aquaporin 4 (AQP4), the most abundant water channel protein in the brain, is involved in brain edema induced by ischemic insults. To evaluate whether the neuroprotective effects of estrogen are associated with AQP4 expression and edema formation, changes in AQP levels and ischemic edema were examined in the brains of male and female mice subjected to transient middle cerebral artery occlusion. Infarct volume and edema formation were markedly less in females than in males. AQP4 expression in the ischemic cortex of females was relatively well preserved, whereas it was significantly decreased in males. These effects disappeared in ovariectomized females but were reversed by estrogen replacement. Furthermore, AQP4 expression was decreased with increased brain edema in females treated with ICI182,780, an estrogen receptor antagonist. These findings suggest that the estrogen effect on the reduction of ischemic brain edema is associated with the preserved level of AQP4 that is partly mediated by estrogen receptors.► Vasogenic edema of the ischemic brain was reduced in female mice compared males. ► AQP4 levels in the ischemic cortex were maintained in female mice. ► Down-regulated AQP4 levels by ovariectomy were reversed by estrogen replacement. ► An ERs antagonist reduced AQP4 levels with an increase of vasogenic edema. ► Estrogen receptors may regulate AQP4 levels to prevent the ischemic brain edema.
Keywords: Aquaporin 4; Brain edema; Estrogen; Estrogen receptor; Ischemic stroke; Sex difference;

Fat-1 transgenic mice with elevated omega-3 fatty acids are protected from allergic airway responses by Sueleyman Bilal; Oliver Haworth; Lijun Wu; Karsten H. Weylandt; Bruce D. Levy; Jing X. Kang (1164-1169).
Omega-3 polyunsaturated fatty acids (n-3 PUFA) have been implicated in the alleviation of asthma. Recent studies have demonstrated that the n-3 PUFA derived lipid mediators, protectin D1 and resolvin E1, may act as potent resolution agonists in airway inflammation. The effects of the n-3 PUFA tissue status itself on asthma pathogenesis remains to be further investigated. In this study allergic airway inflammation induced by allergen sensitization and aerosol challenge in Fat-1 and wild-type mice was investigated. Fat-1 transgenic mice displayed increased endogenous lung n-3 PUFA. When allergen-sensitized and aerosol-challenged, these animals had decreased airway inflammation with decreased leukocyte accumulation in bronchoalveolar lavage fluid and lung parenchyma. The Fat-1 mice had a shift to the right in the dose–response relationship for methacholine induced bronchoconstriction with a significant increase in the log ED200. The Fat-1 mice had lower BALF concentrations of the pro-inflammatory cytokines IL-1α, IL-2, IL-5, IL-9, IL-13, G-CSF, KC and RANTES. Furthermore, increased lung tissue amounts of the counter-regulatory mediators protectin D1 and resolvin E1 were found in Fat-1 mice after bronchoprovocative challenge. These results therefore demonstrate a direct protective role for lung n-3 PUFA in allergic airway responses and an increased generation of protectin D1 and resolvin E1 in this context.► Fat-1 mice had increased endogenous lung omega-3 polyunsaturated fatty acids. ► These mice showed decreased allergen-induced airway inflammation. ► This was associated with alleviated methacholine-induced bronchoconstriction. ► Lavage fluid concentrations of IL-1α, IL-2, IL-5, IL-9, IL-13, G-CSF, KC and RANTES were decreased. ► Anti-inflammatory protectin D1 and resolvin E1 were increased in fat-1 lungs.
Keywords: Omega-3; Resolvins; Protectins; Inflammation; Asthma; Fat-1 mice;

Hyaluronan reduces inflammation in experimental arthritis by modulating TLR-2 and TLR-4 cartilage expression by Giuseppe M. Campo; Angela Avenoso; Giancarlo Nastasi; Antonio Micali; Vera Prestipino; Mario Vaccaro; Angela D'Ascola; Alberto Calatroni; Salvatore Campo (1170-1181).
Previous studies have reported that low molecular mass HA and highly polymerized HA respectively elicited pro- and anti-inflammatory responses by modulating the toll-like receptor 4 (TLR-4) and the TLR-2. The activation of TLR-4 and TLR-2 mediated by collagen-induced arthritis (CIA) induces the myeloid differentiation primary response protein (MyD88) and the tumor necrosis factor receptor-associated factor 6 (TRAF6), and ends with the liberation of NF-kB which, in turn, stimulates pro-inflammatory cytokine production. The aim of this study was to investigate the influence of high molecular weight HA at different concentrations on TLR-4 and TLR-2 modulation in CIA in mice. Arthritis was induced in mice via intradermal injection of an emulsion containing bovine type II collagen in complete Freund's adjuvant. Mice were treated with HA intraperitoneally daily for 30 days. CIA increased TLR-4, TLR-2, MyD88 and TRAF6 mRNA expression and the related protein in the cartilage of arthritic joints. High levels of both mRNA and related protein were also detected for tumor necrosis factor alpha (TNF-α), interleukin 1-beta (IL-1-β), interleukin-17 (IL-17), matrix metalloprotease-13 (MMP-13) and inducible nitric oxide synthase (iNOS) in the joint of arthritic mice. HA treatment significantly limited CIA incidence and decreased all the parameters up-regulated by CIA. The improvement of biochemical parameters was also supported by histological analysis, plasma and synovial fluid HA levels. These results suggest that the TLR-4 and TLR-2 play an important role in the arthritis mechanism and the interaction/block of HA at high molecular mass may reduce inflammation and cartilage injury.► We evaluated the effect of high polymerized HA on TLR4 and TLR2 activity in CIA mice. ► Arthritic mice were treated with HA intraperitoneally for 30 days. ► CIA increased TLR-4, TLR-2, MyD88 and TRAF6 mRNA expression in the joint cartilage. ► High levels of TNF-α, IL-1-β, IL-17, MMP-13 and iNOS were also detected in joints. ► HA treatment significantly limited CIA and decreased all the parameters up-regulated.
Keywords: NF-kB; Toll-like receptor; Cytokine; TNF-alpha; Arthritis; Hyaluronan;

The purpose of our study was to assess mitochondrial biogenesis and distribution in murine primary neurons. Using 5-bromo-2-deoxyuridine (BrdU) incorporation and primary neurons, we studied the mitochondrial biogenesis and mitochondrial distribution in hippocampal neurons from amyloid beta precursor protein (AβPP) transgenic mice and wild-type (WT) neurons treated with oxidative stressors, rotenone and H2O2. We found that after 20 h of labeling, BrdU incorporation was specific to porin-positive mitochondria. The proportion of mitochondrial area labeled with BrdU was 40.3 ± 6.3% at 20 h. The number of mitochondria with newly synthesized DNA was higher in AβPP neuronal cell bodies than in the cell bodies of WT neurons (AβPP, 45.23 ± 2.67 BrdU-positive/cell body; WT, 32.92 ± 2.49 BrdU-positive/cell body; p = 0.005). In neurites, the number of BrdU-positive mitochondria decreased in AβPP cultures compared to WT neurons (AβPP, 0.105 ± 0.008 BrdU-positive/μm neurite; WT, 0.220 ± 0.036 BrdU-positive/μm neurite; p = 0.010). Further, BrdU in the cell body increased when neurons were treated with low doses of H2O2 (49.6 ± 2.7 BrdU-positive/cell body, p = 0.0002 compared to untreated cells), while the neurites showed decreased BrdU staining (0.122 ± 0.010 BrdU-positive/μm neurite, p = 0.005 compared to the untreated). BrdU labeling was increased in the cell body under rotenone treatment. Additionally, under rotenone treatment, the content of BrdU labeling decreased in neurites. These findings suggest that Aβ and mitochondrial toxins enhance mitochondrial fragmentation in the cell body, and may cause impaired axonal transport of mitochondria leading to synaptic degeneration.► Using BrdU incorporation, mitochondrial biogenesis was assessed in primary neurons from amyloid beta precursor protein transgenic mice. ► A-beta and mitochondrial toxins enhanced mitochondrial fragmentation, and defective mitochondria were not able to transport to terminals. ► Mitochondria-targeted antioxidant, SS31 serve to promote intact and healthy mitochondria. ► We did not observe mitochondrial division in distal regions of neurons.
Keywords: Primary neuron; Alzheimer's disease; Mitochondrial DNA synthesis; 5-Bromo-2-deoxyuridine; Cell body mitochondria; Mitochondria-targeted antioxidant;

The melanocortin-4 receptor (MC4R) is a G protein-coupled receptor critically involved in regulating energy balance. MC4R activation results in decreased food intake and increased energy expenditure. Genetic and pharmacological studies demonstrated that the MC4R regulation of energy balance is conserved from fish to mammals. In humans, more than 150 naturally occurring mutations in the MC4R gene have been identified. Functional study of mutant MC4Rs is an important component in proving the causal link between MC4R mutation and obesity as well as the basis of personalized medicine. In this article, we studied 20 MC4R mutations that were either not characterized or not fully characterized. We showed that 11 mutants had decreased or absent cell surface expression. D126Y was defective in ligand binding. Three mutants were constitutively active but had decreased cell surface expression. Eleven mutants had decreased basal signaling, with two mutants defective only in this parameter, suggesting that impaired basal signaling might also be a cause of obesity. Five mutants had normal functions. In summary, we provided detailed functional data for further studies on identifying therapeutic approaches for personalized medicine to treat patients harboring these mutations.► We performed detailed functional studies on 20 naturally occurring MC4R mutations. ► More than half of the mutants had decreased cell surface expression. ► We identified a mutant defective in ligand binding per se. ► Constitutively active mutations had decreased cell surface expression. ► Some mutants have normal functions.
Keywords: Melanocortin-4 receptor; Naturally occurring mutation; Intracellular retention; Binding; Signaling;