Introduction:Immune-checkpoint inhibitors are effective in various advanced cancers. Type 1 diabetes mellitus induced by them (ICI-T1DM) is a serious complication requiring prompt insulin treatment, but the immunological mechanism behind it is unclear.Methods:We examined amino acid polymorphisms in human histocompatibility leukocyte antigen (HLA) molecules and investigated proinsulin epitope binding affinities to HLA molecules.Results and Discussion:Twelve patients with ICI-T1DM and 35 patients in a control group without ICI-T1DM were enrolled in the study. Allele and haplotype frequencies of HLA-DRB1*04:05, DQB1*04:01, and most importantly DPB1*05:01 were significantly increased in patients with ICI-T1DM. In addition, novel amino acid polymorphisms in HLA-DR (4 polymorphisms), in DQ (12 polymorphisms), and in DP molecules (9 polymorphisms) were identified. These amino acid polymorphisms might be associated with the development of ICI-T1DM. Moreover, novel human proinsulin epitope clusters in insulin A and B chains were discovered in silico and in vitro peptide binding assays to HLA-DP5. In conclusion, significant amino acid polymorphisms in HLA-class II molecules, and conformational alterations in the peptide-binding groove of the HLA-DP molecules were considered likely to influence the immunogenicity of proinsulin epitopes in ICI-T1DM. These amino acid polymorphisms and HLA-DP5 may be predictive genetic factors for ICI-T1DM.

Background: Research has revealed the crucial roles of inflammasomes in various central nervous system disorders. However, the role of inflammasomes in secondary damage following spinal cord injury (SCI) remains incompletely understood. Methods: Here, we investigated the role of apoptosis-associated speck-like protein (ASC), an adaptor protein for inflammasome formation, after contusion SCI in ASC homozygous knockout (ASC(-/-)) mice. Contusion SCI was induced using a force of 60 kdyn, and recovery of open-field locomotor performance was evaluated using the nine-point Basso Mouse Scale (BMS). Bone marrow transplantation (BMT) was performed to create mice chimeric for ASC expression in bone marrow cells. Results: Western blot analysis revealed that protein expression of NLRP3, ASC, Caspase-1, and IL-beta were increased in injured spinal cords compared with sham-control spinal cords at 1 day post injury (dpi). Double immunostaining showed that ASC expression was co-localized to cellular constituents of the spinal cord, including NeuN(+) neurons, CD11b(+) microglia/macrophages, GFAP(+) astrocytes, and MOG(+) oligodendrocytes. ASC(-/-) mice had significantly better locomotor function assessed by BMS than wildtype (WT) mice. ASC(-/-) mice also had significantly reduced levels of Nlrp3, Casp1, IL1b, Il-6, Tnfa, Cxcl1, and Ly6g mRNA compared with WT mice. BMT (WT -> ASC(-/-)) mice had significantly better BMS scores than BMT (WT -> WT) mice. BMT (ASC(-/-)-> WT) mice also had significantly better BMS scores than BMT (WT -> WT) mice. However, the statistical significance was limited to time points between 7 and 21 dpi. Conclusions: These results suggest that ASC-dependent inflammasome formation, especially in resident cells of the spinal cord, plays a pivotal role in the progression of secondary damage following SCI. (C) 2020 The Japanese Orthopaedic Association. Published by Elsevier B.V. All rights reserved.

This paper aims to detect compositional features from room images and present appropriate compositions for attractive photographic assistance. In this paper, we define the boundaries between walls and floor, the boundaries between walls and ceiling as compositional feature lines and vanishing points as compositional feature points. The proposed method was able to properly detect the composition of 27 images out of 36 room images. Especially for the two vanishing point images, the detection was successful in more than 80% of the images. Vanishing points were detected in all the room images with one and two vanishing points. We proposed a method that can easily take attractive indoor images by presenting an appropriate composition by detecting the compositional features. With this composition and features, a photography support system would be promoted to take effective room images easily. As a feature works, this method will be optimized to real time and interactive support for shooting seen.

Acetaminophen (APAP) overdose is a common cause of drug-induced acute liver failure. Although hepatocyte cell death is considered to be the critical event in APAP-induced hepatotoxicity, the underlying mechanism remains unclear. Ferroptosis is a newly discovered type of cell death that is caused by a loss of cellular redox homeostasis. As glutathione (GSH) depletion triggers APAP-induced hepatotoxicity, we investigated the role of ferroptosis in a murine model of APAP-induced acute liver failure. APAP-induced hepatotoxicity (evaluated in terms of ALT, AST, and the histopathological score), lipid peroxidation (4-HNE and MDA), and upregulation of the ferroptosis maker PTGS2 mRNA were markedly prevented by the ferroptosis-specific inhibitor ferrostatin-1 (Fer-1). Fer-1 treatment also completely prevented mortality induced by high-dose APAP. Similarly, APAP-induced hepatotoxicity and lipid peroxidation were prevented by the iron chelator deferoxamine. Using mass spectrometry, we found that lipid peroxides derived from n-6 fatty acids, mainly arachidonic acid, were elevated by APAP, and that auto-oxidation is the predominant mechanism of APAP-derived lipid oxidation. APAP-induced hepatotoxicity was also prevented by genetic inhibition of acyl-CoA synthetase long-chain family member 4 or α-tocopherol supplementation. We found that ferroptosis is responsible for APAP-induced hepatocyte cell death. Our findings provide new insights into the mechanism of APAP-induced hepatotoxicity and suggest that ferroptosis is a potential therapeutic target for APAP-induced acute liver failure.

Pyroptosis is a form of regulated cell death that is characterized by gasdermin processing and increased membrane permeability. Caspase-1 and caspase-11 have been considered to be essential for gasdermin D processing associated with inflammasome activation. In the present study, we found that NLRP3 inflammasome activation induces delayed necrotic cell death via ASC in caspase-1/11-deficient macrophages. Furthermore, ASC-mediated caspase-8 activation and subsequent gasdermin E processing are necessary for caspase-1-independent necrotic cell death. We define this necrotic cell death as incomplete pyroptosis because IL-1β release, a key feature of pyroptosis, is absent, whereas IL-1α release is induced. Notably, unprocessed pro-IL-1β forms a molecular complex to be retained inside pyroptotic cells. Moreover, incomplete pyroptosis accompanied by IL-1α release is observed under the pharmacological inhibition of caspase-1 with VX765. These findings suggest that caspase-1 inhibition during NLRP3 inflammasome activation modulates forms of cell death and permits the release of IL-1α from dying cells.

Introduction: Kawasaki disease (KD) is a systemic febrile syndrome which causes coronary arteritis. Candida albicans water-soluble fraction (CAWS) is a mannoprotein-β-glucan complex obtained from the culture supernatant of Candida albicans , and frequently used as a murine KD model, because administration ofCAWS induces coronary arteritis. NLRP3 inflammasome is a large multiprotein complex (NLRP3, ASC and caspase-1), and regulates the release of the potent inflammatory cytokine IL-1β. We hypothesized that NLRP3 inflammasome-mediated IL-1β had a pivotal role in the pathogenesis of CAWS-induced vasculitis. Methods and Results: In vivo : The protein level of IL-1β in the hearts was increased by CAWS administration. Activation of caspase-1 was detected around the coronary arteries by FLICA assay. Histological study showed that incidence and severity of CAWS-induced vasculitis, macrophage infiltration, and fibrosis were suppressed in NLRP3-, ASC- and IL-1β-deficient mice (Fig. A). In vitro : Bone marrow-derived dendritic cells (BMDCs) produced IL-1β by CAWS stimulation, which was suppressed by the anti-Dectin-2 antibody or caspase-1 inhibitor. The CAWS-induced IL-1β production was also suppressed in NLRP3-, ASC-, and caspase-1-deficient BMDCs (Fig. B). CAWS induced mitochondrial reactive oxygen species (mtROS) production, and Mito-TEMPO, a mtROS inhibitor, suppressed CAWS-induced caspase-1 activation (Fig. C) and IL-β production. In addition, CAWS-induced mtROS production was suppressed by Syk or JNK inhibitor. Furthermore, Syk or JNK inhibitor suppressed CAWS-induced NF-κB activation and subsequent IL-1β production. Conclusion: These findings demonstrated that NLRP3 inflammasome activation through Dectin-2/Syk/JNK-mediated mtROS plays a pivotal role in CAWS-induced vasculitis. CAWS also induced priming signal through Dectin-2/Syk/JNK/NF-κB pathway. Our finding indicates NLRP3 inflammasome as a new therapeutic target of KD.

Background: Platelets are critical mediators of vascular homeostasis and thrombosis, and also contribute to the development of inflammation. NLRP3 inflammasome is a cytosolic multi-protein complex that consists of NLRP3, ASC and caspase-1, and regulates IL-1 beta-mediated inflammation. Method and Results: Using two mouse models of thrombosis (i.e., occlusion of the middle cerebral artery and inferior vena cava), we found that thrombus formation was significantly enhanced in ASC-deficient (ASC(-/-)) mice, compared to that in wild-type (WT) and IL-1 beta(-/-) mice. ASC deficiency had no effects on blood coagulation parameters (i.e., prothrombin time [PT] and activated partial thromboplastin time [APTT]). Platelets from WT mice express ASC, but neither NLRP3 nor caspase-1. ASC deficiency significantly enhanced the expression of P-selectin and GPIIb/IIIa in response to a GPVI agonist (collagen-related peptide [CRP]), but not to thrombin, in platelets. CRP induced ASC speck formation in WT platelets. ASC deficiency also enhanced cytosolic Ca2+ elevation and phosphorylation of ERK1/2 and Akt in platelets. Conclusion: Our results demonstrate that ASC negatively regulates GPVI signaling in platelets and enhances thrombus formation, independent of NLRP3 inflammasome and IL-1 beta, and provide novel insights into the link between inflammation and thrombosis. (C) 2020 Elsevier Inc. All rights reserved.

Hepatic ischemia-reperfusion (I/R) injury is a major problem in liver transplantation (LT). Although hepatocyte cell death is the initial event in hepatic I/R injury, the underlying mechanism remains unclear. In the present study, we retrospectively analyzed the clinical data of 202 pediatric living donor LT and found that a high serum ferritin level, a marker of iron overload, of the donor is an independent risk factor for liver damage after LT. Since ferroptosis has been recently discovered as an iron-dependent cell death that is triggered by a loss of cellular redox homeostasis, we investigated the role of ferroptosis in a murine model of hepatic I/R injury, and found that liver damage, lipid peroxidation, and upregulation of the ferroptosis marker Ptgs2 were induced by I/R, and all of these manifestations were markedly prevented by the ferroptosis-specific inhibitor ferrostatin-1 (Fer-1) or α-tocopherol. Fer-1 also inhibited hepatic I/R-induced inflammatory responses. Furthermore, hepatic I/R injury was attenuated by iron chelation by deferoxamine and exacerbated by iron overload with a high iron diet. These findings demonstrate that iron overload is a novel risk factor for hepatic I/R injury in LT, and ferroptosis contributes to the pathogenesis of hepatic I/R injury.

Intestinal ischemia/reperfusion (I/R) injury is a life-threatening complication that leads to inflammation and remote organ damage. The NLRP3 inflammasome regulates the caspase-1-dependent release of IL-1β, an early mediator of inflammation after I/R injury. In this study, we investigated the role of the NLRP3 inflammasome in mice with intestinal I/R injury. Deficiency of NLRP3, ASC, caspase-1/11, or IL-1β prolonged survival after intestinal I/R injury, but neither NLRP3 nor caspase-1/11 deficiency affected intestinal inflammation. Intestinal I/R injury caused acute lung injury (ALI) characterized by inflammation, reactive oxygen species generation, and vascular permeability, which was markedly improved by NLRP3 deficiency. Bone marrow chimeric experiments showed that NLRP3 in non-bone marrow-derived cells was the main contributor to development of intestinal I/R-induced ALI. The NLRP3 inflammasome in lung vascular endothelial cells is thought to be important to lung vascular permeability. Using mass spectrometry, we identified intestinal I/R-derived lipid mediators that enhanced NLRP3 inflammasome activation in lung vascular endothelial cells. Finally, we confirmed that serum levels of these lipid mediators were elevated in patients with intestinal ischemia. To our knowledge, these findings provide new insights into the mechanism underlying intestinal I/R-induced ALI and suggest that endothelial NLRP3 inflammasome-driven IL-1β is a novel potential target for treating and preventing this disorder.

Although the intimate linkage between hypoxia and inflammation is well known, the mechanism underlying this linkage has not been fully understood. Nucleotide‐binding oligomerization domain‐like receptor (NLR) family pyrin domain containing 3 (NLRP3) inflammasome is an intracellular multiprotein complex that regulates interleukin‐1β (IL‐1β) secretion and pyroptosis, and is implicated in the pathogenesis of sterile inflammatory diseases. Here, we investigated the regulatory mechanism of NLRP3 inflammasome activation in response to hypoxia in macrophages. Severe hypoxia (0.1% O 2 ) induced the processing of pro‐IL‐1β, pro‐caspase‐1, and gasdermin D, as well as the release of IL‐1β and lactate dehydrogenase in lipopolysaccharide (LPS)‐primed murine macrophages, indicating that hypoxia induces NLRP3 inflammasome‐driven inflammation and pyroptosis. NLRP3 deficiency and a specific caspase‐1 blockade inhibited hypoxia‐induced IL‐1β release. Hypoxia‐induced IL‐1β release and cell death were augmented under glucose deprivation, and an addition of glucose in the media negatively regulated hypoxia‐induced IL‐1β release. Under hypoxia and glucose deprivation, hypoxia‐induced glycolysis was not driven and subsequently, the intracellular adenosine triphosphates (ATPs) were depleted. Atomic absorption spectrometry analysis showed a reduction of intracellular K + concentrations, indicating the K + efflux occurring under hypoxia and glucose deprivation. Furthermore, hypoxia and glucose deprivation‐induced IL‐1β release was significantly prevented by inhibition of K + efflux and K ATP channel blockers. In vivo experiments further revealed that IL‐1β production was increased in LPS‐primed mice exposed to hypoxia (9.5% O 2 ), which was prevented by a deficiency of NLRP3, an apoptosis‐associated speck‐like protein containing a caspase recruitment domain, and caspase‐1. Our results demonstrate that NLRP3 inflammasome can sense intracellular energy crisis as a danger signal induced by hypoxia and glucose deprivation, and provide new insights into the mechanism underlying hypoxia‐induced inflammation.

Kawasaki disease (KD) is a systemic febrile syndrome during childhood that is characterized by coronary arteritis. The etiopathogenesis of KD remains to be elucidated. NLRP3 inflammasome is a large multiprotein complex that plays a key role in IL-1β-driven sterile inflammatory diseases. In the present study, we investigated the role of NLRP3 inflammasome in a murine model of KD induced by Candida albicans water-soluble fraction (CAWS) and found that NLRP3 inflammasome is required for the development of CAWS-induced vasculitis. CAWS administration induced IL-1β production, caspase-1 activation, leukocyte infiltration, and fibrotic changes in the aortic root and coronary arteries, which were significantly inhibited by a deficiency of IL-1β, NLRP3, and ASC. In vitro experiments showed that among cardiac resident cells, macrophages, but not endothelial cells or fibroblasts, expressed Dectin-2, but did not produce IL-1β in response to CAWS. In contrast, CAWS induced caspase-1 activation and IL-1β production in bone marrow-derived dendritic cells (BMDCs), which were inhibited by a specific caspase-1 inhibitor and a deficiency of NLRP3, ASC, and caspase-1. CAWS induced NLRP3 and pro-IL-1β expression through a Dectin-2/Syk/JNK/NF-κB pathway, and caspase-1 activation and cleavage of pro-IL-1β through Dectin-2/Syk/JNK-mediated mitochondrial ROS generation, indicating that CAWS induces the priming and activation of NLRP3 inflammasome in BMDCs. These findings provide new insights into the pathogenesis of KD vasculitis, and suggest that NLRP3 inflammasome may be a potential therapeutic target for KD.

Long-term peritoneal dialysis (PD) therapy leads to peritoneal inflammation and fibrosis. However, the mechanism underlying PD-related peritoneal inflammation and fibrosis remains unclear. NLRP3 inflammasome regulates the caspase-1-dependent release of interleukin-1β and mediates inflammation in various diseases. Here, we investigated the role of NLRP3 inflammasome in a murine model of PD-related peritoneal fibrosis induced by methylglyoxal (MGO). Inflammasome-related proteins were upregulated in the peritoneum of MGO-treated mice. MGO induced parietal and visceral peritoneal fibrosis in wild-type mice, which was significantly reduced in mice deficient in NLRP3, ASC, and interleukin-1β (IL-1β). ASC deficiency reduced the expression of inflammatory cytokines and fibrotic factors, and the infiltration of macrophages. However, myeloid cell-specific ASC deficiency failed to inhibit MGO-induced peritoneal fibrosis. MGO caused hemorrhagic ascites, fibrin deposition, and plasminogen activator inhibitor-1 upregulation, but all of these manifestations were inhibited by ASC deficiency. Furthermore, in vitro experiments showed that MGO induced cell death via the generation of reactive oxygen species in vascular endothelial cells, which was inhibited by ASC deficiency. Our results showed that endothelial NLRP3 inflammasome contributes to PD-related peritoneal inflammation and fibrosis, and provide new insights into the mechanisms underlying the pathogenesis of this disorder.

Background: Intestinal ischemia/reperfusion (I/R) injury is a life-threatening complication that leads to inflammation and remote organ damage. However, the underlying mechanism is not yet fully understood. Toll-like receptor 5 (TLR5) is highly expressed in mucosa and recognizes flagellin, the main component of the bacterial flagella. Here, we investigated the role of TLR5 in inflammation and tissue damage after intestinal I/R injury using TLR5-deficient mice. Methods and results: Intestinal levels of TLR5 mRNA and flagellin protein were elevated in wild-type mice subjected to intestinal I/R. Although TLR5 deficiency had no effect on intestinal flagellin levels, it significantly attenuated intestinal injury and inflammatory responses after intestinal I/R. TLR5 deficiency also markedly improved survival in mice after intestinal I/R injury. In wild-type mice, intestinal I/R injury induced remote organ damage, particularly in the lung, which was attenuated by TLR5 deficiency. Furthermore, TLR5 deficiency prevented lung inflammatory responses and vascular permeability after intestinal I/R injury. Conclusion: These findings demonstrate a novel role of TLR5 and provide new insights into the mechanism underlying inflammation and tissue damage after intestinal I/R injury. (C) 2019 Elsevier Inc. All rights reserved.

Inflammation plays a pivotal role in the pathophysiology of gastric aspiration-induced acute lung injury (ALI). However, its mechanism remains unclear. In this study, we investigated the role of NLRP3 inflammasome-driven IL-1β production in a mouse model of acid aspiration-induced inflammation and ALI. Acid aspiration-induced inflammatory responses and ALI in wild-type mice were significantly attenuated in IL-1β-/- mice, but not NLRP3-/- mice. In vitro experiments revealed that severe acidic stress (pH 1.75) induced the processing of pro-IL-1β into its 18-kDa mature form (p18-IL-1β), which was different from the caspase-1-processed 17-kDa form (p17-IL-1β), in human THP-1 macrophages and primary murine macrophages. Deficiency of NLRP3 and caspase-1 had no effect on acidic stress-produced IL-1β. The production of IL-1β by severe acidic stress was prevented by inhibitors of serine proteases [4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride], but not of cysteine proteases (E-64), cathepsin G, or inflammasome. The cathepsin D inhibitor pepstatin A inhibited IL-1β production induced by mild acidic stress (pH 6.2) or lactic acid, but not severe acidic stress. Using mass spectrometry and processing-site mutants of pro-IL-1β, we identified D109 as a novel cleavage site of pro-IL-1β in response to severe acidic stress and calculated the theoretical molecular mass of the mature form to be 18.2 kDa. The bioactivity of acidic stress-produced IL-1β was confirmed by its ability to promote p38 phosphorylation and chemokine upregulation in alveolar epithelial cells. These findings demonstrate a novel mechanism of acid-induced IL-1β production and inflammation independent of NLRP3 inflammasome and provide new insights into the therapeutic strategies for aspiration pneumonitis and ALI.

Background Kawasaki disease (KD), which is a common paediatric heart disease, is characterised by coronary vasculitis and subsequently aneurysm formation. Although the administration of intravenous immunoglobulin (IVIG) is effective for reducing aneurysm formation, approximately 10%–20% of patients are resistant to this therapy. Therefore, additional therapeutic approaches for treating the IVIG-resistant patients need to be developed. Candida albicans water-soluble fraction (CAWS)-induced vasculitis on coronary arteries and root of aorta is a frequently used murine model of KD. It has been considered that C-type lectin receptor Dectin-2 recognises CAWS. Recent studies showed CAWS-resistant strains of mice have higher serum IL-10 levels, which suggested that IL-10 might negatively regulate the development of CAWS-induced vasculitis. Objectives The aim of the study is to investigate the therapeutic effect of IL-10 in CAWS-induced vasculitis and elucidate the underlying pathogenesis of KD. Methods To induce the expression of IL-10 in vivo, Adeno-associated virus (AAV) vectors encoding IL-10 were injected into DBA/2 mice. After the induction of IL-10, the mice were treated intraperitoneally with CAWS to induce vasculitis. Cardiac functions by echocardiography, inflammation and fibrosis by histological analyses, gene expression of inflammatory cytokines and fibrosis-related factors in the heart, and infiltrating cells by flow cytometry were assessed to evaluate the effects of IL-10. For in vitro study, bone marrow-derived macrophages (BMDM) were stimulated with CAWS in presence or absence of IL-10. TNF-α and IL-6 produced by the BMDM and Dectin-2 expressions on the BMDM were assessed. Results AAV-mediated induction of IL-10 significantly attenuated CAWS-induced cardiac functions (%FS and LVEDD). Histological analyses revealed that IL-10 markedly attenuated the vascular inflammation and fibrosis in the aortic root and coronary artery. Accordingly, increased gene expressions of inflammatory cytokines or fibrosis-related factors in the heart of CAWS-treated mice were significantly reduced by IL-10. The predominant infiltrating inflammatory cells in vascular walls were Dectin-2+CD11b+ macrophages, and they were also decreased by IL-10. Furthermore, we showed GM-CSF induced Dectin-2 expression on BMDM, and the GM-CSF-treated BMDM produced TNF-α and IL-6 upon CAWS-stimulation. IL-10 had no effect on the Dectin-2 expression but significantly inhibited the production of the cytokines. Finally, the AAV-mediated induction of IL-10 prevented the expression of TNF-α and IL-6 in the heart of the mice treated with CAWS for 24 hours (at the early phase), but not GM-CSF and Dectin-2. These results suggest that GM-CSF mediates CAWS-induced vasculitis via Dectin-2 upregulation and IL-10 inhibits the downstream of GM-CSF and Dectin-2 signalling. Conclusions Our study has shown that IL-10 may have therapeutic application in the prevention of coronary vasculitis and aneurysm formation, and provided new insights into the mechanism underlying the pathogenesis of KD. Disclosure of Interest None declared

Objective— Inflammation provoked by the imbalance of fatty acid composition, such as excess saturated fatty acids (SFAs), is implicated in the development of metabolic diseases. Recent investigations suggest the possible role of the NLRP3 (nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain containing 3) inflammasome, which regulates IL-1β (interleukin 1β) release and leads to inflammation, in this process. Therefore, we investigated the underlying mechanism by which SFAs trigger NLRP3 inflammasome activation. Approach and Results— The treatment with SFAs, such as palmitic acid and stearic acid, promoted IL-1β release in murine primary macrophages while treatment with oleic acid inhibited SFA-induced IL-1β release in a dose-dependent manner. Analyses using polarized light microscopy revealed that intracellular crystallization was provoked in SFA-treated macrophages. As well as IL-1β release, the intracellular crystallization and lysosomal dysfunction were inhibited in the presence of oleic acid. These results suggest that SFAs activate NLRP3 inflammasome through intracellular crystallization. Indeed, SFA-derived crystals activated NLRP3 inflammasome and subsequent IL-1β release via lysosomal dysfunction. Excess SFAs also induced crystallization and IL-1β release in vivo. Furthermore, SFA-derived crystals provoked acute inflammation, which was impaired in IL-1β–deficient mice. Conclusions— These findings demonstrate that excess SFAs cause intracellular crystallization and subsequent lysosomal dysfunction, leading to the activation of the NLRP3 inflammasome, and provide novel insights into the pathogenesis of metabolic diseases.

Background: Inflammatory Bowel Disease (IBD) is most often diagnosed in the second through fourth decades of life which, for female patients, coincides with their childbearing years.Pregnant women with IBD are at higher risk for developing gestational diabetes, preterm delivery, and cesarean delivery.Disease monitoring is crucial in this population to prevent adverse events -yet imaging and instrumental procedures are often contraindicated.Fecal lactoferrin and calprotectin have been shown to have high sensitivity and specificity for detecting active disease in IBD patients.However, no studies currently exist for validating the use of the clinical cut-offs for defining active IBD in pregnant women.In this study, the aim is to determine the baseline levels of lactoferrin and calprotectin during pregnancy of healthy women.Methods: Stool samples were collected over 3 trimesters (T=1, 2 and 3) from 47 healthy pregnant women with a mean age of 26 years.The intestinal inflammation biomarkers, fecal lactoferrin and calprotectin, as well as hemoglobin, which was used as an indicator of intestinal bleeding, were measured by enzyme-immunoassay (EIA) and results are reported as µg/g feces.A normal level was defined by cut-offs < 7.25 µg/g for lactoferrin; <50 µg/g for calprotectin; and < 15 µg/g for Hemoglobin.Results: A total of 75 stool samples were collected over three trimesters of pregnancy, 15 at T=1, 27 at T=2 and 33 at T=3 from 47 patients.Mean/median levels for lactoferrin, calprotectin and hemoglobin for all trimesters were 2.6/0.5, 19.7/0.0 and 0.7/0.0,respectively.There were 7 patients (15% including 8 stool samples) that had elevated calprotectin (74.6, 226.9, 53.8, 50.8, 87.6, 300.1, 83.5, 127.0) of whom 2 also had increased lactoferrin, one at T= 1 and the other at T=3 (27.6 and 86.0).Lactoferrin was slightly elevated (7.5 and 7.3) in two additional patients for a total of 4 patients (9%).There was a single patient with increased hemoglobin (36.2) at T= 1 who had normal levels for T=2 and 3.For patients who had more than a single trimester sampled, there were no increasing or decreasing trends identified for the fecal biomarkers.The two patients who had both markedly elevated lactoferrin and calprotectin suffered pregnancies complicated by gestational hypertension, maternal obesity, and type II diabetes.

Kawasaki disease (KD), which is the leading cause of pediatric heart disease, is characterized by coronary vasculitis and subsequent aneurysm formation. Although intravenous immunoglobulin therapy is effective for reducing aneurysm formation, a certain number of patients are resistant to this therapy. Because interleukin-10 (IL-10) was identified as a negative regulator of cardiac inflammation in a murine model of KD induced by Candida albicans water-soluble fraction (CAWS), we investigated the effect of IL-10 supplementation in CAWS-induced vasculitis. Mice were injected intramuscularly with adeno-associated virus (AAV) vector encoding IL-10, then treated with CAWS. The induction of AAV-mediated IL-10 (AAV-IL-10) significantly attenuated the vascular inflammation and fibrosis in the aortic root and coronary artery, resulting in the improvement of cardiac dysfunction and lethality. The predominant infiltrating inflammatory cells in the vascular walls were Dectin-2+CD11b+ macrophages. In vitro experiments revealed that granulocyte/macrophage colony-stimulating factor (GM-CSF) induced Dectin-2 expression in bone marrow-derived macrophages and enhanced the CAWS-induced production of tumor necrosis factor-α (TNF-α) and IL-6. IL-10 had no effect on the Dectin-2 expression but significantly inhibited the production of cytokines. IL-10 also inhibited CAWS-induced phosphorylation of ERK1/2, but not Syk. Furthermore, the induction of AAV-IL-10 prevented the expression of TNF-α and IL-6, but not GM-CSF and Dectin-2 at the early phase of CAWS-induced vasculitis. These findings demonstrate that AAV-IL-10 may have therapeutic application in the prevention of coronary vasculitis and aneurysm formation, and provide new insights into the mechanism underlying the pathogenesis of KD.