Atsuo Maemoto

Functional analysis of the α-defensin disulfide array in mouse cryptdin-4

The α-defensin antimicrobial peptide family is defined by a unique tridisulfide array. To test whether this invariant structural feature determines α-defensin bactericidal activity, mouse cryptdin-4 (Crp4) tertiary structure was disrupted by pairs of site-directed Ala for Cys substitutions. In a series of Crp4 disulfide variants whose cysteine connectivities were confirmed using NMR spectroscopy and mass spectrometry, mutagenesis did not induce loss of function. To the contrary, the in vitro bactericidal activities of several Crp4 disulfide variants were equivalent to or greater than those of native Crp4. Mouse Paneth cell α-defensins require the proteolytic activation of precursors by matrix metalloproteinase-7 (MMP-7), prompting an analysis of the relative sensitivities of native and mutant Crp4 and pro-Crp4 molecules to degradation by MMP-7. Although native Crp4 and the α-defensin moiety of proCrp4 resisted proteolysis completely, all disulfide variants were degraded extensively by MMP-7. Crp4 bactericidal activity was eliminated by MMP-7 cleavage. Thus, rather than determining α-defensin bactericidal activity, the Crp4 disulfide arrangement confers essential protection from degradation by this critical activating proteinase.

Quantitative analysis for human glucocorticoid receptor α/β mRNA in IBD ☆

We have previously reported that in peripheral blood mononuclear cells (PBMC), the augmented expression of the β isoform of the human glucocorticoid receptor (hGRβ), as a putative dominant negative regulator of glucocorticoid action, is associated with glucocorticoid (GC) unresponsiveness of UC patients. In this study, we quantified the levels and serial changes of hGR transcripts in PBMC of IBD patients by a real-time fluorescence monitoring of PCR. As results, relative hGRβ mRNA expression was significantly higher in the active stage of UC than in inactive periods of UC or CD patients. Longitudinal analysis revealed that hGRβ mRNA expression in UC was increased after the relapse of inflammation, suggesting that the overproduction of cytokines during inflammation may be responsible. In in vitro culture experiments of human lymphoid cell (CEM) and human PBMC, IL-7, and IL-18 increased hGRβ mRNA expression in these cells but GC itself did not. Through these analyses, it is indicated that the inflammatory cytokines altered the splicing condition of the primary transcript of hGR gene in IBD patients.

Sonic hedgehog derived from human pancreatic cancer cells augments angiogenic function of endothelial progenitor cells

Hedgehog signaling is important in the pathogenesis of pancreatic cancer. Several recent observations suggest the involvement of sonic hedgehog (SHH) in postnatal neovascularization. We identified a novel role for SHH in tumor‐associated angiogenesis in pancreatic cancer. Immunohistochemical analysis revealed that patched homolog 1 (PTCH1), both a receptor for and transcriptional target of hedgehog signaling, was expressed in a small fraction of endothelial cells within pancreatic cancer, but not in normal pancreatic tissue. When endothelial progenitor cells (EPC) isolated from human peripheral blood were cultured with supernatant from SHH‐transfected 293 cells or pancreatic cancer cells, mRNA levels of vascular endothelial growth factor (VEGF), stromal cell‐derived factor‐1 and angiopoietin‐1 were significantly increased, whereas no such induction was observed in human umbilical vein endothelial cell (HUVEC) and human dermal microvascular endothelial cell (HMVEC). HUVEC tube formation was stimulated when cocultured with EPC, and preconditioning EPC with supernatant from KP‐1 N pancreatic cancer cells highly expressing SHH significantly enhanced the effect. The effect was partially attenuated by specific inhibition of SHH with cyclopamine or a neutralizing antibody. These findings suggest that tumor‐derived SHH can induce angiogenesis, and this is mediated by its effects on EPC specifically. Targeting SHH would be a novel therapeutic approach that can inhibit not only proliferation of cancer cells but also EPC‐mediated angiogenesis. (Cancer Sci 2008; 99: 1131–1138)

Minute findings by magnifying colonoscopy are useful for the evaluation of ulcerative colitis

BACKGROUND Colonoscopy has an important role in the diagnosis of ulcerative colitis. However, colonoscopic findings are inadequate for the prediction of relapse without histologic examination. In this study, the role of magnifying colonoscopy in ulcerative colitis was evaluated. METHODS One hundred sixteen magnifying colonoscopy observations were made in 61 patients with ulcerative colitis between January 1994 and October 1998. A simple classification of magnifying colonoscopic findings into 5 categories was devised as follows: regularly arranged crypt openings, villous-like, minute defects of epithelium, small yellowish spots, and coral reef-like appearance. The colonoscopic findings by classification were compared with histopathologic findings, and the usefulness of the classification for predicting relapse was prospectively analyzed in 18 patients. RESULTS Compared with grade as determined by conventional colonoscopy, there was a better correlation between the classification of findings by magnifying colonoscopy and histopathologic findings (r(2) = 0.665, 0.807, respectively). Of 18 patients studied prospectively, 7 of 9 with minute defects of epithelium relapsed within 6 months, and the cumulative nonrelapsing rate was significantly lower in patients with minute defects of epithelium compared with those without minute defects of epithelium (p = 0.0059). Moreover, minute defects of epithelium was found to be a significant independent predictive factor for relapse (multivariate analysis, Cox proportional hazards model; p = 0.0203). CONCLUSIONS Our proposed classification of magnifying colonoscopic findings in patients with ulcerative colitis is useful for the evaluation of disease activity and for the prediction of periods of remission.

Clinicopathologic Implications of Genetic Instability in Intestinal-Type Gastric Cancer and Intestinal Metaplasia as a Precancerous Lesion Proof of Field Cancerization in the Stomach

To clarify field cancerization in the stomach by genetic alterations, we studied 83 cases of intestinal-type gastric cancer (GC) and paired intestinal metaplasia (IM) distant from GC and 39 cases of chronic gastritis with IM (CG-IM) for genetic instability (GIN). Microsatellite instability (MSI) and loss of heterozygosity (LOH) were evaluated at 5 microsatellite loci. The incidence of GIN was 21% (8/39) in CG-IM, 48% (40/83) in GC-IM, and 65% (54/83) in GC and showed a significant difference among these 3 categories. By tumor location, MSI showed the highest incidence in GC and GC-IM with the tumor located in the upper third of the stomach. GIN in GC and GC-IM significantly increased with the progression of tumor invasion from mucosal to advanced cancer. GIN, especially LOH, was more frequently detected in cases with vs without lymphatic or vascular invasion and lymph node involvement in GC and GC-IM. The GIN of GC and GC-IM was significantly similar in relation to clinicopathologic features. Biologic detection of GIN in IM may be a surrogate marker for GC risk and for clinical evaluation of malignant potential. The condition is consistent with the hypothesis of field cancerization in the stomach.

Expression of the antimicrobial peptide α‐defensin/cryptdins in intestinal crypts decreases at the initial phase of intestinal inflammation in a model of inflammatory bowel disease, IL‐10‐deficient mice

Background: The etiology of inflammatory bowel disease (IBD) is associated with an altered microflora due to a failure of the immune system. This study investigated the expression of the intestinal antimicrobial peptide &agr;‐defensin, which plays a pivotal role in the regulation of the intestinal microflora in a representative model of IBD, interleukin (IL)‐10‐deficient mice. Methods: The expression of &agr;‐defensin/cryptdins in IL‐10‐deficient mice was assessed by real‐time polymerase chain reaction (PCR) and acid/urea polyacrylamide gel (AU‐PAGE). The alteration of &agr;‐defensin/cryptdins expression was compared with the inflammatory grade of mice intestine at various weeks from birth. Results: The weight, length, and inflammation grade of the mouse intestines were assessed at 5, 7, 9, 11, 13, and 15 weeks from birth. While the weight of the large intestine was heavier at 15 weeks after birth in the IL‐10‐deficient mice than in the control mice, histological inflammation began from 7 weeks after birth. Real‐time PCR and AU‐PAGE identified a significant decrease in the expression of &agr;‐defensin/cryptdins at 7 weeks after birth in the IL‐10 knockout mice, thus illustrating the involvement of &agr;‐defensin/cryptdins in the etiology of the intestinal inflammation in IBD. This study also identified the expression of &agr;‐defensin/cryptdins to be inversely proportional to age until 11 weeks, suggesting a relationship between the formation of the intestinal microflora and a reduction in the expression of &agr;‐defensin/cryptdins. Conclusions: The altered expression of antimicrobial peptide &agr;‐defensin may cause the onset of intestinal inflammation due to a failure to regulate intestinal microflora. (Inflamm Bowel Dis 2010)

Precursor Processing of Human Defensin-5 Is Essential to the Multiple Functions in vitro and in vivo

Human defensin-5 (HD-5) is one of the major antimicrobial peptides secreted by Paneth cells in the human small intestine. HD-5 is produced and stored as a propeptide in Paneth cell granules, secreted in response to stimulation by cholinergic reagents or bacterial antigens. The activation process by trypsin occurs in the intestinal lumen to produce mature HD-5. This study evaluated the difference between proHD-5 and mature HD-5 in bactericidal activity and induction of chemokine secretion in vitro. Mature HD-5 showed bactericidal activities against all bacterial strains. Though, proHD-5 without enzymatic cleavage possessed less antimicrobial ability against Salmonella typhimurium and Escherichia coli but not against Staphylococcus aureus. Mature HD-5 also induced intestinal epithelial cells to increase the protein and mRNA levels of interleukin-8. Furthermore, the peptides were applied to dextran sulfate sodium-induced mouse colitis. The expression of endogenous mouse defensins was not changed in the small intestine, and the additional injection of exogenous HD-5 improved mortality (p < 0.05). This study demonstrated the multifunctional roles of the activation process in human defensin and the possibility of using antimicrobial peptides for the treatment of inflammatory bowel diseases in future applications.

In Vitro Activation of the Rhesus Macaque Myeloid α-Defensin Precursor proRMAD-4 by Neutrophil Serine Proteinases

α-Defensins are mammalian antimicrobial peptides expressed mainly by cells of myeloid lineage or small intestinal Paneth cells. The peptides are converted from inactive 8.5-kDa precursors to membrane-disruptive forms by post-translational proteolytic events. Because rhesus myeloid pro-α-defensin-4 (proRMAD-4(20–94)) lacks bactericidal peptide activity in vitro, we tested whether neutrophil azurophil granule serine proteinases, human neutrophil elastase (NE), cathepsin G (CG), and proteinase-3 (P3) have in vitro convertase activity. Only NE cleaved proRMAD-4(20–94) at the native RMAD-4 N terminus to produce fully processed, bactericidal RMAD-4(62–94). The final CG cleavage product was RMAD-4(55–94), and P3 produced both RMAD-4(55–94) and RMAD-4(57–94). Nevertheless, NE, CG, and P3 digests of proRMAD4 and purified RMAD-4(62–94), RMAD-4(55–94), and RMAD-4(57–94) peptides had equivalent in vitro bactericidal activities. Bactericidal peptide activity assays of proRMAD-4(20–94) variants containing complete charge-neutralizing D/E to N/Q or D/E to A substitutions showed that (DE/NQ)-proRMAD-4(20–94) and (DE/A)-proRMAD-4(20–94) were as active as mature RMAD-4(62–94). Therefore, proregion Asp and Glu side chains inhibit the RMAD-4 component of full-length proRMAD-4(20–94), perhaps by a combination of charge-neutralizing and hydrogen-bonding interactions. Although native RMAD-4(62–94) resists NE, CG, and P3 proteolysis completely, RMAD-4(62–94) variants with disulfide pairing disruptions or lacking disulfide bonds were degraded extensively, evidence that the disulfide array protects the α-defensin moiety from degradation by the myeloid converting enzymes. These in vitro analyses support the conclusion that rhesus macaque myeloid pro-α-defensins are converted to active forms by serine proteinases that co-localize in azurophil granules.

Transnasal ultrathin endoscopy for placement of a long intestinal tube in patients with intestinal obstruction.

BACKGROUND The technical difficulties related to the insertion of a long intestinal tube into the jejunum under fluoroscopy present a considerable problem in patients with an intestinal obstruction. OBJECTIVE To evaluate the usefulness of endoscopic long intestinal-tube placement with the ultrathin esophagogastroduodenoscope (UT-EGD). DESIGN A prospective randomized clinical trial was conducted. PATIENTS Twenty-eight consecutive patients who presented with an intestinal obstruction were included in the study. INTERVENTION The UT-EGD was inserted nasally into at least the second portion of the duodenum or beyond. After a guidewire was introduced through the working channel, with fluoroscopic guidance, the UT-EGD itself was carefully removed with the guidewire left in place. Next, a hydrophilic intestinal tube was advanced over the guidewire into the jejunum, and then the guidewire was removed. MAIN OUTCOME MEASUREMENTS Primary end points are the total procedure time, the radiation exposure time, and the rate of complications, all compared with the conventional method. RESULTS The mean (+/-SD) total procedure time was 18.7 +/- 8.4 minutes for the UT-EGD method and 39.5 +/- 15.0 minutes for the conventional method, with a significant time difference between the 2 methods (P < .0005). The mean (+/-SD) radiation exposure time was also shorter with the UT-EGD method (11.1 +/- 6.0 minutes) than with the conventional method (30.3 +/- 13.7 minutes) (P < .0005). There were no complications, except for mild nasal bleeding with each method. CONCLUSIONS The UT-EGD method has definite advantages in the placement of a long intestinal tube for patients with an intestinal obstruction in comparison with the conventional method.

K‐ras mutations and cell kinetics in Helicobacter pylori associated gastric intestinal metaplasia: a comparison before and after eradication in patients with chronic gastritis and gastric cancer

Background:Helicobacter pylori related gastric intestinal metaplasia (IM) is considered to be a precancerous lesion. Aims: To identify the effects of H pylori eradication on K-ras mutations, cell kinetics in IM and histological changes in patients with and without gastric cancers in a one-year prospective study. Methods: Patients included group A (n = 39), chronic gastritis, and group B (n = 53), intestinal-type early gastric cancer patients who had all undergone endoscopic mucosal resection (n = 25) or surgical resection (n = 28). K-ras codon 12 mutations in IM were examined, followed by DNA sequencing analysis. Proliferating and apoptotic cells were detected with anti-Ki-67 antibody and using the TUNEL method, respectively. Results: The incidence of K-ras mutations in the cancer was only 3.8%. The mutant K-ras in IM was observed more frequently in group A (46.2%) than in group B patients (1.9%) (p<0.005). After eradication, the K-ras mutations significantly declined to 12.8% in group A (p<0.005). The mutation pattern of K-ras codon 12 before eradication was that GGT was mainly changed to AGT (50%) in group A. AGT transformation was not affected by treatment. Apoptosis in IM showed an increase after H pylori eradication in both groups (p<0.05 in group A) although no histological improvement in IM was observed. The monocyte score was significantly higher in group A than in group B (p<0.05); the score improved significantly after eradication. Conclusions: K-ras mutations in IM do not always play a role in gastric carcinogenesis but cell kinetics, especially apoptosis, in IM may contribute to it. There are early events in K-ras mutations which are influenced by H pylori infection; some mutations may also be selected by eradication. These unstable K-ras mutations in IM may be related to lymphocyte infiltration caused by H pylori infection.