Search

Objectives: Using a murine model of systemic Kawasaki disease (KD)–like vasculitis induced by Candida albicans cell–wall–derived mannan · β–glucan · protein complexes, the objective was to elucidat...

Summary Uveitis (intraocular inflammation) is a leading cause of loss of vision. Although its aetiology is largely speculative, it is thought to arise from complex genetic–environmental interactions that break immune tolerance to generate eye-specific autoreactive T cells. Experimental autoimmune uveitis (EAU), induced by immunization with the ocular antigen, interphotoreceptor retinoid binding protein (IRBP), in combination with mycobacteria-containing complete Freund's adjuvant (CFA), has many clinical and histopathological features of human posterior uveitis. Studies in EAU have focused on defining pathogenic CD4+ T cell effector responses, such as those of T helper type 17 (Th17) cells, but the innate receptor pathways precipitating development of autoreactive, eye-specific T cells remain poorly defined. In this study, we found that fungal-derived antigens possess autoimmune uveitis-promoting function akin to CFA in conventional EAU. The capacity of commensal fungi such as Candida albicans or Saccharomyces cerevisae to promote IRBP-triggered EAU was mediated by Card9. Because Card9 is an essential signalling molecule of a subgroup of C-type lectin receptors (CLRs) important in host defence, we evaluated further the proximal Card9-activating CLRs. Using single receptor-deficient mice we identified Dectin-2, but not Mincle or Dectin-1, as a predominant mediator of fungal-promoted uveitis. Conversely, Dectin-2 activation by α-mannan reproduced the uveitic phenotype of EAU sufficiently, in a process mediated by the Card9-coupled signalling axis and interleukin (IL)-17 production. Taken together, this report relates the potential of the Dectin-2/Card9-coupled pathway in ocular autoimmunity. Not only does it contribute to understanding of how innate immune receptors orchestrate T cell-mediated autoimmunity, it also reveals a previously unappreciated ability of fungal-derived signals to promote autoimmunity.

Kawasaki disease (KD) is a paediatric vasculitis whose pathogenesis remains unclear. Based on experimental studies using a mouse model for KD, we report here that proline-rich protein tyrosine kinase 2 (Pyk2) plays a critical role in the onset of KD-like murine vasculitis. The mouse model for KD was prepared by administrating a Candida albicans water-soluble fraction (CAWS). Unlike CAWS-treated WT mice, CAWS-treated Pyk2-Knockout (Pyk2-KO) mice did not develop apparent vasculitis. A sustained increase in MIG/CXCL9 and IP-10/CXCL10, both of which have potent angiostatic activity, was observed in CAWS-treated Pyk2-KO mice. CAWS-induced activation of STAT3, which negatively regulates the expression of these chemokines, was also attenuated in macrophages derived from Pyk2-KO mice. The present study suggests that defects in Pyk2 suppress KD-like experimental vasculitis, presumably through CXCL9- and CXCL10-dependent interference with neo-angiogenesis. Since Pyk2-KO mice show no life-threatening phenotype, Pyk2 may be a promising therapeutic molecular target for KD.

Background: Kawasaki’s disease is a pediatric medium vessel vasculitis causing coronary arteritis and potentially coronary artery aneurysms. Higher systemic levels of Interleukin-6 (IL-6) in the first week of disease has been shown to be associated with a higher risk of coronary artery aneurysms (1, 2). Mice injected with Candida albicans water-soluble fraction (CAWS) develop coronary and aortic arteritis like that seen in Kawasaki’s disease. Our prior work has demonstrated that mast cell degranulation can inhibit lipopolysaccharide (LPS)-induced IL-6 gene expression in the aorta and systemic IL-6 production (3). Objectives: The objective of this study was to determine if mast cells play a role in the IL-6 homeostasis in an established mouse model of Kawasaki’s disease (CAWS model). Methods: 8-10-week-old male wild type controls (WT) and kitW-sh/W-sh mice (mast cell deficient, MCD) of C57Bl/6 background were randomly distributed into four groups (WT-PBS, WT-CAWS, MCD-PBS, MCD-CAWS). They were either injected intraperitoneally with PBS or CAWS (2 mg/mouse) daily for a period of 5 days. Eight animals from each group were sacrificed at either 7, 14 or 30 days after the last injection and blood, aorta and heart specimens were harvested. A pathologist (OT), blinded to treatment groups, examined aortic root and adjacent myocardium specimens and assigned a score for the degree of inflammation (0-6). Results: Seven MCD-CAWS mice died unexpectedly within 24 hours of the first CAWS injection. Autopsies reviewed marked liver sinusoidal dilation in all and patchy necrosis of the liver in 4. Similar, liver findings were seen in 75% of MCD-CAWS after 7 days, but not in other groups. MCD-CAWS mice had higher systemic IL-6 compared to WT-CAWS at both 7 days (208± 21 vs 152 ± 12 pg/ml; p Conclusion: Mast cell deficiency resulted in higher systemic levels of IL-6, TNFα and INFγ in the CAWS mouse model of Kawasaki’s disease. Similarly, mast cell deficiency resulted in more intense inflammation at the root of the aorta in this model. These results support the novel concept that mast cells play a protective role in reducing the initial systemic inflammatory response in Kawasaki’s disease. References: [1] Ueno Y, Takano N, Kanegane H, Yokoi T, Yachie A, Miyawaki T, et al. The acute phase nature of interleukin 6: studies in Kawasaki disease and other febrile illnesses. Clinical and experimental immunology. 1989;76(3):337-42. [2] Chow YM, Lin CY, Hwang B. Serum and urinary interleukin-6 (IL-6) levels as predicting factors of Kawasaki disease activity. Zhonghua Minguo xiao er ke yi xue hui za zhi [Journal] Zhonghua Minguo xiao er ke yi xue hui. 1993;34(2):77-83. [3] Springer J, Raveendran V, Smith D, Maz M, Dileepan K. OP0138 Novel Role of Mast Cells in the Regulation of Large Vessel Vasculitis. EULAR Paris, France: Ann Rheum Dis; 2014. p. 112-3. Acknowledgement: Acknowledgements: Joseph & Elizabeth Carey Endowment Fund and Lied Basic Science Research Development Award from University of Kansas Medical Center Disclosure of Interests: None declared

Recognition of (1→3)-β-d-glucans (BGs) by invertebrate β-1,3-d-glucan recognition protein (BGRP) plays a significant role in the activation of Toll pathway and prophenoloxidase systems in insect host defense against fungal invasion. To examine the structure diversity of BGRPs for the recognition of physiochemically different BGs, the binding specificity of BGRPs cloned from four different insects to structure different BGs was characterized using ELISA. Recombinant BGRPs expressed as Fc-fusion proteins of human IgG1 bound to the solid phase of BGs. Based on the binding specificities, the BGRPs were categorized into two groups with different ultrastructures and binding characters; one group specifically binds BGs with triple-helical conformation, while the other group recognizes BGs with disordered conformations like single-helical or partially opened triple helix. The BGRPs from the silkworm and the Indian meal moth bound to the BGs with a triple-helical structure, whereas BGRPs from the red flour beetle and yellow mealworm beetle showed no binding to triple-helical BGs, but bound to alkaline-treated BGs that have a partially opened triple-helical conformation. This evidence suggests that the insect BGRPs can distinguish between different conformations of BGs and are equipped for determining the diversity of BG structures.

Background We have previously demonstrated that mast cell degranulation acutely downregulates lipopolysaccharide induced aortic expression and serum levels of IL-6 in vivo. This is accompanied by aortic upregulation of suppressor of cytokine signaling-1 (SOCS-1) gene expression1. This effect is not seen in histamine H1 receptor-knockout mice suggesting that mast cell-derived histamine is a key mediator involved in IL-6 homoeostasis2. Mice injected with Candida albican water-soluble-fraction (CAWS) have been shown to develop coronary and aortic vasculitis3. Our long-term objective is to determine the pathogenic mechanism of large vessel vasculitis (LVV). Objectives The aim of this pilot study was to replicate and develop a working mouse model to determine the regulatory role of mast cells in LVV. Methods Eight to ten weeks-old male C57Bl6/J mice were randomly distributed into two groups [CAWS, N=8; and Control, N=8] and were injected i.p. daily for 5 days with either CAWS in normal saline (2 mg/day/mouse) or normal saline alone (controls). All mice were sacrificed 30 days after the 5th injection. We examined serum levels of IL-6 and TNF-α, as well as aortic tissue expressions of IL-6, TNF-a, IL-10 and SOCS-1 mRNA. Heart and aortic sections were evaluated for inflammation and mast cells after staining with H & E and toluidine blue, respectively. Results Treatment of mice with CAWS for 5-consecutive days led to overexpression of IL-6, TNF-a and IL-10 genes in the aortic tissue with modest upregulation of SOCS-1. At the root of the aorta, all animals in the CAWS group had intense inflammatory infiltrates composed of mixed acute and chronic inflammatory cells. There is also evidence of vasculitis in the coronary arteries. In contrast, none of the control mice had any evidence of aortic inflammation or vasculitis. Serum IL-6 concentrations were below detectable levels in both controls and CAWS-treated mice whereas TNF-a levels were elevated in 3 out of 8 mice in the CAWS group. There were no signs or increased presence of intact or degranulating mast cells in the area of inflammation. Conclusions These results suggest that CAWS-induced LVV involves acute and chronic inflammatory response and vascular tissue expression of both pro- and anti-inflammatory cytokines and SOC-1. Detailed kinetic studies are warranted to determine the optimum windows of peak inflammatory response and the expression of these genes to understand the pathobiology of CAWS-induced LVV. References Springer J, Raveendan V, Smith D, et al. [abstract] Ann Rheum Dis 2014;73:112–113. Raveendran V, Springer J, Smith D, et al. [abstract]. Arthritis Rheumatol 2015;67(suppl 10). Miura N, Shingo Y, Adachi Y, et al. Immunopharmacol Immunotoxicol 2004;26(4):527–43. Acknowledgements Supported by Basic Science Development Award from Department of Medicine at the University of Kansas. Disclosure of Interest None declared

Kawasaki disease (KD) is a paediatric idiopathic vasculitis. In this study, on the basis of studies using an established animal model for KD, we report that mannose-binding lectin (MBL) is involved in the pathogenesis of the disease. KD-like experimental murine vasculitis was induced by intraperitoneally administering a Candida albicans water-soluble extract (CAWS). MBL-A gradually increased in the serum of the model mice treated with CAWS. Deposition of MBL-A and MBL-C was observed in the aortic root, including the coronary arteries, which is a predilection site in experimental vasculitis. Corresponding to the distribution patterns of MBLs, marked deposition of C3/C3-derived peptides was also observed. Regarding the self-reactivity of MBLs, we observed that MBLs interacted with core histones to activate the lectin pathway. These results suggest that some types of pathogens provoke the MBL-dependent complement pathway (lectin pathway) to cause and/or exacerbate KD-like vasculitis.

Recognition of (1&rarr, 3)-&beta, d-glucans (BGs) by invertebrate &beta, 1,3-d-glucan recognition protein (BGRP) plays a significant role in the activation of Toll pathway and prophenoloxidase systems in insect host defense against fungal invasion. To examine the structure diversity of BGRPs for the recognition of physiochemically different BGs, the binding specificity of BGRPs cloned from four different insects to structure different BGs was characterized using ELISA. Recombinant BGRPs expressed as Fc-fusion proteins of human IgG1 bound to the solid phase of BGs. Based on the binding specificities, the BGRPs were categorized into two groups with different ultrastructures and binding characters, one group specifically binds BGs with triple-helical conformation, while the other group recognizes BGs with disordered conformations like single-helical or partially opened triple helix. The BGRPs from the silkworm and the Indian meal moth bound to the BGs with a triple-helical structure, whereas BGRPs from the red flour beetle and yellow mealworm beetle showed no binding to triple-helical BGs, but bound to alkaline-treated BGs that have a partially opened triple-helical conformation. This evidence suggests that the insect BGRPs can distinguish between different conformations of BGs and are equipped for determining the diversity of BG structures.

Patients with chronic granulomatous disease (CGD) have mutated phagocyte NADPH oxidase, resulting in reduced production of reactive oxygen species (ROS). While the mechanism underlying hyperinfection in CGD is well understood, the basis for inflammatory disorders that arise in the absence of evident infection has not been fully explained. This study aimed to evaluate the effect of phagocyte NADPH oxidase deficiency on lung inflammation induced by nonviable Candida albicans (nCA). Mice deficient in this enzyme (CGD mice) showed more severe neutrophilic pneumonia than nCA-treated wild-type mice, which exhibited significantly higher lung concentrations of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and keratinocyte-derived chemokine (KC). Neutralization of these proinflammatory mediators significantly reduced neutrophil infiltration. In vitro, production of IL-1β and TNF-α from neutrophils and that of KC from macrophages was enhanced in nCA-stimulated neutrophils from CGD mice. Expression of IL-1β mRNA was higher in the stimulated CGD neutrophils than in the stimulated wild-type cells, concomitant with upregulation of nuclear factor (NF)-κB and its upstream regulator extracellular-signal regulated kinase (ERK) 1/2. Pretreatment with an NADPH oxidase inhibitor significantly enhanced IL-1β production in the wild-type neutrophils stimulated with nCA. These results suggest that lack of ROS production because of NADPH oxidase deficiency results in the production of higher levels of proinflammatory mediators from neutrophils and macrophages, which may at least partly contribute to the exacerbation of nCA-induced lung inflammation in CGD mice.

Amino acids 37 to 53 (RAARISLGPRCIKAFTE) of C5a anaphylatoxin form an essential region for C5a function. To target this sequence, we generated a complementary peptide (ASGAPAPGPAGPLRPMF) designated PepA which has a potent inhibitory effect on C5a activity. By introducing an acetyl group at the N-terminal alanine of PepA, an acetylated form was generated which was designated AcPepA. The acetylation resulted in increased inhibition of C5a stimulation of neutrophils as determined by Ca influx. Furthermore, AcPepA partially inhibited the lethal shock induced in mice by intravenous administration of Candida albicans water-soluble mannoprotein-beta-glucan complex. In addition, local skin inflammation in rats caused by an anti-Crry monoclonal antibody was suppressed when AcPepA and the antibody were injected together, while PepA had little inhibitory capacity. The potent inhibitory capacity of AcPepA was also confirmed by a skin reaction of guinea pigs inoculated with recombinant human C5a together with AcPepA.

Introduction: Although etiology of Kawasaki disease (KD) remains elusive, a line of recent experimental studies implies that some kinds of infectious stimuli are implicate in the vasculitis through uncontrolled innate immune systems such as pattern recognition receptor (PPR)-mediated inflammatory signaling. It has already known that Candida albicans water-soluble fraction (CAWS) inducing KD-like vasculitis in mice function through PRP. Furthermore, it is reported that proline-rich tyrosine kinase (Pyk2), which is molecule involved in the PRPs-dependent signaling pathways, plays an important role in activation of NF-κB. Therefore, we investigated a possible relevance of Pyk2 in the pathogenesis of KD. Methods: Pyk2-knock out (Pyk2-KO) and wild-type C57BL/6 mice (WT) were administered CAWS to induce KD-like vasculitis. Extension of the experimental vasculitis was immunohistochemically determined with anti-MPO antibody. CAWS-stimulated NF-κB activation was evaluated by quantifying nuclear translocation of NF-κB p65 subunit in peritoneal macrophages isolated from PYK2-KO and wild-type mice in vitro. Cytokines and chemokines across each mice were compared by cytokine array. Results: Pyk2-KO mice didn’t show any apparent defective phenotype. While marked inflammation was observed in the aortic root of CAWS-treated WT mice, such vasculitis was barely detected in CAWS-treated Pyk2-KO mice. CAWS-induced NF-κB activation was also less observed in macrophages from Pyk2-KO mice. There were differences in some cytokines and chemokines production between mice. Conclusion: We speculate that Pyk2 is involved in the pathogenesis of KD. Pyk2 might be a potential therapeutic target for KD.

Background: The cause of Kawasaki disease (KD) has been still unknown though the intestinal flora was reported as one of the candidate etiology of KD. Candida is known as one of the intestinal flora. Intraperitoneal injection of Candida albicans water-soluble fractions (CAWS) that compose of polysaccharide of cell wall of the yeast can induce systemic vasculitis in mice. It is evaluated as animal model of Kawasaki disease vasculitis because this experimental vasculitis is similar to those observed in KD patients. However it is uncertain whether or not oral administration of CAWS can induce vasculitis in mice. Aim: The present study aimed to elucidate whether or not oral administration of CAWS can induce vasculitis. Materials and methods: Mice, DBA/2, male, 4 weeks of age were used. CAWS suspended in PBS was administered perorally to mice for 28 consecutive days. As a priming agent, 20 micrograms of LPS was injected to mice intraperitoneally before administration of CAWS. Experimental groups are as follows. Group-I : Peroral administration of 800 micrograms of CAWS with LPS, Group-II: Peroral administration of 800 micrograms of CAWS without LPS, Group-III : Peroral administration of 250 micrograms of CAWS with LPS. Mice injected CAWS intraperitoneally (conventional procedure) were used as control group. Vasculitis was evaluated by routine histological techniques. This study conformed with regulations of position of the American Heart Association on research animal use and Toho University’s Animal Ethics Committee. Result: The incidence of vasculitis in each group were as follows : Group-I : 1/6, Group-II:0/6, Group-III:1/6, Control : 3/3. Dense infiltration of neutrophils and macrophages was observed in coronary artery and/or aortic root. Severity of inflammatory cell infiltration and extent of the lesion in Group-I and Group-III were milder and smaller than those of control. Conclusion: Present study revealed that peroral administration of CAWS could induce vasculitis in mice though the incidence of vasculitis was lower than that of control. This model is considered to be useful to clarify the relation between intestinal immunity and vasculitis. We should clarify the mechanism that oral administration of CAWS induced vasculitis.

CAWS, a water-soluble extracellular polysaccharide fraction obtained from the culture supernatant of Candida albicans, is one of the fungal pathogen-associated molecular patterns (PAMPs). It has been reported to show potent activity inducing arteritis and coronaritis in mice. Especially, CAWS-induced arteritis has a 100% incidence and severe mortality in the DBA/2 mouse strain. This artificial vasculitis was reported to provide a good murine model of Kawasaki disease and other inflammatory vascular disease. However, severe mortality was observed only in DBA/2 mice, which is a CAWS-sensitive strain. In this study, to clarify the mechanisms of CAWS-induced arteritis and mortality, we investigated microscopic histopathological changes in cardiovascular tissues in DBA/2 mice. Severe inflammatory infiltration was observed from the external elastic lamina in the aorta and proximal coronary arteries within 1 week after CAWS administration. Severe stenosis of the aorta and coronary arteries was observed more than 3 weeks after CAWS administration. Fibrinoid necrosis was observed in these vessel walls. All CAWS-treated mice died between the fifth and twelfth week after administration. Severe inflammatory change with aortic valve transformation suggested that CAWS-treated mice died of valvular endocarditis or cardiac dysfunction. Based on the simple induction method and complete incidence, these data suggest that CAWS-induced arteritis is a good model of not only Kawasaki disease but also other cardiovascular diseases such as valvular endocarditis.

Silica-doped poly(lactic acid) (PLA) composite hollow spheres containing calcium carbonates (Si-CCPC spheres) were prepared using aminopropyltriethoxysilane (APTES) for injectable bone fillers combined with a cell-delivery system. Si-CCPC spheres have a hollow spherical shape of ~1 mm in the external diameter and an open channel in the shell, which is selfformed. The channel size is about 500 *m in diameter. X-ray energy dispersive spectroscopy (EDS) analysis showed incorporation of silicon in Si-CCPC spheres. After soaking Si-CCPC spheres in simulated body fluid (SBF), hydroxycarbonate apatite formed on the Si-CCPC spheres. Inductively coupled plasma atomic emission spectroscopy (ICP-AES) showed that the Si4+ ion is released from Si-CCPC spheres in SBF.

Novel hollow spheres for bone fillers incorporating cells were prepared using composites consisting of poly(lactic acid) and calcium carbonates. An open channel of ~800 µm in diameter was easily formed using a chemical etching method to provide a pathway to the interior of the sphere. Cells could migrate through the open channel into the interior of the sphere. Bonelike apatite coating on the surface of the sphere was prepared by soaking in calcium chloride solution to supply excess Ca2+ ions on the surface and subsequently by soaking in simulated body fluid. The hollow spheres with an open channel may be one of the great potential candidates as novel bone fillers combined with a cell-delivery system.

Central Research Institute of Electric Power Industry (CRIEPI) has been conducting a research project on the verification of evaluation method for structural integrity of high-temperature components. As a part of the framework, we developed a flaw evaluation system on PC to predict creep-fatigue crack propagation, which was considered to be an essential crack driving force at high temperature especially in case where stresses caused by thermal loading were significant. The system is featured by easy and dialogical operation with graphical user interface. Fatigue and creep crack propagation behavior can be predicted using fatigue J-integral range and creep J-integral, respectively, which are the variations of the non-linear fracture mechanics parameter, J-integral. Both fatigue J-integral range and creep J-integral are calculated by the reference stress method with revised solutions developed by detailed finite element analysis.

Ishimaki and Tahara limestones occur as exotic blocks juxtaposed in the Mesozoic (Jurassic) accretionary complex of Southern Chichibu Terrane in eastern Aichi Prefecture, central Japan. They are supposed to be of the seamount-type limestone, since they have no terrigenous materials and are intimately associated with greenstones. REE (rare earth elements) and Sr isotopic studies for the limestones have been made in order to know their geochemical characteristics, ages and origins. Their 87Sr/86Sr ratios, when referred to the seawater 87Sr/86Sr curve and relevant geological data, suggest that Ishimaki and Tahara limestones are the late Permian and the Carboniferous to the Early Permian, respectively. Two greenstone fragments found inside the Ishimaki limestone block and one greenstone sample associated with Tahara limestone block, resemble the Hawaiian alkali basalt in their REE and Y patterns. This is supporting the idea that the limestone blocks may be parts of reef limestones on ancient volcanic seamounts. All the limestone samples, except three unusual Tahara ones, show seawater REE and Y signatures in their chondrite-normalized patterns. Their REE/Ca ratios, however, are 102-104 times as high as those ratios of modern biogenic carbonates like corals and the seawater. Accordingly, seawater REE and Y were incorporated into the limestones, when originally biogenic carbonates transformed into inorganic calcite and its secondary growths occurred in diagenesis in contact with sufficient seawater. This view is favored by the reported REE partition experiment between calcite overgrowths and seawater solution. The seawater Ce anomaly as a function of water depth in the modern ocean is a key to infer the water depth of the REE and Y incorporation. The Ce anomalies given by log(Ce/Ce∗) for about a half of Ishimaki samples and most of Tahara ones are between -0.5 and -0.2, which are compatible with the shallow water origin. Another half of Ishimaki samples, however, have log(Ce/Ce∗) values between -0.7 and -1.0, suggesting moderately deep waters (ca. 500-1000 m deep or more). This may reflect such a situation that water depths of REE incorporation into the seamount-type limestones are generally greater than the depositional water depths of original biogenic carbonates because of the fate of limestone-capped volcanic seamounts decided by the oceanic plate motion.

Sparassis crispa Fr. is an edible mushroom recently cultivable in Japan. It contains a remarkably high content of 6-branched 1,3-beta-D-glucan showing antitumor activity. Using ion-exchange chromatography, a purified beta-glucan preparation, SCG, was prepared. In this study, we examined the hematopoietic response by SCG in cyclophosphamide (CY)-induced leukopenic mice. SCG enhanced the hematopoietic response in CY induced leukopenic mice by intraperitoneal routes over a wide range of concentrations. SCG enhanced the hematopoietic response in CY-treated mice by prior or post administration. Analyzing the leukocyte population by flow cytometry, monocytes and granulocytes in the peritoneal cavity, liver, spleen and bone marrow (BM) recovered faster than in the control group. The ratio of natural killer cells and gammadelta T cells in the liver, spleen and peritoneal cavity was also increased. In contrast, CD4+ CD8+ cells in the thymus were temporarily significantly decreased by the administration of SCG. Interleukin-6 (IL-6) production of CY+SCG-treated peritoneal exdated cells (PECs), spleen cells and bone marrow cells (BMCs) were higher than that of the CY-treated group. By in vitro culture of CY-treated PEC and spleen cells, IL-6 production was enhanced by the addition of SCG. These facts suggested the possibility that IL-6 might be a key cytokine for the enhanced hematopoietic response by SCG.

Various inflammatory cytokines, including tumor necrosis factor-α (TNF-α), have been reported to play roles in Kawasaki disease (KD). Recently, anti-TNF-α therapy was reported to show efficacy in patients who do not respond to high-dose intravenous immunoglobulin therapy. However, there are many gaps in our understanding of the role that TNF-α plays in the development of KD arteritis as well as whether anti-TNF-α therapy causes any histological changes in the arteritis. Accordingly, the present histopathological study was carried out to elucidate the inhibitory effect of anti-TNF-α therapy on vasculitis as well as the role of TNF-α in the development of vasculitis in a murine model of KD vasculitis.We used two anti-TNF-α drugs (etanercept and infliximab) to treat a Candida albicans-induced murine model of KD vasculitis. We investigated the histopathological changes in terms of the incidence of vasculitis, the scope of lesions and the degree of inflammation.Administration of etanercept to the mice reduced not only the incidence of vasculitis but also the scope of lesions and the degree of inflammation.Based on the histological findings, TNF-α is deeply involved in the development of vasculitis.

We have studied the mobility of rare earth elements (REE) and yttrium during dolomitization of marine limestone in reaction with MgCl2 solution at 150°C for 10 and 20 days. On the experiment using the pulverized limestone of 7.5 g and 25 ml of 2 M MgCl2 solution, 90% of the calcite transformed into dolomite at 150°C within 20 days. The dolomitized products showed that there is no significant change of REE abundances of original marine limestone. This is experimental evidence for the field observation that dolostones sometimes preserve the REE and Y signatures of limestone precursors.

Distribution coefficients of REE between Fe oxyhydroxide precipitates and NaCl solutions doped with NaHCO3 have been determined in pH = 8.1-8.6 and at room temperature and pressure. The coefficient is defined as Kd(REE: ppt./sol.), where REE designates each lanthanide (Ln), Y or Sc. The NaHCO3 concentration was changed in the range of (0∼12) × 10-3 M under a constant NaCl concentration (0.45 M). Kd(Sc) rapidly approaches Kd(Lu) as [NaHCO3] increases. Kd(Y) is lower than Kd(Ho) even in solutions with high [NaHCO3]. The convex tetrad effect in logKd(Ln) becomes less conspicuous with increasing [NaHCO3]. We proposed a method to determine REE-carbonate complexation constants from observed variations of Kd(REE) with increasing [CO32-, aq]. In solutions with [NaHCO3] ≥ 1 × 10-2 M, the dominant dissolved REE species are REE(CO3)2-(aq) except for La. We have tentatively determined the stability constants of β2 for REE(CO3)2-(aq) from our preliminary data set, although β1 for REECO3+(aq) could not be estimated. The series variation of logβ2 are compatible with literature values. On the basis of the refined spin-pairing energy theory (RSPET), we have analyzed the series variations of logβ2 and logKd(Ln) values with and without Ln(III)-carbonate complexation effect. Racah E1 parameter is approximately the same between Ln(OH)3·nH2O as the precipitate and Ln(CO3)2-(aq), whereas Racah E3 parameter of Ln(CO3)2-(aq) is only slightly larger than that of Ln(OH)3·nH2O. This is the reason that the convex tetrad effect of logKd(Ln) diminishes as Ln(III)-carbonate complexation proceeds. Our experimental logKd(Ln) values and apparent logKd(Ln) ones from marine Mn-Fe deposit/seawater pairs suggest that reported log(β1/β2) values for light Ln are slightly smaller than those ought to be.

REE data for a GSJ reference carbonate sample (JDo-1) by ICP-AES, when normalized by reliable seawater REE data, exhibit fairly smooth abundance patterns except for Ce. This allows us to estimate the monoisotopic REE (Pr, Tb, Ho, and Tm) concentrations compatible with the widely accepted ID-MS data for seawater REE. For the same purpose, reported seawater REE data by ICP-MS have also been used. The monoisotopic REE concentrations estimated by the two methods are in good agreement. Eventually, the seawater REE patterns normalized by chondrite or average shale show obvious concave tetrad effects of W-type. There is no room for doubt as to the seawater tetrad effect, but its origin is open to debate. We proposed here that the tetrad effect originates from REE partitioning described by the ligand-exchange reactions of REE3+ between particulate REE and REE(CO3)2-(aq) in seawater. The particulate REE must be REE(OH)3-like ones rather than REEO1.5. The refined spin-pairing energy theory explains that the logarithmic equilibrium constants for the reactions exhibit a tetrad effect when Racah parameters for 4f electron repulsion in REE3+ ions are different between the pair of REE(III) species. We inferred that Racah parameters of REE(OH)3 and REEO1.5 are similar but they are commonly much smaller than those parameters of REE(CO3)2-(aq). When the reactions occur in the particulate-seawater system with high particulate concentration such as coastal seawater and estuarine water, the signature of concave tetrad effect is inevitably given to the seawater solution. Subsequent removal of excessive particulate matter from the system leaves the detectable tetrad effect in the seawater. Shale-normalized seawater REE patterns correspond roughly to the logarithmic equilibrium concentration ratios by the ligand-exchange reactions. Our proposal gives a consistent explanation for the seawater-like tetrad effects in REE patterns of marine carbonate rocks and the absence of such tetrad effects in those of deep-sea nodules. This is also related to a theoretical interpretation of the Masuda-Coryell plot in REE geochemistry.

医用材料は,滅菌処理並びに表面の改質がなされているが,これらの材料が細胞に与える影響を検討した.すなわち,γ-線及びアルゴン(Ar)プラズマをポリスチレンに照射し,接触角により表面の物理化学的変化を測定するとともに,好中球様HL-60細胞が材料に接触した際に産生される活性酸素を,ルミノール依存性化学発光により測定した.ポリスチレンにγ-線あるいはArプラズマ照射を施すことで,接触角は減少し,表面の親水性化が確認された。又,ポリスチレンに接触した細胞から産生される活性酸素産生量に,照射による影響が認められ,好中球様HL-60細胞を用いた生物学的方法により照射したポリスチレン表面の微小な変化をとらえることができた.

This study aimed to evaluate the effect of myeloperoxidase (MPO) deficiency on lung inflammation induced by nonviable Candida albicans (nCA).Mice were inoculated intranasally with nCA, and accumulation of neutrophils and macrophages in the bronchoalveolar lavage fluid was analyzed by flow cytometry. The levels of macrophage inflammatory protein 2 (MIP-2), keratinocyte-derived chemokine (KC), tumor necrosis factor (TNF)-α, and interleukin (IL)-1β in the lung were measured by ELISA. Production of MIP-2 and KC from neutrophils and macrophages was quantified in vitro. MIP-2 mRNA expression in the neutrophils was analyzed by real-time reverse transcription-PCR, and the extent of phosphorylation of ERK1/2 and Syk in the neutrophils was analyzed by Western blotting.The MPO(-/-) mice that received nCA showed more severe pneumonia than wild-type mice. Within 12 h of nCA administration, MPO(-/-) mice had significantly higher numbers of alveolar neutrophils and increased production of MIP-2 and KC relative to the responses seen in wild-type mice. Neutralization of MIP-2 and KC in vivo significantly reduced neutrophil infiltration. In vitro, production of MIP-2, but not that of KC, was enhanced in the nCA-stimulated neutrophils from MPO(-/-) mice, concomitant with up-regulation of Syk and ERK1/2. At 1 and 3 days after nCA administration, MPO(-/-) mice had significantly higher lung concentrations of TNF-α and IL-1β than wild-type mice.Pulmonary administration of nCA produced an altered inflammatory response in MPO(-/-) mice relative to wild-type mice. Enhanced MIP-2 production by MPO(-/-) neutrophils may at least partly contribute to exacerbated inflammation in mutant mice.

Background Serum interleukin-6 (IL-6) has been shown to closely follow disease activity in large vessel vasculitis 1 . Prior research by our group has demonstrated that mast cell degranulation downregulates lipopolysaccharide (LPS) induced aortic expression of IL-6 in vivo. This is accompanied by aortic upregulation of SOCS-1 2 . This effect is lost in histamine 1 receptor knockout mice suggesting that histamine is a primary mediator of aortic IL-6 inhibition. Prior studies have also demonstrated that mice injected with Candida albicans water-soluble fraction (CAWS) develop coronary and aortic arteritis 3 . The mechanism through which arteritis is induced by CAWS remains poorly understood. Objectives The objective of this study is to determine the effects of histamine and CAWS on IL-6 production by human aortic endothelial cells (HAEC) in vitro. Methods HAEC were cultured with histamine (10μM), LPS (100ng/mL), CAWS (10μg/mL), CAWS (1μg/mL) or a combination of these. After 22 hours, levels of IL-6 and 6-ketoprostaglandin-F1α, a breakdown product of prostacyclin, were measured. Microculture tetrazolium (MTT) assays were used to determine cell viability. Results IL-6 levels were significantly lower in cells treated with CAWS 10μg/mL compared to controls (175.2±11.9 vs 270.0 pg/mL, p=0.006). Similarly, IL-6 levels were significantly lower with the addition 10μg/mL CAWS to LPS compared to LPS alone (359.8±37.74 vs 2906.5±318.02, p=0.0001). No significant differences were seen between the 10μg/mL and the 1μg/mL CAWS concentrations. Histamine synergistically enhanced IL-6 levels over LPS alone (3758.0±38.18 vs 2906.5±318.0, p=0.028). CAWS had no effect on prostacyclin levels compared to controls. MTT assays demonstrated no differences in cell viability in CAWS or histamine treated cells compared to controls. Conclusions Our experiments suggest that CAWS inhibits IL-6 production in HAEC. Since IL-6 is a key cytokine in the development of large vessel vasculitis we suspect that endothelial cells do not play a role in CAWS mediated arteritis. Similarly, the inhibitory effects of histamine do not appear to be mediated through endothelial cells. Future research will focus on the in vivo effects of histamine on the development of CAWS mediated vasculitis. References Weyand CM, Fulbright JW, Hunder GG, Evans JM, Goronzy JJ. Treatment of giant cell arteritis: interleukin-6 as a biologic marker of disease activity. Arthritis and rheumatism. May 2000;43(5):1041–1048. Springer J, Raveendran V., Smith D., Maz M., Dileepan K. Novel role of mast cells in the regulation of large vessel vasculitis. Paper presented at: European League Against Rheumatism 2014 meeting; June 12th, 2014, 2014; Paris, France. Nagi-Miura N, Shingo Y, Adachi Y, et al. Induction of coronary arteritis with administration of CAWS (Candida albicans water-soluble fraction) depending on mouse strains. Immunopharmacology and immunotoxicology. 2004;26(4):527–543. Acknowledgement Supported by Basic Science Development Award from Department of Medicine at the University of Kansas. Disclosure of Interest None declared

It has become more serious concern to quantify Low-k damage in Cu/Low-k interconnects. We investigated the relationship between the amount of OH group in Low-k material obtained from micro-beam IR method and the interconnect capacitance. And it was found that there is good correlation among them. Additionally, the micro-beam IR method sensitively detected modifications of amount of OH group for several kinds of treatment processes except for the NH 3 plasma treatment prior to Cu diffusion barrier dielectric deposition.

beta-Glucan is a major component of the cell walls and pathogen-associated microbial patterns of fungi. We previously reported the presence of an antibody which reacts to beta-glucan, anti-beta-glucan (BG) antibody, in human sera. In livestock and domestic pets, the antibody's response to fungal cell wall beta-glucan is little understood. In this study, we examined the existence and reactivity of anti-BG antibody in various animal species. We demonstrated the presence of the anti-BG antibody in each animal's serum. Individual differences in the titer existed. The antibody was highly reactive to Candida solubilized cell wall beta-glucan (CSBG) while reacting little to grifolan (GRN) from Grifola frondosa. This suggested that the anti-BG antibody interacted with fungal cell wall beta-glucan and participated in the immune-response to pathogenic fungi.

Zymosan activates phagocytes through the innate immune system and causes inflammatory responses in animals. Because of the complexity of the active substances included in Zymosan preparations, simplifying the active moiety actually responsible for innate immune recognition is needed. One way to remove possible active substances from commercially available Zymosan preparations is to wash then with pyrogen-free water to obtain a ZWIS (Zymosan water insoluble fraction), ethanol insoluble (EtIS), or chloroform/methanol insoluble (CMIS) preparation. The effects of various washed Zymosan preparations on nuclear factor (NF)-kappaB activation and binding to beta-glucan recognition protein were examined. Significant NF-kappaB activation by Toll-like receptor (TLR) 2-expressing HEK293 cells and enhanced NF-kappaB activity via the co-expression of TLR2 and Dectin-1, a functional beta-glucan receptor, was only observed in response to ZWIS. However, the ability of Zymosan preparations to bind Dectin-1 protein was not altered even after treatment with the organic solvents by which the TLR2-mediated NF-kappaB activity was abolished. Another NF-kappaB activation pathway involving CARD9/Bcl10 was triggered by these Zymosan preparations in the presence of Dectin-1. The results suggest that the beta-glucan-dependent characteristics of Zymosan were not affected by the washing with chloroform/methanol or ethanol, and that TLR2-mediated activity was easily eliminated with these organic solvents. This treatment might be useful for distinguishing natural ligands for TLR2 and beta-glucan receptors when studying the innate immune response to fungal macromolecules.

This paper describes process optimization of UV curing for ultra Low-k SiOC (ULK-SiOC, k=2.65) and High stress silicon nitride (HS-SiN) liner. We have investigated the impact of UV curing using different UV bulbs (UV-X and UV-Y) on BEOL process damages to the ULK-SiOC. The k-value variations of ULK-SiOC modified with UV-X with higher photon energy than UV-Y are greater than those of the pristine ULK-SiOC and the ULK-SiOC modified with UV-Y. These results are consistent with the previously reported FT-IR and NMR analyses that the UV-X generates 3-fold Si-O ring defects and Si-H bonds in the ULK-SiOC. In order to discuss the pore structure in UV-modified ULK-SiOC, the solvent diffusion of toluene and ethanol in ULK-SiOC were measured. The solvent diffusion of the UV-modified ULK-SiOC with UV-X is faster than that of the UV-modified ULK-SiOC with UV-Y. This result indicates that the pore size of UV-modified ULK-SiOC with UV-X is larger than that of UV- modified ULK-SiOC with UV-Y. The dependence of the stress increase in HS-SiN liner on different UV bulbs (UV-X and UV-Y) has been investigated by using stress measurement and FT-IR. The UV-modified HS-SiN liner with UV-Y achieves higher tensile stress, compared to the UV-X. FT-IR results have revealed that UV-Y is more effective for the dehydrogenation and SiN crosslinking, resulting in the greater increase in the tensile stress. In conclusion, UV-Y bulb with medium photon energy yields the desired UV modifications for ULK-SiOC and HS-SiN applications.

This paper describes film characterization of Ultra Low-k (ULK) dielectrics modified by UV curing with different wavelength bands. We have demonstrated UV hardening of ULK-SiOC (k=2.65) with two types of UV bulbs (UV-X and UV-Y) and the UV modifications of ULK-SiOC film properties are characterized by using FT-IR spectroscopy, 29Si Solid-state NMR spectroscopy and Raman spectroscopy. FT-IR and NMR analyses reveal that UV-Y curing is preferable for UV curing modification of ULK-SiOC. UV-Y curing increases Q mode peak in NMR, resulting in the enhanced Si-O crosslinking, while UV-X curing increases TH mode and TOR mode peaks. Spin lattice relaxation time T1 for 29Si is decreased with UV curing. This result indicates that UV curing enhances molecular motion in Si-O network. Raman analysis shows that UV curing increases amorphous carbon groups, which corresponds to the enhanced molecular motion in Si-O network.

In this paper, we discuss the possibility of stress engineering in the Cu/low-k interconnect reliability. We mention the film characteristics of UV cured SiCN and SiCO. A large stress change from compressive to tensile stress was observed. Through TEG demonstration, it was found that UV-cured SiCN and SiCO film make it possible to reduce SIV failure without degradation to other interconnect reliability

A UV-hardened high-modulus ULK (ultra low-k) material is proposed for 45-nm-node Cu/low-k interconnects with homogeneous dielectric structures. An elastic modulus as high as 16 GPa was achieved for the ULK material with k=2.65. By combining this material with an advanced dielectric barrier (k=3.7), interconnect test devices with 65-nm-node dimensions were fabricated. The UV-hardened high-modulus ULK material is shown to be effective in improving electrical performance while maintaining sufficient mechanical integrity.

The animal biopolymers prepared from hen eggshell membrane and broiler chicken feathers, which are byproducts of the poultry-processing industry, were evaluated for the removal of the oxyanions selenium [Se(IV) and Se(VI)] and arsenic [As(III) and As(V)] from aqueous solutions. The biopolymers were found to be effective at removing Se(VI) from solution. Optimal Se(IV) and Se(VI) removal was achieved at pH 2.5-3.5. At an initial Se concentration of 100 mg/L (1.3 mM), the eggshell membrane removed approx 90% Se(VI) from the solution. Arsenic was removed less effectively than Se, but the chemical modification of biopolymer carboxyl groups dramatically enhanced the As(V) sorption capacity. Se(VI) and As(V) sorption isotherms were developed at optimal conditions and sorption equilibrium data fitted the Langmuir isotherm model. The maximum uptakes by the Langmuir model were about 37.0 mg/g and 20.7 mg/g of Se(VI) and 24.2 mg/g and 21.7 mg/g of As(V) for eggshell membrane and chicken feathers, respectively.

Sparassis crispa Fr. is an edible mushroom recently cultivable in Japan. It contains a remarkably high content of 6-branched 1,3-beta-D-glucan showing antitumor activity. Using ion-exchange chromatography, a purified beta-glucan preparation, SCG, was prepared. In this study, we examined the hematopoietic response by SCG in cyclophosphamide (CY)-induced leukopenic mice. SCG enhanced the hematopoietic response in CY induced leukopenic mice by intraperitoneal routes over a wide range of concentrations. SCG enhanced the hematopoietic response in CY-treated mice by prior or post administration. Analyzing the leukocyte population by flow cytometry, monocytes and granulocytes in the peritoneal cavity, liver, spleen and bone marrow (BM) recovered faster than in the control group. The ratio of natural killer cells and gammadelta T cells in the liver, spleen and peritoneal cavity was also increased. In contrast, CD4+ CD8+ cells in the thymus were temporarily significantly decreased by the administration of SCG. Interleukin-6 (IL-6) production of CY+SCG-treated peritoneal exdated cells (PECs), spleen cells and bone marrow cells (BMCs) were higher than that of the CY-treated group. By in vitro culture of CY-treated PEC and spleen cells, IL-6 production was enhanced by the addition of SCG. These facts suggested the possibility that IL-6 might be a key cytokine for the enhanced hematopoietic response by SCG.

Enhancement of n-channel metal–oxide–semiconductor field-effect transistor (nMOSFET) performance with a carbon-doped source/drain (Si:C-S/D) was approached analytically for the first time. Si:C-S/D was formed by molecular carbon (C7H x ) ion implantation and laser annealing. C7H x implantation forms a smooth interface between Si:C layers and Si substrates, and laser annealing also achieves a high carbon concentration of substitution. The channel strain with Si:C-S/D was successfully measured by UV Raman spectroscopy using a particular test pattern. The thick Si:C-S/D layer and the high carbon concentration of substitution produce a large strain at the channel region. It was confirmed that the performance of nMOSFETs is effectively improved by strained Si:C-S/D. These analytical approaches are quite valuable for promoting the development of strained nMOSFETs with Si:C-S/D.