Sachiko Nakajima

Extracellular ATP mediates mast cell-dependent intestinal inflammation through P2X7 purinoceptors

Mast cells are known effector cells in allergic and inflammatory diseases, but their precise roles in intestinal inflammation remain unknown. Here we show that activation of mast cells in intestinal inflammation is mediated by ATP-reactive P2X7 purinoceptors. We find an increase in the numbers of mast cells expressing P2X7 purinoceptors in the colons of mice with colitis and of patients with Crohns disease. Treatment of mice with a P2X7 purinoceptor-specific antibody inhibits mast cell activation and subsequent intestinal inflammation. Similarly, intestinal inflammation is ameliorated in mast cell-deficient KitW-sh/W-sh mice, and reconstitution with wild-type, but not P2x7−/− mast cells results in susceptibility to inflammation. ATP-P2X7 purinoceptor-mediated activation of mast cells not only induces inflammatory cytokines, but also chemokines and leukotrienes, to recruit neutrophils and subsequently exacerbate intestinal inflammation. These findings reveal the role of P2X7 purinoceptor-mediated mast cell activation in both the initiation and exacerbation of intestinal inflammation.

Serum leucine-rich alpha-2 glycoprotein is a disease activity biomarker in ulcerative colitis

Background: Reliable biomarkers for monitoring disease activity have not been clinically established in ulcerative colitis (UC). This study aimed to investigate whether levels of serum leucine‐rich alpha‐2 glycoprotein (LRG), identified recently as a potential disease activity marker in Crohns disease and rheumatoid arthritis, correlate with disease activity in UC. Methods: Serum LRG concentrations were determined by enzyme‐linked immunosorbent assay (ELISA) in patients with UC and healthy controls (HC) and were evaluated for correlation with disease activity. Expression of LRG in inflamed colonic tissues from patients with UC was analyzed by western blotting and immunohistochemistry. Interleukin (IL)‐6‐independent induction of LRG was investigated using IL‐6‐deficient mice by lipopolysaccharide (LPS)‐mediated acute inflammation and dextran sodium sulfate (DSS)‐induced colitis. Results: Serum LRG concentrations were significantly elevated in active UC patients compared with patients in remission (P < 0.0001) and HC (P < 0.0001) and were correlated with disease activity in UC better than C‐reactive protein (CRP). Expression of LRG was increased in inflamed colonic tissues in UC. Tumor necrosis factor alpha (TNF‐&agr;), IL‐6, and IL‐22, serum levels of which were elevated in patients with active UC, could induce LRG expression in COLO205 cells. Serum LRG levels were increased in IL‐6‐deficient mice with LPS‐mediated acute inflammation and DSS‐induced colitis. Conclusions: Serum LRG concentrations correlate well with disease activity in UC. LRG induction is robust in inflamed colons and is likely to involve an IL‐6‐independent pathway. Serum LRG is thus a novel serum biomarker for monitoring disease activity in UC and is a promising surrogate for CRP. (Inflamm Bowel Dis 2012;)

IgG Oligosaccharide Alterations Are a Novel Diagnostic Marker for Disease Activity and the Clinical Course of Inflammatory Bowel Disease

BACKGROUND AND AIMS:Patients with inflammatory bowel disease (IBD) share several immunologic similarities with rheumatoid arthritis (RA). Patients with RA have significantly increased levels of serum agalactosyl immunoglobulin G (IgG). Our aim was to investigate the clinical significance of analyzing the oligosaccharide structure of serum IgG in patients with IBD.METHODS:Serum IgG oligosaccharide structures were analyzed using high-performance liquid chromatography in 60 patients with Crohns disease (CD), 58 patients with ulcerative colitis (UC), 27 healthy volunteers (HV), and 15 disease controls (DC). The activity and mRNA level of beta-1,4-galactosyltransferase (Beta4GalT) in antibody-secreting cells were investigated in these subjects.RESULTS:The agalactosyl fraction of the fucosylated IgG oligosaccharides (G0F/G2F) in CD and UC was significantly greater than that in HV and DC (P < 0.001). The percentage of subjects with a high G0F/G2F in CD, UC, HV, and DC was 72%, 33%, 0%, and 0%, respectively. G0F/G2F, which is significantly correlated with disease severity in both CD and UC, had higher sensitivity to diagnose IBD compared with anti-Saccharomyces cerevisiae antibody. Moreover, G0F/G2F was significantly correlated with the prognosis of UC patients: patients with a high G0F/G2F did not maintain long-term remission. The activity and mRNA level of Beta4GalT were significantly elevated in UC but not in CD.CONCLUSIONS:G0F/G2F is a potentially effective diagnostic marker of disease activity in both CD and UC, and of the clinical course in UC. A pathophysiologic difference between CD and UC was also demonstrated.

Altered Oligosaccharide Structures Reduce Colitis Induction in Mice Defective in β-1,4-Galactosyltransferase

BACKGROUND & AIMS Oligosaccharide modifications induce various functional changes in immune cells. The galactose-deficient fraction of fucosylated IgG oligosaccharides is increased, whereas that of β-1,4-galactosyltransferase I (B4GalTI) is reduced, in patients with Crohns disease. We investigated the role of oligosaccharide modification in the pathophysiology of colitis using B4galt1-deficient mice. METHODS Colitis severity was compared between B4galt1(+/-) and B4galt1(+/+) mice. B cells isolated from B4galt1(+/-) and B4galt1(+/+) mice were adoptively transferred to recombination activating gene 2(-/-) mice, in which colitis was induced by administration of CD4(+)CD62L(+) T cells. Cell-surface glycan profiles were determined by lectin microarray analysis. Cytokine production was determined in a coculture of various types of cells isolated from either B4galt1(+/-) or B4galt1(+/+) mice. RESULTS Colitis induction by dextran sodium sulfate or trinitrobenzene sulfonic acid was significantly reduced in B4galt1(+/-) mice, which had galactose deficiency in IgG oligosaccharides (similar to patients with Crohns disease) compared with B4galt1(+/+) mice. Amelioration of colitis was associated with increased production of interleukin-10 by macrophages in B4galt1(+/-) mice. Colitis induction in recombination activating gene 2(-/-) mice by administration of CD4(+)CD62L(+) T cells was reduced by cotransfer of B cells isolated from B4galt1(+/-), but not from B4galt1(+/+) mice. Lectin microarray analysis revealed increased expression of polylactosamines on B4galt1(+/-) B cells and macrophages, compared with B4galt1(+/+) cells. The production of interleukin-10 from macrophages was induced via their direct interaction with B4galt1(+/-) B cells. CONCLUSIONS Altered oligosaccharide structures on immune cells modulate mucosal inflammation. Oligosaccharides in immune cells might be a therapeutic target for inflammatory bowel diseases.

Functional analysis of agalactosyl IgG in inflammatory bowel disease patients

Background: Agalactosyl immunoglobulin (Ig) G is increased in inflammatory bowel disease (IBD) similarly to rheumatoid arthritis (RA). The lectin complement pathway is shown to be activated through association of agalactosyl IgG with mannan‐binding lectin (MBL) in RA. Functional changes of IgG agalactosylation in IBD, however, have not yet been clarified. Methods: The ratio of the agalactosyl/non‐agalactosyl fraction in fucosylated IgG oligosaccharides (G0F/G2F) and serum MBL levels were analyzed in 59 patients with Crohns disease (CD), 64 ulcerative colitis (UC), and 39 healthy volunteers (HV). The MBL levels associated with serum IgG were analyzed by enzyme‐linked immunosorbent assay. MBL expression in the intestinal mucosa was analyzed by immunohistochemistry. Phagocytosis of sheep red blood cells (SRBC) reacted with either an agalactosyl or non‐agalactosyl SRBC‐specific IgG antibody was determined by flow cytometry. Results: The serum MBL levels were not significantly different among CD, UC, or HV. In patients with CD, the serum MBL levels were negatively correlated with the Crohns Disease Activity Index (CDAI). The levels of MBL associated with agalactosyl IgG were not different from those associated with non‐agalactosyl IgG. Immunoreactivity to MBL was less in the inflamed mucosa compared with the noninflamed mucosa. Phagocytic activity of SRBC was significantly higher in the presence of agalactosyl IgG compared to non‐agalactosyl IgG. Conclusions: Agalactosyl IgG oligosaccharides enhanced antibody‐dependent phagocytosis in vitro but did not activate the lectin complement pathway. Oligosaccharide alterations of IgG are not only a marker of IBD but also functionally modulate the immune function of IBD. (Inflamm Bowel Dis 2010;)

Deficiency of N‐acetylgalactosamine in O‐linked oligosaccharides of IgA is a novel biologic marker for Crohn's disease

Background: Ideal biomarkers are required to be developed for the diagnosis and prediction of the treatment of inflammatory bowel disease (IBD). We have reported that alteration of N‐linked oligosaccharides of immunoglobulin (Ig) G is a novel diagnostic marker of IBD. Oligosaccharide alterations of IgA, however, have not been investigated in IBD patients. Methods: N‐ and O‐linked oligosaccharides of serum IgA purified from 32 patients with Crohns disease (CD), 30 patients with ulcerative colitis (UC), and 30 healthy volunteers (HV) were analyzed with high‐performance liquid chromatography and mass spectrometry. Enzymes related to oligosaccharide attachment were investigated. Results: N‐linked oligosaccharides of IgA were not different between IBD and HV. In contrast, the number of N‐acetylgalactosamines per hinge glycopeptide (GalNAc/HP) in the O‐linked oligosaccharides of IgA was significantly decreased in patients with CD compared with UC and HV. GalNAc/HP had high sensitivity and specificity for discriminating between CD and HV based on receiver operating characteristic analysis. Lower GalNAc/HP was associated with more severe disease activity of CD. Changes in GalNAc/HP levels in 6 weeks after treatment with infliximab were associated with the clinical activity of CD at 30 weeks. GalNAc transferase expression of naïve B cells and extent of GalNAc attachment in IgA were significantly decreased by interleukin‐21 in vitro. Conclusions: The number of GalNAc attached in the IgA O‐linked glycans of CD patients was significantly decreased, and strongly correlated with the clinical activity. Alterations of GalNAc attachment in IgA could be useful as a novel diagnostic and prognostic marker of CD. (Inflamm Bowel Dis 2012;)

Upregulation of GRAIL is associated with remission of ulcerative colitis

Abrogating tolerance against unidentified antigens is a critical step in the pathogenesis of ulcerative colitis (UC). T cell anergy, one of the main mechanisms of tolerance, has been shown to be induced by E3 ubiquitin ligases, such as gene related to anergy in lymphocytes (GRAIL), Itch, and c-Cbl in mice. However, it is not well known whether these E3 ligases play roles in human diseases. The pathophysiological role of the E3 ligases in patients with UC was investigated. At first, the expression of GRAIL, Itch, and c-Cbl in human anergic T cells was analyzed by quantitative RT-PCR and Western immunoblotting. Next, the mRNA expression of the E3 ligases was analyzed in peripheral CD4+ T cells of 20 patients with UC and 10 healthy volunteers (HV). mRNA expression was analyzed in patients with active UC before and after treatment with prednisolone and leukocytapheresis. Anergic human CD4+ T cells expressed significantly higher levels of GRAIL, Itch, and c-Cbl than nonanergic cells. GRAIL expression was significantly higher in patients with UC in remission than in patients with active disease and in HV (P < 0.01). The level of GRAIL expression was also significantly increased in patients with active disease whose clinical activity index scores improved after treatment (P < 0.05). There were no significant differences in Itch and c-Cbl expression among patients with active UC, patients with UC in remission, and HV. These data suggest that GRAIL plays an important role in maintaining remission in patients with UC.

Vitamin K deficiency leads to exacerbation of murine dextran sulfate sodium-induced colitis

BackgroundPatients with inflammatory bowel disease (IBD) often exhibit vitamin K deficiency. Vitamin K has been shown to inhibit inflammation via interleukin (IL)-6 suppression. This study aimed to evaluate the effect of vitamin K in a murine model of colitis.MethodsColitis was induced using dextran sulfate sodium (DSS) in mice fed either a vitamin K-deficient (K-def) or a vitamin K-supplemented (K-sup) diet. The clinical and histological severity of colitis was assessed, and levels of cytokine production from the spleen and colonic lamina propria were measured by enzyme-linked immunosorbent assay and quantitative real-time reverse transcription polymerase chain reaction. Cytokine expression levels in CD4+, CD11b+, and CD19+ cells in the presence and absence of vitamin K [menatetrenone (MK-4)] were measured in vitro and apoptosis was determined by caspase 3/7 activity and Annexin V staining.ResultsDSS administration resulted in significantly more severe body weight loss, shorter colon length, and higher histological scores in mice fed a K-def diet than those fed a K-sup diet. IL-6 expression in lamina propria mononuclear cells was significantly higher in the K-def group than in the K-sup group. IL-6 expression was significantly decreased in the presence of MK-4 in CD19+ cells, but not in the CD4+ and CD11b+ subpopulations. Apoptotic cell population in CD19+ cells was increased in the presence of MK-4 in vitro and in vivo.ConclusionsVitamin K exerts a protective effect against DSS colitis; this effect is associated with IL-6 downregulation. Vitamin K could be a potential treatment target for IBD.

Frequency of coexistent carcinoma in sessile serrated adenoma/polyps and traditional serrated adenomas removed by endoscopic resection.

Background and study aims: Sessile serrated adenoma/polyps (SSA/Ps) have a different potential than traditional adenomatous polyps for developing into malignant colorectal cancer. However, little is known about the coexistent cancer rate. Here, we evaluate the frequency of carcinoma in serrated polyps removed by endoscopic resection (ER). Patients and methods: This was a retrospective single-center cohort study of consecutive patients with colorectal polyps who underwent ER from March 2003 to October 2014. We determined the frequency of serrated polyps among all resected colorectal polyps and analyzed the clinicopathological findings as well as the frequency and characteristics of coexistent carcinoma in the serrated polyps resected by ER based on pathology reports. Results: A total of 21,048 polyps from 15,326 patients were identified, including 15,984 traditional adenomatous polyps (75.9 %), 621 SSA/Ps (3.0 %), 136 traditional serrated adenomas (TSAs) (0.6 %), 1,121 hyperplastic polyps (5.3 %), and 3,186 polyps of other types (15.1 %). The clinical and endoscopic findings of SSA/Ps revealed a male predominance (68.6 %), with 61.7 % of the polyps located in the proximal colon. Males accounted for 77.2 % of all patients with TSAs, and 77.2 % of these polyps were located in the distal colon. The mean sizes of the SSA/Ps and TSAs were 8.8 and 10.7 mm, respectively. Among the SSA/Ps, 8 (1.3 %) cases had coexistent carcinoma, and 1 (0.7 %) patient with TSA showed coexistent carcinoma. In the patients with SSA/Ps, female sex and a tumor size ≥ 10 mm were predictive factors for coexistent carcinoma. Conclusions: The frequency of SSA/Ps with carcinoma was lower than that for traditional adenoma. Female sex and tumor size ≥ 10 mm were significant predictive factors for coexistent carcinoma.

M1620 Caldesmon Suppresses Cancer Cell Invasion By Regulating Podosome/Invadopodium Formation

The podosome and invadopodium are dynamic cell-adhesion structures that degrade the extracellular matrix (ECM) and promote cell invasion. We recently reported that the actin-binding protein caldesmon is a pivotal regulator of podosome formation. Here, we analyzed the caldesmons involvement in podosome/invadopodium-mediated invasion by transformed and cancer cells. The ectopic expression of caldesmon reduced the number of podosomes/invadopodia and decreased the ECM degradation activity, resulting in the suppression of cell invasion. Conversely, the depletion of caldesmon facilitated the formation of podosomes/invadopodia and cell invasion. Taken together, our results indicate that caldesmon acts as a potent repressor of cancer cell invasion.