Katsuyuki Kitoh

Elevated Circulating Platelet-Derived Microparticles in Patients with Active Inflammatory Bowel Disease

OBJECTIVES:Platelet-derived microparticles (PDMPs) are active molecules involved in the hemostatic and inflammatory responses. To evaluate the changes in the platelet function in patients with inflammatory bowel disease (IBD), we measured circulating PDMP levels.METHODS:Twenty-five healthy controls, 44 patients with ulcerative colitis (UC), and 43 patients with Crohns Disease (CD) were studied. The PDMP and soluble P-selectin (sP-selectin) levels were measured by enzyme-linked immunosorbent assay (ELISA).RESULTS:In the healthy controls, the PDMP levels were 17.2 ± 6.2 U/mL. Significant differences were not observed between the healthy controls and inactive UC patients (20.8 ± 9.5 U/mL, n = 25) or between the healthy controls and inactive CD patients (17.6 ± 7.8 U/mL, n = 24). In contrast, the PDMP levels were significantly higher in both active UC (49.2 ± 33.6 U/mL, n = 19) and active CD (48.6 ± 42.8 U/mL, n = 19) patients than in the healthy controls. A significant correlation was found between the PDMP levels and the clinical activity indexes (CAI) of UC patients (r = 0.65, p < 0.01, n = 44), and between the PDMP levels and Crohns disease activity indexes (CDAI) (r = 0.72, p < 0.01, n = 43). Elevated PDMP levels in active patients were significantly reduced after remission. A significant correlation was observed between the PDMP levels and the sP-selectin levels (r = 0.60, p < 0.01, n = 122).CONCLUSION:Elevated circulating PDMPs in active IBD patients suggest a role for platelets in the pathogenesis of IBD.

Interleukin-17 augments tumor necrosis factor-α-induced granulocyte and granulocyte/macrophage colony-stimulating factor release from human colonic myofibroblasts

BackgroundInterleukin (IL)-17 is a newly identified T-cell-specific cytokine. In this study, we investigated the effects of IL-17 on colony-stimulating factor (CSF) release in human colonic subepithelial myofibroblasts (SEMFs).MethodsCSF release and mRNA expression were determined by enzyme-linked immunosorbent assay (ELISA) and Northern blotting, respectively. Nuclear factor (NF)-κB- and activating protein (AP-1)-DNA binding activities were evaluated by electrophoretic gel mobility shift assays (EMSAs).ResultsUnstimulated cells secreted a small amount of granulocyte G- and granulocyte/macrophage (GM)-CSF, and a considerable amount of M-CSF. IL-17 weakly enhanced G-CSF release, but did not affect GM- and M-CSF release. IL-17 selectively enhanced tumor necrosis factor (TNF)-α-induced G- and GM-CSF release. The combination of IL-17 plus TNF-α induced a marked increase in NF-κB- and AP-1-DNA binding activities. The adenovirus-mediated transfer of a stable form of IκBα and/or a dominant negative mutant of c-Jun markedly inhibited the IL-17 plus TNF-α-induced G- and GM-CSF mRNA expression. Furthermore, a stability study showed that IL-17 plus TNF-α markedly enhanced the stability of G- and GM-CSF mRNA.ConclusionsIL-17 augments TNF-α-induced G- and GM-CSF release via transcriptional and posttranscriptional mechanisms.

Increased aggregation response of platelets in patients with inflammatory bowel disease.

BackgroundPlatelets play an important role in hemostatic and inflammatory responses. To evaluate any potential enhancement of platelet activity in patients with inflammatory bowel disease (IBD), we measured the platelet aggregation responses to various stimuli.MethodsTwenty-two healthy controls, 24 patients with ulcerative colitis (UC) and 25 patients with Crohns Disease (CD) were studied. The aggregation responses induced by three agonists (epinephrine, collagen, and ADP) were measured by an 8-channel aggregometer. The platelet-derived microparticles (PDMP) levels were measured by an enzyme-linked immunosorbent assay.ResultsTwenty-one out of the 22 healthy controls did not respond to epinephrine (0.1 µg/ml), collagen (0.2 µg/ml), or ADP (1.0 µM). Eight out of the 12 active UC patients were sensitive to all agonists, and 4 patients showed increased sensitivity to epinephrine/collagen or epinephrine/ADP. Three out of the 12 inactive UC patients were normal, but 9 of these patients showed increased sensitivity, mainly to epinephrine. Ten out of the 12 active CD patients were sensitive to all agonists, and 2 active CD patients were sensitive to epinephrine/collagen or epinephrine/ADP. Eight out of the 13 inactive CD patients were sensitive to two or all agonists. Even after remission, almost all of the UC and CD patients showed some increased sensitivity to the agonists. The platelet number and the plasma PDMP levels were significantly higher in the active IBD patients than in the control group.ConclusionsPlatelet aggregation responses are enhanced in IBD, even in inactive-phase patients. This increased sensitivity of the platelets may play an important role in the pathophysiology of IBD.

Increased Expression of Interleukin-36, a Member of the Interleukin-1 Cytokine Family, in Inflammatory Bowel Disease.

Background:Interleukin (IL)-36 (IL-36&agr;, IL-36&bgr;, and IL-36&ggr;) is a recently reported member of the IL-1 cytokine family. In this study, we investigated IL-36 expression in the inflamed mucosa of patients with inflammatory bowel disease and characterized the proinflammatory actions of IL-36 cytokines in human colonic epithelial cells. Methods:IL-36 mRNA expression was evaluated using real-time PCR. IL-36 protein expression was analyzed using immunoblotting and immunohistochemical technique. Intracellular signaling pathways were evaluated by immunoblotting and by specific siRNA-transfected cells. Results:The mRNA expression of IL-36&agr; and IL-36&ggr;, but not of IL-36&bgr;, was enhanced in the inflamed mucosa of patients with inflammatory bowel disease, in particular, in ulcerative colitis. Immunohistochemical analysis showed that T cells, monocytes, and plasma cells are the source of IL-36&agr; and IL-36&ggr; in colonic mucosa. DNA microarray analysis indicated that IL-36&agr; induces the mRNA expression of CXC chemokines and acute phase proteins in intestinal epithelial cell line, HT-29 cells. IL-36&agr; and IL-36&ggr; dose-dependently and time-dependently induced the mRNA and protein expression of CXC chemokines (CXCL1, CXCL2, CXCL3 etc.) in HT-29 and Widr cells. Stimulation with IL-36&agr; and IL-36&ggr; assembled MyD88 adaptor proteins (MyD88, TRAF6, IRAK1, and TAK1) into a complex and induced the activation of NF-&kgr;B and AP-1 and also the phosphorylation of MAPKs. MAPK inhibitors and siRNAs specific for NF-&kgr;B and c-Jun AP-1 significantly reduced IL-36–induced CXC chemokine expression. Conclusions:IL-36&agr; and IL-36&ggr; may play a proinflammatory role in the pathophysiology of inflammatory bowel disease through induction of CXC chemokines and acute phase proteins.

Suppression of inflammatory cytokine secretion by granulocyte/monocyte adsorptive apheresis in active ulcerative colitis.

Abstract:  To elucidate the molecular mechanisms involved in the therapeutic effects of granulocyte/monocyte adsorption apheresis, changes were investigated in the cytokine responses of peripheral blood mononuclear cells (PBMC) before and after granulocyte/monocyte adsorptive apheresis in ulcerative colitis (UC) patients. Four patients with active UC were enrolled. All patients responded to granulocyte/monocyte adsorptive apheresis. A total of 20 sessions of four patients were analyzed. Peripheral blood mononuclear cells were isolated from peripheral venous blood within 5 min before and after each session of granulocyte/monocyte adsorptive apheresis. The cells were stimulated with interleukin (IL)‐1β and tumor necrosis factor (TNF)‐α for 24 h, and the secreted IL‐8 and IL‐6 levels were determined by enzyme‐linked immunosorbent assay (ELISA). IL‐1β‐induced IL‐8 and IL‐6 secretion was significantly decreased after granulocyte/monocyte adsorptive apheresis. TNF‐α‐induced IL‐8 secretion was also significantly decreased after apheresis, but there was no significant difference in TNF‐α‐induced IL‐6 secretion (P = 0.052). In conclusion, granulocyte/monocyte adsorptive apheresis down‐regulates the IL‐1β‐ and TNF‐α‐induced inflammatory responses in PBMC. The induction of hyporesponsiveness to pro‐inflammatory cytokines may be an important factor mediating the clinical effects of granulocyte/macrophage adsorptive apheresis in UC patients.

Small intestinal perforation due to cytomegalovirus infection in patients with non-Hodgkin's lymphoma

We describe two patients with non-Hodgkins lymphoma (NHL) who suffered cytomegalovirus (CMV)-related small intestinal perforations during the course of chemotherapy. Surgical specimens from both patients revealed histologic evidence of occlusive vasculitis and tissue destruction caused by CMV-affected cells in the submucosa and muscular walls, that may have played an important role in the pathogenesis of these perforations. Although such intestinal perforations are rare complications in NHL patients, CMV infection should be recognized as a primary etiological factor in acute abdominal crises when treating NHL patients with pharmaceutical agents including steroids. Emergency surgery and the anti-CMV agent, ganciclovir, would improve the prognoses of such patients.

Trisomy 4 in a case of acute lymphocytic leukemia (L1)

Trisomy 4 has been identified previously as a chromosome abnormality associated with acute nonlymphocytic leukemia (ANLL) with myelomonocytic lineage and in myelodysplastic syndromes (MDS). We report a case of acute lymphocytic leukemia (ALL) (French-American-British, FAB L1) in a 42-year-old Japanese man, with trisomy 4 as the sole chromosomal anomaly. Immunophenotypically, the leukemic blasts demonstrated reactivity with CD2, CD5, and CD7 and indicated on early stage of T cell.

Interleukin (IL)-1β Is a Strong Inducer of IL-36γ Expression in Human Colonic Myofibroblasts

Backgrounds and aims Interleukin (IL)-36 cytokines are members of the IL-1 cytokine family. In this study, we investigated the expression of IL-36γ in human colonic myofibroblasts to explore the molecular mechanisms underlying IL-36γ induction. Materials and methods IL-36 mRNA was analyzed by real-time PCR method. Secretion of IL-36γ protein was evaluated by Western blot and ELISA analyses. Molecular mechanism of IL-36γ induction was evaluated by siRNA analyses and immunofluorescence experiments. Results IL-36γ mRNA expression was scarcely detected in the cells without stimulation. IL-1β induced a marked increase of IL-36γ mRNA expression. TNF-α markedly enhanced IL-1β-induced IL-36γ mRNA expression. These responses were confirmed at the protein levels. The inhibitors for ERK1/2 (PD98059 and U0216) and a p38 MAPK (SB203580) significantly reduced the IL-1β-induced IL-36γ mRNA expression. In addition, the siRNAs specific for NF-κB p65 and AP-1 (c-Jun) significantly reduced the expression of IL-1β-induced IL-36γ mRNA. Conclusions Colonic myofibroblasts are cellular source of IL-36γ in the intestine. IL-36γ expression was induced by the combination of IL-1β and TNF-α via activation of MAPKs and transcription factors, NF-κB and AP-1.

A case of Crohn’s disease that developed anti-infliximab and anti-adalimumab antibodies

There are few reports about the rapid appearance of anti-adalimumab antibodies in patients with Crohn’s disease positive for anti-infliximab antibodies. We report the case of a 29-year-old female patient with a diagnosis of Crohn’s disease who revealed a loss of response to infliximab due to high levels of antibodies to infliximab, and did not respond to the subsequent therapy by adalimumab, with a rapid appearance of antibodies to adalimumab. As one of the possible mechanisms of non-response to adalimumab, immunologic reactivity of infliximab to adalimumab was suspected, since the patient’s IgG that was obtained just before the induction of adalimumab reacted with infliximab and adalimumab. We should pay attention to the easy appearance of anti-adalimumab antibodies in association with reactivity of anti-infliximab antibodies to adalimumab in patients with high levels of anti-infliximab antibodies.

Magnified single-balloon enteroscopy in the diagnosis of intestinal follicular lymphoma: a case series

The objective of this study was to evaluate the magnified endoscopic findings in the diagnosis of follicular lymphoma in the small intestine in comparison with those of intestinal follicular lymphoma and lymphangiectasia. Four patients with follicular lymphoma and 3 with lymphangiectasia in the small intestine were retrospectively analyzed. A prototype magnifying singleballoon enteroscope was used. The findings of the intestinal follicular lymphoma and lymphangiectasia were retrospectively analyzed to determine the magnified endoscopic findings of follicular lymphoma in the small intestine. Opaque white granules were observed in 3 of the 4 patients with follicular lymphoma. Magnified narrow-band imaging (NBI) of the opaque white granules showed stretched microvessels, which had a diminutive tree-like appearance. The remaining patient had no opaque white granules and only displayed whitish villi. Magnified NBI observation of the whitish villi revealed the absence of marginal villus epithelium, which was confirmed by histology. The magnified NBI enteroscopy revealed the diminutive tree-like appearance on the opaque white granules and the absence of marginal villus epithelium of the whitish villi in intestinal follicular lymphoma. These findings may be useful in diagnosing follicular lymphoma.