Toshimitsu Araki

Incidence and Risk Factors

Epidemiological data indicate that inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn’s disease (CD), is the third highest risk condition for the development of colorectal cancer (CRC), namely, colitis-associated cancer (CAC). CD is also associated with an increased risk of small-bowel adenocarcinoma, in response to chronic inflammation of the small intestine. In studies published in the 1990s, the risk for CAC in IBD was approximately 7 % at 20 years after diagnosis. In recent studies, the overall incidence of CAC in IBD is lower, less than 5 % at 20 years. However, several factors, such as the longer duration of colitis, extensive or severer colitis, and coexistent primary sclerosing cholangitis, have continued to be important in the development of CRC in patients with IBD. Despite clinical and experimental investigations, the molecular mechanisms by which chronic inflammation promotes cancer progression are still unknown.

The expression patterns of Toll-like receptors in the ileal pouch mucosa of postoperative ulcerative colitis patients.

The aim of this study was to evaluate the expression pattern of Toll-like receptors (TLRs) in the pouch mucosa of ulcerative colitis patients in comparison with that in the ileum mucosa of noninflammatory bowel disease patients. Pouch mucosal biopsy specimens were collected from postoperative patients who had undergone surgery for ulcerative colitis. Normal ileum specimens were collected from colon cancer patients. The specimens were assessed by immunofluorescence histochemistry using TLR2, TLR3, TLR4, and TLR5 polyclonal antibodies. The normal ileal mucosa constitutively expressed TLR3 and TLR5, whereas TLR2 and TLR4 were barely detectable. In the mucosa of active pouchitis, TLR2 and TLR4 was strongly upregulated, and TLR4 was upregulated even in a noninflamed pouch. No TLR3 or TLR5 expression was detectable. These data suggest that pouchitis may be associated with distinctive changes in selective TLR expression in the pouch mucosa, and that TLR4 alterations in the innate response system may contribute to the pathogenesis of these disorders in particular.

In vivo characterization of neutrophil extracellular traps in various organs of a murine sepsis model.

Neutrophil extracellular traps (NETs) represent extracellular microbial trapping and killing. Recently, it has been implicated in thrombogenesis, autoimmune disease, and cancer progression. The aim of this study was to characterize NETs in various organs of a murine sepsis model in vivo and to investigate their associations with platelets, leukocytes, or vascular endothelium. NETs were classified as two distinct forms; cell-free NETs that were released away from neutrophils and anchored NETs that were anchored to neutrophils. Circulating cell-free NETs were characterized as fragmented or cotton-like structures, while anchored NETs were characterized as linear, reticular, membranous, or spot-like structures. In septic mice, both anchored and cell-free NETs were significantly increased in postcapillary venules of the cecum and hepatic sinusoids with increased leukocyte-endothelial interactions. NETs were also observed in both alveolar space and pulmonary capillaries of the lung. The interactions of NETs with platelet aggregates, leukocyte-platelet aggregates or vascular endothelium of arterioles and venules were observed in the microcirculation of septic mice. Microvessel occlusions which may be caused by platelet aggregates or leukocyte-platelet aggregates and heterogeneously decreased blood flow were also observed in septic mice. NETs appeared to be associated with the formation of platelet aggregates or leukocyte-platelet aggregates. These observational findings may suggest the adverse effect of intravascular NETs on the host during a sepsis.

Intravital imaging of DSS-induced cecal mucosal damage in GFP-transgenic mice using two-photon microscopy

BackgroundTwo-photon laser-scanning microscopy (TPLSM) is a powerful diagnostic tool for real-time, high-resolution structural imaging. However, obtaining high-quality in vivo TPLSM images of intra-abdominal organs remains technically challenging.Materials and methodsAn organ-stabilizing system was applied to high-quality TPLSM imaging. Real-time imaging of visceral organs, such as the liver, spleen, kidney and intestine, of transgenic green fluorescent protein (GFP) mice was performed in vivo using TPLSM. The bacterial translocation model using dextran sodium sulfate (DSS)-induced colitis was also investigated in prepared GFP mice following simple surgery. This allowed the capture of morphological real images using in vivo TPLSM. Immunohistochemical analysis of ZO-1 was performed to support the morphological findings of TPLSM.Results and conclusionsWe established an organ-stabilizing system to evaluate the real-time imaging of visceral organs in actin–GFP transgenic mice using in vivo TPLSM. DSS-induced colitis showed irregularity of crypt architecture, disappearance of crypts, inflammatory cell infiltration and increased rolling of white blood cells along the vasculature. In addition, the intercellular distance of mucosal cells in the crypt and vascular endothelial cells in the intestinal wall was increased in the intestinal mucosa during DSS colitis. In DSS colitis, there was remarkable loss of mucosal and vascular endothelial ZO-1 expression, as could be seen by a decrease in ZO-1 staining. In conclusion, our observations suggested the possibility that our TPLSM imaging system can be used to clarify the pathophysiological changes in various diseases using longitudinal studies of microscopic changes in the same animal over long periods of time.

Brain-derived neurotrophic factor (BDNF)-induced tropomyosin-related kinase B (Trk B) signaling is a potential therapeutic target for peritoneal carcinomatosis arising from colorectal cancer.

Tropomyosin-related receptor kinase B (TrkB) signaling, stimulated by brain-derived neurotrophic factor (BDNF) ligand, promotes tumor progression, and is related to the poor prognosis of various malignancies. We sought to examine the clinical relevance of BDNF/TrkB expression in colorectal cancer (CRC) tissues, its prognostic value for CRC patients, and its therapeutic potential in vitro and in vivo. Two hundred and twenty-three CRC patient specimens were used to determine both BDNF and TrkB mRNA levels. The expression of these proteins in their primary and metastatic tumors was investigated by immunohistochemistry. CRC cell lines and recombinant BDNF and K252a (a selective pharmacological pan-Trk inhibitor) were used for in vitro cell viability, migration, invasion, anoikis resistance and in vivo peritoneal metastasis assays. Tissue BDNF mRNA was associated with liver and peritoneal metastasis. Tissue TrkB mRNA was also associated with lymph node metastasis. The co-expression of BDNF and TrkB was associated with liver and peritoneal metastasis. Patients with higher BDNF, TrkB, and co-expression of BDNF and TrkB had a significantly poor prognosis. BDNF increased tumor cell viability, migration, invasion and inhibited anoikis in the TrkB-expressing CRC cell lines. These effects were suppressed by K252a. In mice injected with DLD1 co-expressing BDNF and TrkB, and subsequently treated with K252a, peritoneal metastatic nodules was found to be reduced, as compared with control mice. BDNF/TrkB signaling may thus be a potential target for treating peritoneal carcinomatosis arising from colorectal cancer.

Preoperative steroid-related complications in Japanese pediatric patients with ulcerative colitis.

PurposeThis study was designed to clarify a limit for steroid therapy in patients with ulcerative colitis through analyzing the preoperative major steroid-related complications and to define when alternative therapies, including surgery, should be performed in pediatric ulcerative colitis patients.MethodsThe medical records of 28 pediatric and 57 adult patients with ulcerative colitis who underwent total proctocolectomy and ileal J-pouch-anal anastomosis were reviewed. The relationship between the preoperative dose of glucocorticoids and major steroid-related complications, as well as the surgery variables, was evaluated.ResultsSignificantly higher incidences of growth retardation, osteoporosis, glaucoma, and cataracts were noted in pediatric patients than in adult patients. In pediatric patients, major steroid-related complications occurred at a significantly lower preoperative total dosage of glucocorticoids/body weight (mg/kg) or preoperative total dosage of glucocorticoids/body surface area (mg/m2) than in adult patients. A similar surgical procedure was performed in both pediatric and adult patients. The presence of major steroid-related complications can lower a patients long-term quality of life.ConclusionsEvidence-based guidelines for the recommended dose ofglucocorticoids according to body weight or body surface area are needed. To allow patients to feel well and maintain a good quality of life, early introduction of alternative treatments, including surgery, should be considered.

In Vivo Time-Course Imaging of Tumor Angiogenesis in Colorectal Liver Metastases in the Same Living Mice Using Two-Photon Laser Scanning Microscopy

In vivo real-time visualization of the process of angiogenesis in secondary tumors in the same living animals presents a major challenge in metastasis research. We developed a technique for intravital imaging of colorectal liver metastasis development in live mice using two-photon laser scanning microscopy (TPLSM). We also developed time-series TPLSM in which intravital TPLSM procedures were performed several times over periods of days to months. Red fluorescent protein-expressing colorectal cancer cells were inoculated into the spleens of green fluorescent protein-expressing mice. First- and second-round intravital TPLSM allowed visualization of viable cancer cells (red) in hepatic sinusoids or the space of Disse. Third-round intravital TPLSM demonstrated liver metastatic colonies consisting of viable cancer cells and surrounding stroma with tumor vessels (green). In vivo time-course imaging of tumor angiogenesis in the same living mice using time-series TPLSM could be an ideal tool for antiangiogenic drug evaluation, reducing the effects of interindividual variation.

The effect on morbidity of mesentery lengthening techniques and the use of a covering stoma after ileoanal pouch surgery

PurposeA tension-free anastomosis in a restorative proctocolectomy requires sufficient length of small-bowel mesentery. To ensure adequate length, it has been proposed that the superior mesenteric artery be divided and the right colon marginal vascular arcade be preserved. This study was designed to evaluate the influence of mesenteric lengthening techniques on the need for a stoma and on early outcomes after restorative proctocolectomy.MethodsRecords of patients who had a restorative proctocolectomy between January 1998 and October 2003 were reviewed. Patient and disease characteristics, operative techniques and findings, the need for a stoma, and postoperative complications were recorded.ResultsIn one patient a restorative proctocolectomy was not possible. The remaining 220 patients were divided into two groups: Group A (inflammatory bowel disease; n = 123) and Group B (noninflammatory bowel disease; n = 97). Sixty-nine patients (31.4 percent) had major comorbidities. A lengthening technique was performed in 120 patients (54.5 percent) by dividing the ileocecal artery (n = 37) or the superior mesenteric artery (n = 88); 5 patients had only the marginal vascular arcade preserved. An ileostomy was not required in 116 patients (52.7 percent). In multivariate analysis, in Group B the only surgical variable influencing the need for an ileostomy was preservation of the marginal vascular arcade (50 vs. 14.7 percent; P < 0.0005). Complications occurred in 41 patients (18.6 percent), more frequently for those in GroupA and for patients receiving steroids (23.6 vs. 12.4 percent, P = 0.012; 10.4 vs. 6.8 percent, P = 0.0172).ConclusionsThe use of mesentery lengthening techniques allows a restorative proctocolectomy to be performed in almost all patients without increasing morbidity and may reduce the number of covering stomas. Because division of the ileocecal and/or superior mesenteric arteries may be required, preservation of the marginal vascular arcade is essential whenever possible.

Factors predicting postoperative infectious complications and early induction of inflammatory mediators in ulcerative colitis patients.

BackgroundPositive outcomes after restorative proctocolectomy are compromised by a number of specific septic complications. However, there is no useful perioperative marker predicting postoperative infectious complications (PICs) in steroid overdosed patients with ulcerative colitis (UC).MethodsTo determine factors associated with PICs and their relation to circulating levels of pro- and anti-inflammatory cytokines and neutrophil elastase (NE), we obtained perioperative blood samples from 60 UC patients.ResultsPostoperative infectious complications were identified in 47% of cases. Patients who developed PICs had significantly longer disease duration, had been administered a greater total preoperative dosage of prednisolone, and had a higher body mass index. Logistic regression analysis showed that the total preoperative dosage of prednisolone was independently associated with the development of PICs. These patients showed suppressed systemic inflammation and pro- and anti-inflammatory cytokine induction. An early increase in the NE level was found to be predictive of PICs in the high-dose group, whereas there was no significant difference in neutrophil counts between the high- and low-dose groups.ConclusionsCirculating NE levels in the early postoperative period might be a useful predictor of PICs in immune-controlled UC patients who received high doses of steroids.

Systemic Analysis of Predictive Biomarkers for Recurrence in Colorectal Cancer Patients Treated with Curative Surgery

AbstractBackgroundPreoperative serum systemic inflammatory response (SIR) in patients with colorectal cancer (CRC) has been reported to be a predictive biomarker of early recurrence. The molecular status of CRC, including microsatellite instability (MSI), BRAF and KRAS mutations, and tumor-infiltrating lymphocytes (TILs), has also been associated with recurrence in CRC patients treated with curative surgery.AimWe investigated the impacts of SIR status, TILs, and MSI on recurrence in curative CRC patients.MethodsIn this retrospective study, we enrolled 157 patients with stage I–III CRC undergoing curative surgery, for whom preoperative neutrophil/lymphocyte ratio (NLR), platelet/lymphocyte ratio (PLR), and C-reactive protein (CRP) data were available as indicators of SIR status. Molecular status was evaluated by counting TILs as the numbers of intratumoral Foxp3- and CD8-positive T cells by immunohistochemistry. MSI status was determined using five mononucleotide repeat microsatellite markers.ResultsKaplan–Meier analysis of SIR indicators revealed that higher CRP, NLR, and PLR were associated with significantly poorer disease-free survival (DFS). Low levels of infiltrating CD8-positive T cells in CRC tissue was a significant predictor of poor DFS. Multivariate analysis showed that few infiltrating CD8-positive T cells and high serum CRP levels were independent predictive factors for recurrence. Furthermore, the combination of high CRP and few infiltrating CD8-positive T cells increased the predictive accuracy in these patients.ConclusionsThe results of this study suggest that both CRP levels in preoperative serum and CD8 T cells in CRC tissue are useful biomarkers for predicting early relapse in CRC patients treated with curative surgery.