Yuhki Koike

Preoperative C‐reactive protein as a prognostic and therapeutic marker for colorectal cancer

This study aimed to evaluate the significance of preoperative C‐reactive protein (CRP) as a prognostic marker for carcinoembryonic antigen (CEA)‐independent stage I or II colorectal cancer (CRC) patients.

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.

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.

Intraluminal appendiceal fluid is a predictive factor for recurrent appendicitis after initial successful non-operative management of uncomplicated appendicitis in pediatric patients

BACKGROUND The risk factors for recurrent appendicitis in pediatric patients are unclear. This study aimed to identify the predictive factors for recurrent appendicitis in pediatric patients who initially underwent successful non-operative management of uncomplicated appendicitis. METHODS Potential predictive factors for recurrent appendicitis in terms of clinical characteristics, laboratory data, and abdominal ultrasonography and computed tomography findings, were evaluated. RESULTS This study included 125 patients who underwent initial successful non-operative management of appendicitis. The rate of recurrent appendicitis was 19.2%, and the mean time to recurrence was 12.6 months. Univariate analyses found that rebound tenderness, muscle guarding, appendicoliths, appendiceal diameter >9 mm, and intraluminal appendiceal fluid were associated with recurrent appendicitis. Multivariate analysis identified only intraluminal appendiceal fluid as an independent predictor of recurrent appendicitis. CONCLUSIONS Intraluminal appendiceal fluid is a predictive factor for recurrent appendicitis after initial non-operative management. The results of this study provide valuable information that may help to determine the appropriate management during the first episode of appendicitis.

Breast milk-derived exosomes promote intestinal epithelial cell growth

BACKGROUND Breast milk administration prevents necrotizing enterocolitis (NEC). However, the mechanism remains unclear. Exosomes are cell-derived vesicles highly present in human milk and regulate intercellular signaling, inflammation, and immune response. We hypothesized that milk-derived exosomes beneficially affect intestinal epithelial cells. METHODS Rat milk was collected, and exosomes were isolated using ExoQuick reagent and visualized by Nanoparticle Tracking Analysis. Protein was extracted from encapsulating exosomes, and concentration was measured. 2×104 intestinal epithelial cells (IEC-18) were treated for five hours with 0.5-μg/μl exosomes, an equal volume of exosome-free milk, or control solution (PBS). IEC-18 viability was measured using a colorimetric assay (MTT), and gene expression was analyzed by qRT-PCR. Data were compared using one-way ANOVA with Bonferroni post-test. RESULTS Rat milk was collected, and exosome isolation was confirmed. Compared to control, treatment with exosomes significantly increased IEC viability, proliferation, and stem cell activity (all p<0.05). However, administration of exosome-free milk had less significant effects. CONCLUSIONS Rat milk-derived exosomes promote IEC viability, enhance proliferation, and stimulate intestinal stem cell activity. These findings provide insight into the mechanism of action of breast milk in the intestines. Exosome administration is a promising prevention method for infants at risk of developing NEC when breastfeeding is not tolerated.

Efficacy of Seprafilm for preventing adhesive bowel obstruction and cost–benefit analysis in pediatric patients undergoing laparotomy

PURPOSE This aim of the study is to determine whether the use of Seprafilm reduces the incidence and the medical costs of adhesive bowel obstruction (ABO) in children. METHODS Pediatric patients undergoing laparotomy were prospectively assigned to the Seprafilm group, n = 441). A historical control group consisted of children without using Seprafilm (n = 409). The incidence of ABO during a 24-month follow-up period was compared between the groups. To clarify the cost-benefit relations, expenses for Seprafilm and medical costs for hospitalization related to ABO in the Seprafilm group were compared with the ABO-associated hospitalization costs in the control group. RESULTS The cumulative incidence rate of ABO in the control group was significantly higher than in the Seprafilm group (4.9% vs. 2.0%, p = 0.015). Nearly all cases that required adhesiolysis had adhesions to areas other than the incision in both groups. In cost-benefit analysis, cost per patient was

Serum Level of Soluble Vascular Cell Adhesion Molecule 1 Is a Valuable Prognostic Marker in Colorectal Carcinoma

105 higher in the control group than in the Seprafilm group, but this did not reach significance (p = 0.63). CONCLUSIONS Seprafilm reduces the incidence of ABO in the pediatric patients undergoing laparotomy. Although associated medical costs in the Seprafilm group were not significantly reduced, use of Seprafilm did not lead to an increase in cost. Wider range of Seprafilm application or an additional anti-adhesion device may help in preventing adhesion to areas other than the incision.

Non-digestible oligosaccharides directly regulate host kinome to modulate host inflammatory responses without alterations in the gut microbiota

PURPOSE: Vascular cell adhesion molecule 1 plays an important role in solid tumor enlargement and/or metastasis. This study evaluated the clinical significance of measuring serum levels of soluble vascular cell adhesion molecule 1 in colorectal cancer and aimed to clarify the biologic significance of its local expression. METHODS: Serum was collected from 161 patients with colorectal cancer and 26 healthy volunteers. Cancer tissue was collected from 128 patients. The level of soluble vascular cell adhesion molecule 1 in serum and cancer tissue was measured by enzyme-linked immunosorbent assay. RESULTS: The mean soluble vascular cell adhesion molecule 1 level in patients was significantly higher than that in control subjects. Elevated serum soluble vascular cell adhesion molecule 1 was significantly associated with clinicopathologic parameters such as tumor size, lymph node metastasis, distant metastasis, and poor prognosis. In Cox multivariate analysis, distant metastasis and elevated serum soluble vascular cell adhesion molecule 1 level were independent risk factors predicting poor prognosis. The prognosis for Stage 2 patients positive for soluble vascular cell adhesion molecule 1 was comparable to that for Stage 3 patients. In addition, the serum level of soluble vascular cell adhesion molecule 1 level was correlated negatively with the cancer tissue level. CONCLUSION: The preoperative level of soluble vascular cell adhesion molecule 1 level reflected disease progression and was a sensitive biomarker for colorectal cancer, especially Stage 2 disease.

Tumor enucleation with preoperative endoscopic transpapillary stenting for pediatric insulinoma

BackgroundPrebiotics are non-digestible food ingredients that enhance the growth of certain microbes within the gut microbiota. Prebiotic consumption generates immune-modulatory effects that are traditionally thought to reflect microbial interactions within the gut. However, recent evidence suggests they may also impart direct microbe-independent effects on the host, though the mechanisms of which are currently unclear.MethodsKinome arrays were used to profile the host intestinal signaling responses to prebiotic exposures in the absence of microbes. Identified pathways were functionally validated in Caco-2Bbe1 intestinal cell line and in vivo model of murine endotoxemia.ResultsWe found that prebiotics directly regulate host mucosal signaling to alter response to bacterial infection. Intestinal epithelial cells (IECs) exposed to prebiotics are hyporesponsive to pathogen-induced mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) activations, and have a kinome profile distinct from non-treated cells pertaining to multiple innate immune signaling pathways. Consistent with this finding, mice orally gavaged with prebiotics showed dampened inflammatory response to lipopolysaccharide (LPS) without alterations in the gut microbiota.ConclusionsThese findings provide molecular mechanisms of direct host-prebiotic interactions to support prebiotics as potent modulators of host inflammation.