Myeloid Cell Expression of LACC1 Is Required for Bacterial Clearance and Control of Intestinal Inflammation.

BACKGROUND & AIMS: Loss-of-function variants in the laccase domain containing 1 (LACC1) gene are associated with immune-mediated diseases, including inflammatory bowel disease. It is not clear how LACC1 balances defenses against intestinal bacteria vs intestinal inflammation or what cells are responsible for this balance in humans or mice. METHODS: Lacc1-/- mice and mice with myeloid-specific disruption of Lacc1 (Lacc1Δmye) were given oral Salmonella Typhimurium or dextran sodium sulfate. CD45RBhiCD4+T cells were transferred to Lacc1-/-Rag2-/- mice to induce colitis. Organs were collected and analyzed by histology and protein expression. Bone marrow-derived macrophages and dendritic cells, lamina propria macrophages, and mesenteric lymph node dendritic cells were examined. We performed assays to measure intestinal permeability, cell subsets, bacterial uptake and clearance, reactive oxygen species, nitrite production, autophagy, signaling, messenger RNA, and cytokine levels. RESULTS: Lacc1-/- mice developed more severe T-cell transfer colitis than wild-type mice and had an increased burden of bacteria in intestinal lymphoid organs, which expressed lower levels of T helper (Th) 1 and Th17 cytokines and higher levels of Th2 cytokines. Intestinal lymphoid organs from mice with deletion of LACC1 had an increased burden of bacteria after oral administration of S Typhimurium and after administration of dextran sodium sulfate compared with wild-type mice. In macrophages, expression of LACC1 was required for toll-like receptor-induced uptake of bacteria, which required PDK1, and of mitogen-activated protein kinase (MAPK)- and nuclear factor κB-dependent induction of reactive oxygen species, reactive nitrogen species, and autophagy. Expression of LACC1 by dendritic cells was required for increasing expression of Th1 and Th17 cytokines and reducing expression of Th2 cytokines upon coculture with CD4+ T cells. Mice with LACC1-deficient myeloid cells had an increased burden of bacteria and altered T-cell cytokines in intestinal lymphoid organs, similar to Lacc1-/- mice. Complementation of cytokines produced by myeloid cells to cocultures of LACC1-deficient myeloid cells and wild-type CD4+ T cells restored T-cell cytokine regulation. When S Typhimurium-infected Lacc1Δmye mice were injected with these myeloid cell-derived cytokines, intestinal tissues increased production of Th1 and Th17 cytokines, and bacteria were reduced. CONCLUSIONS: Disruption of Lacc1 in mice increases the burden of bacteria in intestinal lymphoid organs and intestinal inflammation after induction of chronic colitis. LACC1 expression by myeloid cells in mice is required to clear bacteria and to regulate adaptive T-cell responses against microbes.

Strategy for mass production of lytic Staphylococcus aureus bacteriophage pSa-3: contribution of multiplicity of infection and response surface methodology.

BACKGROUND: Antibiotic-resistant bacteria have emerged as a serious problem; bacteriophages have, therefore, been proposed as a therapeutic alternative to antibiotics. Several authorities, such as pharmacopeia, FDA, have confirmed their safety, and some bacteriophages are commercially available worldwide. The demand for bacteriophages is expected to increase exponentially in the future; hence, there is an urgent need to mass-produce bacteriophages economically. Unlike the replication of non-lytic bacteriophages, lytic bacteriophages are replicated by lysing host bacteria, which leads to the termination of phage production; hence, strategies that can prolong the lysis of host bacteria in bacteria-bacteriophage co-cultures, are required. RESULTS: In the current study, we manipulated the inoculum concentrations of Staphylococcus aureus and phage pSa-3 (multiplicity of infection, MOI), and their energy sources to delay the bactericidal effect while optimizing phage production. We examined an increasing range of bacterial inoculum concentration (2 × 108 to 2 × 109 CFU/mL) to decrease the lag phase, in combination with a decreasing range of phage inoculum (from MOI 0.01 to 0.00000001) to delay the lysis of the host. Bacterial concentration of 2 × 108 CFU/mL and phage MOI of 0.0001 showed the maximum final phage production rate (1.68 × 1010 plaque forming unit (PFU)/mL). With this combination of phage-bacteria inoculum, we selected glycerol, glycine, and calcium as carbon, nitrogen, and divalent ion sources, respectively, for phage production. After optimization using response surface methodology, the final concentration of the lytic Staphylococcus phage was 8.63 × 1010 ± 9.71 × 109 PFU/mL (5.13-fold increase). CONCLUSIONS: Therefore, Staphylococcus phage pSa-3 production can be maximized by increasing the bacterial inoculum and reducing the seeding phage MOI, and this combinatorial strategy could decrease the phage production time. Further, we suggest that response surface methodology has the potential for optimizing the mass production of lytic bacteriophages.

Genomic characterization of bacteriophage pEt-SU, a novel phiKZ-related virus infecting Edwardsiella tarda.

A bacteriophage infecting Edwardsiella tarda (named pEt-SU) was isolated from freshwater collected in Chung-ju, South Korea. The whole genome of pEt-SU was 276,734 bp in length, representing the first giant phage infecting Edwardsiella reported to date. A total of 284 putative open reading frames were predicted and annotated. Morphology and genome analyses verified that pEt-SU may be distantly related to the phiKZ-like phages, a well-known giant myovirus. The findings in this study provide new insights into the phages infecting E. tarda ads well as fundamental data for the study of giant phages.

Resolvin D1 activates the sphingosine-1-phosphate signaling pathway in murine livers with ischemia/reperfusion injury.

Resolvins (Rvs) are endogenous lipid mediators that promote resolution of inflammation and return to homeostasis. We previously reported that RvD1 both facilitates M2 macrophage polarization of Kupffer cells (KCs) and efferocytosis and modulates thioredoxin 2-mediated mitochondrial quality control in liver ischemia/reperfusion (IR) injury. However, the specific cellular or molecular targets of RvD1 remain poorly understood. Sphingosine-1-phosphate (S1P), the natural sphingolipid ligand for a family of G protein-coupled receptors (S1P1-S1P5), regulates lymphocyte circulation and various immune responses. Here we investigated the role of RvD1 in IR-induced hepatocellular damage with a focus on S1P signaling. Male C57BL/6 mice were subjected to partial hepatic ischemia for 60 min, followed by reperfusion. Mice were pretreated with RvD1 (15 µg/kg, i.p.) 1 h prior to ischemia and immediately before reperfusion. To deplete KCs, liposome clodronate was administered (100 µL/mice, i.v.) 24 h prior to ischemia. Mice were pretreated with VPC23019 (100 µg/kg, i.p.), an antagonist for S1P1/S1P3 10 min prior to initial RvD1 treatment. Exogenous RvD1 attenuated IR-induced hepatocellular damage as evidenced by serum HMGB1 release. RvD1 attenuated the decrease in hepatic S1P concentration induced by IR. KC depletion by liposome clodronate did not alter the effect of RvD1 on sphingosine kinases (SKs) and S1P receptors, suggesting independency of KCs. Moreover, in purified hepatocytes of mice exposed to IR, mRNA expression of SK1, SK2, S1P1, and S1P3 decreased significantly, and this was attenuated by RvD1. Finally, VPC23019 pretreatment abolished the hepatoprotective effects of RvD1 in serum HMGB1 release. Our findings suggest that RvD1 protects the liver against IR injury by activating S1P signaling.

Enhanced bath immersion vaccination through microbubble treatment in the cyprinid loach.

Immunization by bath immersion is likely the simplest method of fish vaccination. Although the route of immunogenicity has not been fully identified, immersion vaccination is clearly a useful labor-saving technique. In this study, microbubble (MB) treatment was assessed for its ability to improve the efficacy of bath immersion vaccination in the cyprinid loach. MBs are commonly defined as minute particles of gas with a diameter of less than 100 µm, which generated free radicals. Here, the efficacy of MB treatment for vaccination enhancement in the cyprinid loach was assessed in direct challenge experiments using the virulent Aeromonas hydrophila JUNAH strain; assessments comprised agglutination titer assay and non-specific parameter analysis. Agglutination titers were high in loaches that were immunized via injection with inactivated cells (FKC group); however, non-specific immune activation parameters (e.g., lysozyme, superoxide dismutase, and phagocytic activity) were more increased in loaches that were immunized via bath immersion with MB treatment. Moreover, MB-treated loaches showed comparable survival rates, relative to loaches immunized via injection with formalin inactivated cells. Thus, higher levels of non-specific immune parameters suggest increased efficacy of this vaccine approach. Improving the effectiveness of bath immersion vaccine will increase its affordability and ease of application in aquaculture.

Immunostimulation of Cyprinus carpio using phage lysate of Aeromonas hydrophila.

Over the last 50 years, various approaches have been established for the development of antigens for immunostimulation. We used phage lysate (PL), composed of inactivated antigens by the lytic bacteriophage pAh 6-c for Aeromonas hydrophila JUNAH strain to develop a vaccine for the prevention of A. hydrophila infection in Cyprinus carpio (common carp). We also assessed the poly D,L lactide-co-glycolic acid (PLGA) microparticles encapsulation method to increase the efficiency of the vaccine. Six groups of vaccines involving encapsulated by PLGA, formalin killed cells, or phage lysate at low or high concentration were prepared for intraperitoneal injection in C. carpio. Blood specimens and head kidney samples were collected at various time points for bacterial agglutination assay and to assess relative expression of immune-related genes interleukin-1 beta (IL-1ß), tumor necrosis factor alpha (TNF-α), lysozyme C, and serum amyloid A (SAA). The vaccine groups using high dose phage lysate antigen showed significantly higher agglutination titers than all other groups at 4- and 6-weeks post vaccination (wpv), with the titer of the PLGA encapsulated vaccine group being highest from 10 wpv to the end of the experiment. The survival rate of fish immunized with the phage lysate vaccines were higher than that of fish immunized with the formailin killed cells vaccine in the challenge experiment conducted 6 wpv. Additionally, the PLGA-encapsulated high dose phage lysate antigen vaccinated groups showed the best protective efficacy in the challenge experiment 12 wpv. Vaccines using the phage lysate antigen also showed higher IL-1ß and lysozyme C gene expression at 7 days post vaccination (dpv) and 2 wpv, and higher TNF-α gene expression was seen at 7 dpv. Higher SAA gene expression was seen in these groups at 1 dpv. These results suggest that phage lysate antigen has the potential to induce robust immune responses than formalin killed cells-based vaccines, and could be more effective as a novel inactivated antigen in preventing A. hydrophila infection in C. carpio.

Resolvin D1 protects the liver from ischemia/reperfusion injury by enhancing M2 macrophage polarization and efferocytosis.

Resolution of inflammation is an active process involving a novel category of lipid factors known as specialized pro-resolving lipid mediators, which includes Resolvin D1 (RvD1). While accumulating evidence suggests that RvD1 counteracts proinflammatory signaling and promotes resolution, the specific cellular targets and mechanisms of action of RvD1 remain largely unknown. In the present study, we investigated the role and molecular mechanisms of RvD1 in ischemia/reperfusion (IR)-induced sterile liver inflammation. Male C57BL/6 mice underwent 70% hepatic ischemia for 60min, followed by reperfusion. RvD1 (5, 10, and 15µg/kg, i.p.) was administered to the mice 1h before ischemia and then immediately prior to reperfusion. RvD1 attenuated IR-induced hepatocellular damage and the proinflammatory response. In purified Kupffer cells (KCs) from mice exposed to IR, the levels of M1 marker genes (Nos2a and Cd40) increased, while those of M2 marker genes (Arg1, Cd206, and Mst1r) decreased, demonstrating a proinflammatory shift. RvD1 markedly attenuated these changes. Depletion of KCs by liposome clodronate abrogated the effects of RvD1 on proinflammatory mediators and macrophage polarization. In addition, RvD1 attenuated increases in myeloperoxidase activity and Cxcl1 and Cxcl2 mRNA expression. RvD1 markedly augmented the efferocytic activity of KCs, as indicated by increases in F4/80(+)Gr-1(+) cells in the liver. However, antagonist pretreatment or gene silencing of the RvD1 receptor, ALX/FPR2, abrogated the anti-inflammatory and pro-resolving actions of RvD1. These data indicate that RvD1 ameliorates IR-induced liver injury, and this protection is associated with enhancement of M2 polarization and efferocytosis via ALX/FPR2 activation.

The role of heme oxygenase-1 in drug metabolizing dysfunction in the alcoholic fatty liver exposed to ischemic injury.

This study was designed to investigate the role of heme oxygenase-1 (HO-1) in hepatic drug metabolizing dysfunction after ischemia/reperfusion (IR) in alcoholic fatty liver (AFL). Rats were fed a Lieber-DeCarli diet for five weeks to allow for development of AFL and were then subjected to 90min of hepatic ischemia and 5h of reperfusion. Rats were pretreated with hemin (HO-1 inducer) or ZnPP (HO-1 inhibitor) for 16h and 3h before hepatic ischemia. After hepatic IR, ethanol diet (ED)-fed rats had higher serum aminotransferase activities and more severe hepatic necrosis compared to the control diet (CD)-fed rats. These changes were attenuated by hemin and exacerbated by ZnPP. The activity and gene expression of HO-1 and its transcription factor (Nrf2) level increased significantly after 5h of reperfusion in CD-fed rats but not in ED-fed rats. After reperfusion, cytochrome P450 (CYP) 1A1, 1A2, and 2B1 activities were reduced to levels lower than those observed in sham group, whereas CYP2E1 activity increased. The decrease in CYP2B1 activity and the increase in CYP2E1 activity were augmented after hepatic IR in ED-fed animals. These changes were significantly attenuated by hemin but aggravated by ZnPP. Finally, CHOP expression and PERK phosphorylation, microsomal lipid peroxidation, and levels of proinflammatory mediators increased in ED-fed rats compared to CD-fed rats after reperfusion. These increases were attenuated by hemin. Our results suggest that AFL exacerbates hepatic drug metabolizing dysfunction during hepatic IR via endoplasmic reticulum stress and lipid peroxidation and this is associated with impaired HO-1 induction.

Enhanced nitric oxide-mediated autophagy contributes to the hepatoprotective effects of ischemic preconditioning during ischemia and reperfusion.

Ischemic preconditioning (IPC) protects against liver ischemia/reperfusion (I/R) injury. Autophagy is an essential cytoprotective system that is rapidly activated by multiple stressors. Nitric oxide (NO) acts as an inducer of IPC. We examined the impact of autophagy in liver IPC and its regulation by NO. Male C57BL/6 mice were subjected to 60 min of hepatic ischemia followed by 6 h of reperfusion. IPC was achieved for 10 min of ischemia followed by 10 min of reperfusion prior to sustained ischemia. N(ω)-Nitro-l-arginine methyl ester (L-NAME, 15 mg/kg, i.v., all NOS inhibitor) and aminoguanidine (AG, 10 mg/kg, i.v., iNOS inhibitor) were injected 10 min before IPC. SB203580 (10 mg/kg, i.p., p38 inhibitor) was injected 30 min before IPC. I/R increased serum alanine aminotransferase activity. IPC attenuated this increase, which was abolished by L-NAME, but not AG. Microtubule-associated protein-1 light chain 3-II levels increased and p62 protein levels decreased after I/R; these changes were augmented by IPC and abolished by L-NAME. I/R increased liver protein expression of autophagy-related protein (Atg)12-Atg5 complex and lysosome-associated membrane protein-2. IPC augmented the expression of these proteins, which were abolished by L-NAME, but not AG. IPC also augmented the level of phosphorylated p38 MAPK induced by I/R and this phosphorylation was abolished by L-NAME. Our findings suggest that IPC-mediated NO protects against I/R-induced liver injury by enhancing autophagic flux.

Melatonin enhances mitophagy and mitochondrial biogenesis in rats with carbon tetrachloride-induced liver fibrosis.

Liver fibrosis leads to liver cirrhosis and failure, and no effective treatment is currently available. Growing evidence supports a link between mitochondrial dysfunction and liver fibrogenesis and mitochondrial quality control-based therapy has emerged as a new therapeutic target. We investigated the protective mechanisms of melatonin against mitochondrial dysfunction-involved liver fibrosis, focusing on mitophagy and mitochondrial biogenesis. Rats were treated with carbon tetrachloride (CCl4) dissolved in olive oil (0.5 mL/kg, twice a week, i.p.) for 8 wk. Melatonin was administered orally at 2.5, 5, and 10 mg/kg once a day. Chronic CCl4 exposure induced collagen deposition, hepatocellular damage, and oxidative stress, and melatonin attenuated these increases. Increases in mRNA and protein expression levels of transforming growth factor ß1 and α-smooth muscle actin in response to CCl4 were attenuated by melatonin. Melatonin attenuated hallmarks of mitochondrial dysfunction, such as mitochondrial swelling and glutamate dehydrogenase release. Chronic CCl4 exposure impaired mitophagy and mitochondrial biogenesis, and melatonin attenuated this impairment, as indicated by increases in mitochondrial DNA and in protein levels of PTEN-induced putative kinase 1 (PINK1); Parkin; peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC-1α); nuclear respiratory factor 1 (NRF1); and transcription factor A, mitochondrial (TFAM). CCl4-mediated decreases in mitochondrial fission- and fusion-related proteins, such as dynamin-related protein 1 (DRP1) and mitofusin 2, were also attenuated by melatonin. Moreover, melatonin induced AMP-activated protein kinase (AMPK) phosphorylation. These results suggest that melatonin protects against liver fibrosis via upregulation of mitophagy and mitochondrial biogenesis, and may be useful as an anti-fibrotic treatment.

Role of Kupffer cells in ischemic injury in alcoholic fatty liver.

BACKGROUND: This study was designed to evaluate the role of Kupffer cells (KCs) in hepatic drug metabolizing dysfunction after hepatic ischemia-reperfusion (IR) in alcoholic fatty liver. MATERIALS AND METHODS: Rats were fed the Lieber-DeCarli diet for 5 wk to develop alcoholic fatty liver, then were subjected to 90 min of hepatic ischemia and 5 h of reperfusion. For ablation of KCs, rats were pretreated with gadolinium chloride (GdCl3) 48 and 24 h before the IR procedure. RESULTS: After the IR procedure, ethanol diet (ED)-fed rats had higher serum aminotransferase activity compared with the control diet-fed rats. These changes were attenuated by GdCl3. The ED-fed rats exhibited increased hepatic microsomal total cytochrome P450 (CYP) content and nicotinamide adenine dinucleotide phosphate-CYP reductase and CYP1A1, 1A2, 2B1, and 2E1 isozyme activity. After hepatic IR, these increases were reduced to lower levels than observed in the sham group, except CYP2E1 activity. Increases in CYP2E1 activity and its expression were augmented after hepatic IR in ED-fed animals, but were attenuated by GdCl3. Finally, toll-like receptor 4 and myeloid differentiation primary response gene 88 protein expression, nuclear translocation of nuclear factor-κB and activator protein 1, and levels of proinflammatory mediators were further increased in ED-fed animals compared with control diet-fed animals after IR. These increases were attenuated by GdCl3. CONCLUSIONS: We suggest that KCs contribute to hepatic drug metabolizing dysfunction during hepatic IR in alcoholic fatty liver via the toll-like receptors 4-mediated inflammatory response.

Melatonin inhibits mTOR-dependent autophagy during liver ischemia/reperfusion.

BACKGROUND: Autophagy is a self-digestion system responsible for maintaining cellular homeostasis and interacts with reactive oxygen species produced during ischemia/reperfusion (I/R). Melatonin (MLT) is a potent and endogenous anti-oxidant that has beneficial effects in liver I/R injury. In this study, we examined the cytoprotective mechanisms of MLT in liver I/R, focusing on autophagic flux and associated signaling pathways. METHODS: Male C57BL/6 mice were subjected to 70% liver ischemia for 60 min followed by reperfusion. MLT (10 mg/kg, i.p.) was injected 15 min prior to ischemia and again immediately before reperfusion. Rapamycin (Rapa, 1 mg/kg, i.p.), which induces autophagy, was injected 1.5 h before ischemia. RESULTS: Liver I/R increased autophagic flux as indicated by the accumulation of LC3-II and degradation of sequestosome1/p62. This increase was attenuated by MLT. Likewise, electron microscopic analysis showed that autophagic vacuoles were increased in livers of mice exposed to I/R, which was attenuated by MLT. I/R decreased phosphorylation of mammalian target of rapamycin (mTOR) and 4E-BP1 and 70S6K, downstream molecules of the mTOR pathway, but increased expression of calpain 1 and calpain 2. MLT attenuated the decrease in mTOR, 4E-BP1 and 70S6K phosphorylation. Pretreatment of Rapa reversed the effect of MLT on autophagic flux as well as mTOR pathway. CONCLUSION: Our findings suggest that MLT downregulates autophagy via activation of mTOR signaling, which may in turn contribute to its protective effects in liver I/R injury.

Impaired expression of caveolin-1 contributes to hepatic ischemia and reperfusion injury.

Caveolae are membrane structures enriched in glycosphingolipids and cholesterol, and caveolin-1 (Cav-1) has been recognized to be pivotal in ischemic tolerance. Sphingosine-1-phosphate (S1P), one of the sphingolipid metabolites, is well known for its anti-apoptotic properties, counteracting ischemia and reperfusion (IR) injury. Here, we investigated the cytoprotective mechanism of Cav-1 against IR injury. Male C57BL/6 mice underwent 70% hepatic ischemia for 60 min, followed by reperfusion. Mice were pretreated with methyl-beta-cyclodextrin (MßCD, 10, 25 and 50mg/kg, i.p.), a caveolae disruptor, or saline 48 and 24h before ischemia. Serum and liver tissues were collected at the end of ischemia, at 0, 1, 4 and 24h of reperfusion. Decreases in the expression of Cav-1 protein and in the number of caveolae of the liver ultrastructure were observed during IR, which were augmented by pretreatment with MßCD. MßCD also augmented the IR-induced increases in serum alanine aminotransferase and tumor necrosis factor-α levels. IR decreased the levels of sphingosine kinase 2 (SK2) and S1P receptor 2 (S1P2) mRNA expressions, while MßCD also augmented these decreases. Moreover, IR resulted in increases of mitochondrial cytochrome c release, caspase 3, 8 activities and Bax/Bcl-xL ratio, and MßCD augmented all of these apoptotic parameters. MßCD also increased p38 MAPK and JNK phosphorylation, but did not affect ERK and PI3K/Akt. Our findings demonstrate that downregulation of Cav-1 mediates IR-induced liver damage by inhibiting SK2/S1P2 signaling and enhancing the apoptotic pathway.

Downregulation of FUSE-binding protein and c-myc by tRNA synthetase cofactor p38 is required for lung cell differentiation.

p38 is associated with a macromolecular tRNA synthetase complex. It has an essential role as a scaffold for the complex, and genetic disruption of p38 in mice causes neonatal lethality. Here we investigated the molecular mechanisms underlying lethality of p38-mutant mice. p38-deficient mice showed defects in lung differentiation and respiratory distress syndrome. p38 was found to interact with FUSE-binding protein (FBP), a transcriptional activator of c-myc. Binding of p38 stimulated ubiquitination and degradation of FBP, leading to downregulation of c-myc, which is required for differentiation of functional alveolar type II cells. Transforming growth factor-beta (TGF-beta) induced p38 expression and promoted its translocation to nuclei for the regulation of FBP and c-myc. Thus, this work identified a new activity of p38 as a mediator of TGF-beta signaling and its functional importance in the control of c-myc during lung differentiation.

Melatonin inhibits type 1 interferon signaling of toll-like receptor 4 via heme oxygenase-1 induction in hepatic ischemia/reperfusion.

The cytoprotective mechanisms of melatonin in hepatic ischemia/reperfusion (I/R) injury associated with heme oxygenase-1 (HO-1) induction and type 1 interferon (IFN) signaling pathway downstream of toll-like receptor 4 (TLR4) were investigated. Rats were subjected to 60min of ischemia followed by 5-hr reperfusion. Melatonin (10mg/kg) or vehicle (5% ethanol in saline) was administered intraperitoneally 15min prior to ischemia and immediately before reperfusion. Rats were pretreated with zinc protoporphyrin (ZnPP, 10mg/kg, i.p.), a HO-1 inhibitor, at 16 and 3hr prior to ischemia. Melatonin attenuated the I/R-induced increase in serum alanine aminotransferase activity, and ZnPP reversed this attenuation. Melatonin augmented the levels of HO activity and HO-1 protein and mRNA expression, and this enhancement was reversed by ZnPP. Melatonin enhanced the level of NF-E2-related factor-2 (Nrf2) nuclear translocation, and ZnPP reversed this increase. Overexpression of TLR4 and its adaptor proteins, toll-receptor-associated activator of interferon (TRIF), and myeloid differentiation factor 88 (MyD88), induced by I/R, was attenuated by melatonin; ZnPP reversed the effect of melatonin on TLR4 and TRIF expression. Melatonin suppressed the increased interaction between TLR4/TRIF and TLR4/MyD88, which was reversed by ZnPP. Melatonin attenuated the increased levels of JAK2 and STAT1 activation as well as IFN-ß, and ZnPP reversed these inhibitory effects of melatonin. Melatonin inhibited the level of chemokine (C-X-C motif) ligand 10 (CXCL-10), and ZnPP reversed this inhibition. Our findings suggest that melatonin protects the liver against I/R injury by HO-1 overexpression, which suppresses the type 1 IFN signaling pathway downstream of TLR4.

Effect of GCSB-5, a Herbal Formulation, on Monosodium Iodoacetate-Induced Osteoarthritis in Rats.

Therapeutic effects of GCSB-5 on osteoarthritis were measured by the amount of glycosaminoglycan in rabbit articular cartilage explants in vitro, in experimental osteoarthritis induced by intra-articular injection of monoiodoacetate in rats in vivo. GCSB-5 was orally administered for 28 days. In vitro, GCSB-5 inhibited proteoglycan degradation. GCSB-5 significantly suppressed the histological changes in monoiodoacetate-induced osteoarthritis. Matrix metalloproteinase (MMP) activity, as well as, the levels of serum tumor necrosis factor-α, cyclooxygenase-2, inducible nitric oxide synthase protein, and mRNA expressions were attenuated by GCSB-5, whereas the level of interleukin-10 was potentiated. By GCSB-5, the level of nuclear factor-κB p65 protein expression was significantly attenuated but, on the other hand, the level of inhibitor of κB-α protein expression was increased. These results indicate that GCSB-5 is a potential therapeutic agent for the protection of articular cartilage against progression of osteoarthritis through inhibition of MMPs activity, inflammatory mediators, and NF-κB activation.

Melatonin protects liver against ischemia and reperfusion injury through inhibition of toll-like receptor signaling pathway.

This study investigated the immunomodulating effect of melatonin on toll-like receptor (TLR)-stimulated signal transduction. Rats were subjected to 60 min of ischemia followed by 1 or 5 hr of reperfusion. Melatonin (10 mg/kg) or the vehicle was administered intraperitoneally 15 min prior to ischemia and immediately before reperfusion. Melatonin treatment significantly reduced the level of serum alanine aminotransferase activity. Increased levels of TLR3 and TLR4 protein expression induced by ischemia/reperfusion (I/R) were attenuated by melatonin. Serum level of high-mobility group box 1 (HMGB1), a potent alarmin of the TLR system, increased significantly in the I/R group, and melatonin inhibited this release. Melatonin suppressed the increase in myeloid differentiation factor 88 (MyD88) protein expression, extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) phosphorylation and nuclear translocation of nuclear factor κB (NF-κB) and phosphorylated c-Jun, a component of activator protein 1. The increased level of toll-receptor-associated activator of interferon (TRIF) expression, phosphorylation of interferon (IFN) regulatory factor 3 (IRF3) and serum IFN-ß was attenuated by melatonin. Melatonin attenuated the levels of tumor necrosis factor alpha (TNF-α), interleukin (IL)-6 and inducible nitric oxide synthase (iNOS) protein and mRNA expression, while the level of heme oxygenase-1 (HO-1) was augmented. Our results suggest that melatonin ameliorates I/R-induced liver damage by modulation of TLR-mediated inflammatory responses.

Reduced brain gray matter concentration in patients with obstructive sleep apnea syndrome.

STUDY OBJECTIVES: To investigate differences in brain gray matter concentrations or volumes in patients with obstructive sleep apnea syndrome (OSA) and healthy volunteers. DESIGNS: Optimized voxel-based morphometry, an automated processing technique for MRI, was used to characterize structural differences in gray matter in newly diagnosed male patients. SETTING: University hospital. PATIENTS AND PARTICIPANTS: The study consisted of 36 male OSA and 31 non-apneic male healthy volunteers matched for age (mean age, 44.8 years). INTERVENTIONS: Using the t-test, gray matter differences were identified. The statistical significance level was set to a false discovery rate P < 0.05 with an extent threshold of k(E) > 200 voxels. MEASUREMENTS AND RESULTS: The mean apnea-hypopnea index (AHI) of patients was 52.5/h. On visual inspection of MRI, no structural abnormalities were observed. Compared to healthy volunteers, the gray matter concentrations of OSA patients were significantly decreased in the left gyrus rectus, bilateral superior frontal gyri, left precentral gyrus, bilateral frontomarginal gyri, bilateral anterior cingulate gyri, right insular gyrus, bilateral caudate nuclei, bilateral thalami, bilateral amygdalo-hippocampi, bilateral inferior temporal gyri, and bilateral quadrangular and biventer lobules in the cerebellum (false discovery rate P < 0.05). Gray matter volume was not different between OSA patients and healthy volunteers. CONCLUSIONS: The brain gray matter deficits may suggest that memory impairment, affective and cardiovascular disturbances, executive dysfunctions, and dysregulation of autonomic and respiratory control frequently found in OSA patients might be related to morphological differences in the brain gray matter areas.

Microstructural and Very High Cycle Fatigue (VHCF) Behavior of Ti6Al4V-A Comparative Study.

In this study, an investigation is carried out to evaluate and compare the material and physical properties of Grade 5 Titanium alloy (Ti6Al4V G5) samples of three different impeller manufacturers. The study aims to identify the efficient impeller core material from different Ti6Al4V G5 manufacturers. Ultrasonic fatigue test for Ti6Al4V samples of 100 horsepower (hp) centrifugal compressor impeller parts is performed before and after heat treatment. The effect of microstructure on Very High Cycle Fatigue (VHCF) behavior of Ti6Al4V is also analyzed and discussed in detail. Optical Microscopy (OM) and Scanning Electron Microscopy (SEM) observation are carried out to investigate the microstructure of different Ti6Al4V material samples. The dynamic elastic properties are measured by the Impulse Excitation Technique (IET) at room temperature. The fracture behavior of the tensile specimens is analyzed by SEM. Post-heat-treatment analysis of Ti6Al4V is also carried out and presented which affects the grain size of the material sample and thus considerable effect in the mechanical properties. Chemical composition investigation of Ti6Al4V using SEM and Energy Dispersive X-ray Spectroscopy (EDS) also included in this study.

Interleukin-32 monoclonal antibodies for immunohistochemistry, Western blotting, and ELISA.

The members of the IL-1 family play important roles in the development and pathogenesis of autoimmune and inflammatory diseases. Especially, IL-1 and IL-18 belong to the IL-1 family because they share structural similarity and require caspase-1 for processing. Currently, IL-18 has been studied for its biological effects in the broad spectrum of Th1- or Th2- related autoimmune diseases. IL-18 also uses a similar signaling pathway as that of IL-1 family members. Taken together these results, IL-18-inducible genes might also contribute to autoimmune and inflammatory diseases. It has recently been reported that an inducer of TNF-alpha was identified as one of IL-18 inducible genes in IL-18 responsible cells and named as a new cytokine IL-32. We have produced novel monoclonal anti IL-32 antibodies in order to help study IL-32 function and to develop improved diagnosis of IL-32-expressing tumors. Several mAbs reactive to IL-32 isoforms were prepared and characterized by the epitope analysis and Western blotting performed using various deletion mutants and IL-32 isoforms (IL-32alpha, beta, gamma, and delta). In order to optimize the sandwich ELISA for IL-32, recombinant IL-32alpha was added, followed by the addition of a biotinylated mAb KU32-52 into the microtiter plate wells pre-coated with a mAb KU32-07 or mAb KU32-56. The bound mAb was probed with a streptavidin conjugated to HRP. The epitope analysis and Western blot analysis revealed that mAb KU32-07 could detect only IL-32alpha and KU32-52 was bound to all isoforms, whereas KU32-56 were reactive to IL-32 alpha, beta, delta isoforms but not gamma isoform. These sandwich ELISAs were highly specific and had a minimal detection limit of 80 pg/ml (mean+3 SD of zero calibrator) and measuring range of up to 3000 pg/ml. An ELISA using a coating mAb KU32-07 and a capturing biotinylated mAb KU32-52 had no cross-reaction with other cytokines such as IL-32beta, IL-32gamma, IL-32delta, hIL-1alpha , IL-1beta , hIL-2, hIL-6, hIL-8, hIL-10, hIL-18, and hTNF-alpha. Intra-assay coefficients of variation were 11 to 6% (n=16) and inter-assay coefficients were 10 to 5% (n=9). Another ELISA using a coating mAb KU32-56 and a capturing biotinylated mAb KU32-52 detected both IL-32alpha and IL-32beta isoforms but not gamma and delta isoforms and had no cross-reaction with other cytokines such as hIL-1alpha , IL-1beta , hIL-2, hIL-6, hIL-8, hIL-10, hIL-18, and hTNF-alpha. One mAb KU32-09 was shown to react strongly on immunohistochemistry. Our newly established mAbs, KU32-07, KU32-09, KU32-52, KU32-56, have different and useful properties for the detection of IL-32 by immunohistochemistry, ELISA, and Western blotting.