Shinichiro Shinzaki

Indigenous opportunistic bacteria inhabit mammalian gut-associated lymphoid tissues and share a mucosal antibody-mediated symbiosis

The indigenous bacteria create natural cohabitation niches together with mucosal Abs in the gastrointestinal (GI) tract. Here we report that opportunistic bacteria, largely Alcaligenes species, specifically inhabit host Peyers patches (PPs) and isolated lymphoid follicles, with the associated preferential induction of antigen-specific mucosal IgA Abs in the GI tract. Alcaligenes were identified as the dominant bacteria on the interior of PPs from naïve, specific-pathogen-free but not from germ-free mice. Oral transfer of intratissue uncultured Alcaligenes into germ-free mice resulted in the presence of Alcaligenes inside the PPs of recipients. This result was further supported by the induction of antigen-specific Ab-producing cells in the mucosal (e.g., PPs) but not systemic compartment (e.g., spleen). The preferential presence of Alcaligenes inside PPs and the associated induction of intestinal secretory IgA Abs were also observed in both monkeys and humans. Localized mucosal Ab-mediated symbiotic immune responses were supported by Alcaligenes-stimulated CD11c+ dendritic cells (DCs) producing the Ab-enhancing cytokines TGF-β, B-cell-activating factor belonging to the TNF family, and IL-6 in PPs. These CD11c+ DCs did not migrate beyond the draining mesenteric lymph nodes. In the absence of antigen-specific mucosal Abs, the presence of Alcaligenes in PPs was greatly diminished. Thus, indigenous opportunistic bacteria uniquely inhabit PPs, leading to PP-DCs-initiated, local antigen-specific Ab production; this may involve the creation of an optimal symbiotic environment on the interior of the PPs.

iTRAQ-based proteomic identification of leucine-rich α-2 glycoprotein as a novel inflammatory biomarker in autoimmune diseases

Objective To identify a novel serum biomarker of disease activity in inflammatory autoimmune disorders. Methods Sera obtained from rheumatoid arthritis (RA) patients before and after anti-TNF therapy were analysed by iTRAQ (isobaric tags for relative and absolute quantitation) quantitative proteomic analysis and further validated by ELISA. Results Of 326 proteins identified by proteomic analysis, increased serum levels of leucine-rich α-2 glycoprotein (LRG) was identified in RA patients before therapy. Serum LRG concentrations were significantly elevated in RA patients compared with healthy controls and decreased after anti-TNF therapy. Furthermore, serum LRG concentrations correlated with disease activity in RA and Crohns disease (CD). Interestingly, in a subpopulation of patients with active CD and normal C-reactive protein levels, serum LRG concentrations were elevated. Conclusions LRG represents a novel serum biomarker for monitoring disease activity during therapy in autoimmune patients, particularly useful in patients with active disease but normal CRP levels.

Fucosylation is a promising target for cancer diagnosis and therapy.

Oligosaccharides, sequences of carbohydrates conjugated to proteins and lipids, are arguably the most abundant and structurally diverse class of molecules. Fucosylation is one of the most important oligosaccharide modifications involved in cancer and inflammation. Recent advances in glycomics have identified several types of glyco-biomarkers containing fucosylation that are linked to certain types of cancer. Fucosylated alpha-fetoprotein (AFP) is widely used in the diagnosis of hepatocellular carcinoma because it is more specific than alpha-fetoprotein. High levels of fucosylated haptoglobin have also been found in sera of patients with various carcinomas. We have recently established a simple lectin-antibody ELISA to measure fucosylated haptoglobin and to investigate its clinical use. Cellular fucosylation is dependent upon fucosyltransferase activity and the level of its donor substrate, guanosine diphosphate (GDP)-fucose. GDP-mannose-4,6-dehydratase (GMDS) is a key enzyme involved in the synthesis of GDP-fucose. Mutations of GMDS found in colon cancer cells induced a malignant phenotype, leading to rapid growth in athymic mice resistant to natural killer cells. This review describes the role of fucosylated haptoglobin as a cancer biomarker, and discusses the possible biological role of fucosylation in cancer development.

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;)

Clinical application of a lectin-antibody ELISA to measure fucosylated haptoglobin in sera of patients with pancreatic cancer

Abstract Background: Recent advanced techniques in glycobiology have produced a number of tumor marker candidates. As a result from the glycomic approach, we found that fucosylated haptoglobin in sera was a possible tumor marker for pancreatic cancer (PC). Although Aleuria aurantia lectin (AAL) blotting can detect fucosylated haptoglobin, it is difficult to quantify fucosylated haptoglobin precisely. To overcome this problem, we developed a fucosylated haptoglobin detection kit as a sandwich enzyme-linked immune sorbent assay (ELISA) using AAL and the Fab portion of anti-haptoglobin antibody. In the present study, we investigated the clinical application of this lectin-antibody ELISA kit to measure fucosylated haptoglobin in PC. Methods: We measured fucosylated haptoglobin in patients with PC with a lectin-antibody ELISA kit. The fucosylated haptoglobin measured with this assay was compared with lectin blotting data, and the discrepancy was analyzed by immunoprecipitation methods. The concentration of fucosylated haptoglobin was investigated with respect to the clinical stage of PC. We also measured fucosylated haptoglobin, using 397 cases of several types of cancers including PC, benign diseases, and normal controls. Results: The sensitivity and specificity for the differential diagnosis of PC from normal controls was 50% and 91%, respectively. The results from lectin-antibody ELISA were significantly correlated with data from previous AAL blotting studies. Positive rates of fucosylated haptoglobin with this method in patients with PC were significantly higher in cases of stage IV compared with other clinical stages. Fucosylated haptoglobin was increased in several types of cancers, in which fucosylated haptoglobin was reported to increase. Conclusions: While certain cases showed a discrepancy in fucosylated haptoglobin concentrations between the lectin-antibody ELISA and conventional lectin blotting, this novel type of lectin-antibody ELISA might be useful for a tumor marker for PC. Clin Chem Lab Med 2010;48:505–12.

Clinical outcomes of endoscopic submucosal dissection for superficial esophageal neoplasms: a multicenter retrospective cohort study

BACKGROUND AND STUDY AIMS The safety and efficacy of endoscopic submucosal dissection (ESD) for superficial esophageal neoplasms (SENs) have not been evaluated in a multicenter survey. The aim of this study was to investigate the clinical outcomes in a multicenter study that included municipal hospitals. PATIENTS AND METHODS Of 312 consecutive patients with 373 esophageal lesions treated by ESD at 11 hospitals from May 2005 to December 2012, a total of 368 SENs in 307 patients were retrospectively analyzed. RESULTS The median tumor size was 18 mm (range 2 - 85 mm). The median procedure time was 90 minutes (range 12 - 450 minutes). The en bloc resection and complete resection rates were 96.7 % (95 % confidence interval [CI] 94.4 % - 98.1 %) and 84.5 % (95 %CI 80.5 % - 87.8 %), respectively. Perforation (including mediastinal emphysema), postoperative pneumonia, bleeding, and esophageal stricture, occurred in 5.2 % (95 %CI 3.3 % - 7.9 %), 1.6 % (95 %CI 0.7 % - 3.5 %), 0 %, and 7.1 % (95 %CI 4.9 % - 10.2 %) of patients, respectively. All of these complications were cured conservatively. No procedure-related mortality occurred. Early treatment periods (odds ratio [OR] = 4.04; P < 0.01) and low volume institutions (OR = 3.03; P  = 0.045) were significantly independent risk factors for perforation. The circumference of the lesion was significantly associated with postoperative stricture (OR = 32.3; P < 0.01). The procedure times significantly decreased in the later period of the study (P < 0.01). Follow-up data (median 35 months; range 4 - 98 months) showed significant differences in overall survival (P = 0.03) and recurrence-free survival (P < 0.01) rates between patients with curative and noncurative resections. CONCLUSIONS Esophageal ESD has become feasible with acceptable complication risks and favorable long term outcomes.

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.

Fucosylated haptoglobin is a novel type of cancer biomarker linked to the prognosis after an operation in colorectal cancer.

Fucosylation is a crucial oligosaccharide modification in cancer and inflammation. Total cellular proteins of cancer cells and the sera of patients with cancer both show increased fucosylation levels. Certain kinds of fucosylated proteins can be applied as novel cancer biomarkers in glyco‐proteomic analyses. We previously identified fucosylated haptoglobin (Fuc‐Hpt) as a serologic marker for pancreatic cancer, and recently developed a lectin‐antibody enzyme‐linked immunosorbent assay system for quantifying Fuc‐Hpt. In the present study, we investigated the clinical outcome of Fuc‐Hpt levels in patients with colorectal cancer (CRC), and examined the mechanisms underlying Fut‐Hpt production using a murine tumor transplantation model.

Synergistic antitumor effects of celecoxib with 5-fluorouracil depend on IFN-γ

Cyclooxygenase‐2 (COX‐2) inhibitors are effective chemopreventive agents against colorectal cancers. For treatment of advanced cancers, combination of COX‐2 inhibitors and chemotherapy has been attempted, but the results are still controversial. In the present study, the effects of the COX‐2 inhibitor celecoxib on the anticancer potential of chemotherapy, and its mechanisms of action were investigated in point of the angiogenesis, using an advanced cancer model in mice. BALB/c mice were inoculated with colon 26 cells. After the allograft grew up to 5 mm in diameter, the animals received celecoxib, 5FU, or a combination of 5FU and celecoxib (5FU/celecoxib). After 21‐days of the treatment, 5FU/celecoxib significantly inhibited the tumor growth and the tumor vessel density compared with the other groups. Celecoxib, 5FU or 5FU/celecoxib significantly suppressed the VEGF content in tumor tissues. 5FU/celecoxib also enhanced IFN‐γ levels in tumor tissue, which could be involved in the potent antitumor effects of 5FU/celecoxib. This hypothesis was proven, because in IFN‐γ knockout (IFN‐γ−/−) mice, the inhibitory effects of 5FU/celecoxib on angiogenesis and tumor growth were significantly impaired compared with that in wild type mice. These results suggest that celecoxib enhances the antitumor effect of 5FU against an advanced colon cancer model by suppressing angiogenesis. In addition to VEGF, IFN‐γ has pivotal roles in tumor suppression induced by celecoxib.

Identification of Fucosylated Haptoglobin as a Novel Tumor Marker for Pancreatic Cancer and Its Possible Application for a Clinical Diagnostic Test

Fucosylation is one of the most important oligosaccharide modifications in cancer and inflammation. The fucosylation level is increased in total cellular proteins of cancer cells as well as in sera of patients with cancer. Recently, on AAL blot analysis, we found a fucosylated glycoprotein of 40 kDa in sera of patients with pancreatic cancer. Based on its N-terminal sequence, this protein was identified as haptoglobin. Fucosylated haptoglobin was increased in sera of patients with several kinds of cancer and the positive rate was higher in pancreatic cancer. The level of fucosylated haptoglobin was not correlated with total haptoglobin, suggesting that a factor other than inflammation could regulate the production of fucosylated haptoglobin. Mass spectrometry analysis revealed the detailed oligosaccharide structure of fucosylated haptoglobin purified from sera of patients with pancreatic cancer. For clinical applications, we developed a lectin-antibody ELISA system for quantifying fucosylated haptoglobin. In this review, we would like to summarize the history of the identification of fucosylated haptoglobin as a marker for pancreatic cancer and its possible application for a clinical diagnostic test.