Kosaku Nanki

Efficient genetic engineering of human intestinal organoids using electroporation

Gene modification in untransformed human intestinal cells is an attractive approach for studying gene function in intestinal diseases. However, because of the lack of practical tools, such studies have largely depended upon surrogates, such as gene-engineered mice or immortalized human cell lines. By taking advantage of the recently developed intestinal organoid culture method, we developed a methodology for modulating genes of interest in untransformed human colonic organoids via electroporation of gene vectors. Here we describe a detailed protocol for the generation of intestinal organoids by culture with essential growth factors in a basement membrane matrix. We also describe how to stably integrate genes via the piggyBac transposon, as well as precise genome editing using the CRISPR-Cas9 system. Beginning with crypt isolation from a human colon sample, genetically modified organoids can be obtained in 3 weeks.

Clinical Efficacy and Safety of Oral Qing-Dai in Patients with Ulcerative Colitis: A Single-Center Open-Label Prospective Study

Background/Aims: Chinese herbal medicine Qing-Dai (also known as indigo naturalis) has been used to treat various inflammatory conditions. However, not much has been studied about the use of oral Qing-Dai in the treatment for ulcerative colitis (UC) patients. Studies exploring alternative treatments for UC are of considerable interest. In this study, we aimed at prospectively evaluating the safety and efficacy of Qing-Dai for UC patients. Methods: The open-label, prospective pilot study was conducted at Keio University Hospital. A total of 20 patients with moderate UC activity were enrolled. Oral Qing-Dai in capsule form was taken twice a day (daily dose, 2 g) for 8 weeks. Results: At week 8, the rates of clinical response, clinical remission, and mucosal healing were 72, 33, and 61%, respectively. The clinical and endoscopic scores, CRP levels, and fecal occult blood results were also significantly improved. We observed 2 patients with mild liver dysfunction; 1 patient discontinued due to infectious colitis and 1 patient discontinued due to mild nausea. Conclusion: This is the first prospective study indicating that oral Qing-Dai is effective for inducing remission in patients with moderate UC activity and can be tolerated. Thus, Qing-Dai may be considered an alternative treatment for patients, although further investigation is warranted.

Early Intervention with Adalimumab May Contribute to Favorable Clinical Efficacy in Patients with Crohn's Disease

Background: We evaluated the clinical efficacy of adalimumab (ADA) for Crohns disease (CD) and analyzed predictive factors for clinical remission and long-term prognosis. Methods: We retrospectively reviewed the medical records of 45 patients treated with ADA for CD at Keio University Hospital between October 2010 and March 2014. Clinical remission was defined as a Harvey-Bradshaw index of ≤4. Results: Twenty-eight of 45 patients (62.2%) achieved clinical remission at week 4. Among these 28 patients, 18 patients (64.3%) maintained clinical remission at week 26, and among these, 16 patients (88.9%) maintained clinical remission at week 52. Absence of a history of bowel resection and absence of prior anti-tumor necrosis factor (anti-TNF) therapy were significant predictive factors for clinical remission at week 4 upon multivariate logistic regression analyses. Younger age and a disease duration of ≤3 years correlated with clinical remission at week 26 upon univariate analyses. Patients without a history of bowel resection showed significantly better long-term prognosis than those with a history of bowel resection (p = 0.01). None of the patients contracted a serious infectious disease. Conclusions: Younger age, shorter duration of disease, being naive to anti-TNF antagonists, and absence of a history of bowel resection were associated with the efficacy of ADA in CD patients.

Ulcerative colitis endoscopic index of severity is associated with long-term prognosis in ulcerative colitis patients treated with infliximab

Ulcerative colitis (UC) is a chronic inflammatory bowel disease that is characterized by periods of remission and episodes of relapse. Mucosal healing is an emerging therapeutic target in UC and various scoring systems have been used. The UC endoscopic index of severity (UCEIS) is the only validated endoscopic index at present, with minimum interobserver variation. Correlation of UCEIS scores after treatment and clinical outcomes of UC has not been examined. In the present study, we aimed to evaluate the usefulness of UCEIS after treatment with infliximab.

Endocytoscopy can be used to assess histological healing in ulcerative colitis

Background and study aim Endocytoscopy (ECS) is used in the in vivo microscopic inspection of microstructural mucosal features and enables endoscopists to assess the histological severity of ulcerative colitis (UC). The aim of this study was to assess histological healing in UC patients by using ECS. Patients and methods A total of 64 patients in clinical and endoscopic remission who underwent ECS were selected. The correlation between the ECS score and Geboes score at the rectum was evaluated in patients with a Mayo endoscopic score (MES) of 0. The diagnostic accuracy of the ECS score for histological remission (Geboes score ≤ 2) was also assessed. Results The ECS score ranged from 0 to 5 in patients with endoscopic remission on conventional white-light images (MES of 0). The agreement between histological remission regarding the ECS score and the Geboes score had a κ value of 0.72, and the ECS score showed high accuracy for histological remission, with a sensitivity of 0.77, a specificity of 0.97, and a diagnostic accuracy of 0.86. Conclusion ECS can be used to assess histological healing in patients with UC without the need for biopsy specimens.

Recent trends and future directions for the medical treatment of ulcerative colitis

Recently, several medical treatments for ulcerative colitis (UC) have been developed, including 5-aminosalicylic acids (5-ASAs), corticosteroids, thiopurine, calcineurin inhibitors, and anti-tumor necrosis factor (TNF) α treatments. Treatment options including calcineurin inhibitors and anti-TNF treatment for refractory UC are discussed in this article. Furthermore, upcoming treatments are introduced, such as golimumab, vedolizumab, AJM300, tofacitinib. Budesonide foamwill be used as one treatment option in patients with distal colitis. Herbal medicine, such as Qing-Dai is also effective for active UC and may be useful for patients who are refractory to anti-TNFα treatments. In the near future, physicians will able to use many different treatments for UC patients. However, we should not forget 5-ASA and corticosteroids as the fundamental treatments for UC patients.

Divergent Routes toward Wnt and R-spondin Niche Independency during Human Gastric Carcinogenesis

Recent sequencing analyses have shed light on heterogeneous patterns of genomic aberrations in human gastric cancers (GCs). To explore how individual genetic events translate into cancer phenotypes, we established a biological library consisting of genetically engineered gastric organoids carrying various GC mutations and 37 patient-derived organoid lines, including rare genomically stable GCs. Phenotype analyses of GC organoids revealed divergent genetic and epigenetic routes to gain Wnt and R-spondin niche independency. An unbiased phenotype-based genetic screening identified a significant association between CDH1/TP53 compound mutations and the R-spondin independency that was functionally validated by CRISPR-based knockout. Xenografting of GC organoids further established the feasibility of Wnt-targeting therapy for Wnt-dependent GCs. Our results collectively demonstrate that multifaceted genetic abnormalities render human GCs independent of the stem cell niche and highlight the validity of the genotype-phenotype screening strategy in gaining deeper understanding of human cancers.

Endoscopic morphologic features of ulcerative colitis–associated dysplasia classified according to the SCENIC consensus statement

BACKGROUND AND AIMS Recent advances in endoscopic equipment and diagnostic techniques have made possible the detection of early dysplasia in the inflamed mucosa of ulcerative colitis (UC). The SCENIC consensus statement recommends the use of unified terminology for the morphology of dysplasia. In this study, we investigated the endoscopic features of high-grade dysplasia (HGD) in a clinical setting. METHODS We retrospectively identified 62 patients with UC who were diagnosed with colitis-associated cancer or HGD between 1997 and 2015. A total of 39 lesions of HGD detected by targeted biopsy sampling in 31 patients were reviewed, and the endoscopic morphology was classified according to the SCENIC guidelines. RESULTS In total, 0 (0%), 6 (15.4%), 19 (48.7%), 12 (30.8%), and 2 (5.1%) lesions with HGD were classified as pedunculated, sessile, superficial elevated, flat, and depressed, respectively. Nearly 80% of the lesions were located in the rectum or sigmoid colon. All flat and depressed lesions were red in color. Typically, sessile/superficial elevated lesions accompanied a flat area (Is+IIb/IIa+IIb). Ulceration was observed in 2 depressed lesions (5.1%). Although the borders were indistinct in 21 lesions (53.8%) without the use of magnifying colonoscopy, all lesions could be distinguished from the surrounding mucosa using magnifying endoscopy. CONCLUSIONS This is the first study to classify the morphologic features of HGD using the SCENIC guidelines in a clinical setting. Based on our findings, endoscopists should recognize that HGD is frequently associated with a flat/superficial elevated area and red discoloration and should inspect particularly carefully in the rectum and sigmoid colon. The findings of chromoendoscopy and magnifying colonoscopy may also be useful in distinguishing lesions from the surrounding mucosa.

Fecal microbiota transplantation for recurrent Clostridium difficile infection in a patient with ulcerative colitis

Fecal microbiota transplantation (FMT) has been reported as a safe and effective therapy in patients with refractory and recurrent Clostridium difficile infection (CDI). FMT has also been reported as a promising therapy in patients with ulcerative colitis (UC). Both, CDI and UC, are believed to be caused by dysbiosis, such as altered compositions or decreased diversity of the intestinal microbiota. This report describes a patient with UC in remission with a second recurrent episode of CDI, who was treated with FMT. A single FMT performed via colonoscopy completely resolved the patients diarrhea and eradicated C. difficile bacteriologically without any severe complications. Molecular biological analysis of the patients fecal microbiota showed that FMT could dramatically change the altered composition of intestinal microbiota and restore its diversity. Despite the restoration of the intestinal microbiota, FMT could not prevent a relapse of UC in this patient. However, it improved the intestinal symptoms of CDI and could prevent further recurrences of CDI.

The risk factor of clinical relapse in ulcerative colitis patients with low dose 5-aminosalicylic acid as maintenance therapy: A report from the IBD registry

Background 5-Aminosalicylic acids (5-ASA) are effective for ulcerative colitis (UC) as a maintenance therapy. It is not clear when and how to reduce a dose of 5-ASA after inducing remission. We aimed to investigate the clinical characteristics and evaluate the risk factors of relapse for UC patients receiving 5-ASA. Methods The medical records of prospectively registered UC patients who received oral 5-ASA as maintenance therapy between January and December 31, 2014, were investigated. The patients’ clinical characteristics in a 2-year follow-up were compared between a relapse group and a remission group. Results Of 527 UC patients receiving only oral 5-ASA, 390 (74.0%) maintained remission and 137 (26.0%) relapsed during the follow-up period. Multivariable analysis indicated that a shorter duration of disease remission (p < 0.001, OR: 1.24, 95% CI: 1.12–1.38) was statistically significant for each comparison between the remission and relapse groups among all the patients. Risk factors for clinical relapse were a shorter duration of disease remission (p <0.001, OR: 1.17, 95% CI: 1.04–1.33) in the high-dose 5-ASA group and a shorter duration of disease remission (p = 0.003, OR: 1.45, 95% CI: 1.13–1.89) and a history of steroid use (p = 0.048, OR: 4.73, 95% CI: 1.01–22.2) in the low-dose group. Conclusions A dose reduction of 5-ASA might be cautiously selected in UC patients with a history of steroid use and a shorter duration of disease remission.