Mutational signature in colorectal cancer caused by genotoxic pks+ E. coli.

Various species of the intestinal microbiota have been associated with the development of colorectal cancer1,2, but it has not been demonstrated that bacteria have a direct role in the occurrence of oncogenic mutations. Escherichia coli can carry the pathogenicity island pks, which encodes a set of enzymes that synthesize colibactin3. This compound is believed to alkylate DNA on adenine residues4,5 and induces double-strand breaks in cultured cells3. Here we expose human intestinal organoids to genotoxic pks+ E. coli by repeated luminal injection over five months. Whole-genome sequencing of clonal organoids before and after this exposure revealed a distinct mutational signature that was absent from organoids injected with isogenic pks-mutant bacteria. The same mutational signature was detected in a subset of 5,876 human cancer genomes from two independent cohorts, predominantly in colorectal cancer. Our study describes a distinct mutational signature in colorectal cancer and implies that the underlying mutational process results directly from past exposure to bacteria carrying the colibactin-producing pks pathogenicity island.

Development and Application of Regenerative Medicine in Inflammatory Bowel Disease.

BACKGROUND: Inflammatory bowel disease (IBD) is characterized by idiopathic and chronic inflammation arising elsewhere within the gastrointestinal tract. Consequently, the mucosal tissue is destroyed during the active phase of the disease, and therefore, spontaneous repair of damaged tissue is required to restore the function and long-term homeostasis of the intestinal mucosa. Also, in patients with refractory Crohn's disease, loss of massive intestinal function can lead to short bowel syndrome or may lead to fatal intestinal failure. SUMMARY: The concept of mucosal healing shares the idea that both regulation of mucosal inflammation and repair of the damaged mucosa are critical to achieve the ideal clinical outcome in patients with IBD. However, current treatments lack the option of those targeted to mucosal repair, and therefore, patients must achieve mucosal healing depending on their intrinsic system. To counteract inflammation-induced mucosal damage, various biologics or cell-based treatments are currently being developed. In the early developmental phase, various growth factors have been tested for their ability to promote mucosal repair. However, most of these factors did not show clinical benefit, except the recombinant glucagon-like peptide-2 (GLP-2). On the contrary, cell-based treatments are rapidly emerging, using both somatic stem cells and pluripotent stem cells. KEY MESSAGES: In this review, we focus on the current state of factor-based or cell-based regenerative medicine in the treatment of IBD. Additionally, we would like to introduce current examples of tissue engineering technologies and provide future prospects for the application of regenerative medicine in IBD.

Small intestinal stem cell identity is maintained with functional Paneth cells in heterotopically grafted epithelium onto the colon.

To develop stem cell therapy for small intestinal (SI) diseases, it is essential to determine whether SI stem cells in culture retain their tissue regeneration capabilities. By using a heterotopic transplantation approach, we show that cultured murine SI epithelial organoids are able to reconstitute self-renewing epithelia in the colon. When stably integrated, the SI-derived grafts show many features unique only to the SI but distinct from the colonic epithelium. Our study provides evidence that cultured adult SI stem cells could be a source for cell therapy of intestinal diseases, maintaining their identity along the gastrointestinal tract through an epithelium-intrinsic mechanism.

Functional analysis of isoflavones using patient-derived human colonic organoids.

Inflammatory bowel disease (IBD) comprises two major subtypes, ulcerative colitis (UC) and Crohn's disease, which are multifactorial diseases that may develop due to genetic susceptibility, dysbiosis, or environmental factors. Environmental triggers of IBD include food-borne factors, and a previous nationwide survey in Japan identified pre-illness consumption of isoflavones as a risk factor for UC. However, the precise mechanisms involved in the detrimental effects of isoflavones on the intestinal mucosa remain unclear. The present study employed human colonic organoids (hCOs) to investigate the functional effect of two representative isoflavones, genistein and daidzein, on human colonic epithelial cells. The addition of genistein to organoid reformation assays significantly decreased the number and size of reformed hCOs compared with control and daidzein treatment, indicating an inhibitory effect of genistein on colonic cell/progenitor cell function. Evaluation of the phosphorylation status of 49 different receptor tyrosine kinases showed that genistein selectively inhibited phosphorylation of epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor (HGFR). We established a two-dimensional wound-repair model using hCOs and showed that genistein significantly delayed the overall wound-repair response. Our results collectively show that genistein may exert its detrimental effects on the intestinal mucosa via negative regulation of stem/progenitor cell function, possibly leading to sustained mucosal injury and the development of UC.

A multicenter study on the clinical characteristics and risk factors of in-hospital mortality in patients with mechanical complications following acute myocardial infarction.

Mechanical complications (MCs) following acute myocardial infarction (AMI), such as ventricular septal rupture (VSR), free-wall rupture (FWR), and papillary muscle rupture (PMR), are fatal. However, the risk factors of in-hospital mortality among patients with MCs have not been previously reported in Japan. The purpose of this study was to evaluate the prognostic factors of in-hospital mortality in these patients. The study cohort consisted of 233 consecutive patients with MCs from the registry of 10 facilities in the Cardiovascular Research Consortium-8 Universities (CIRC-8U) in East Japan between 1997 and 2014 (2.3% of 10,278 AMI patients). The authors conducted a retrospective observational study to analyse the correlation between the subtypes of MCs with in-hospital mortality, clinical data, and medical treatment. We observed a decreasing incidence of MC (1997-2004: 3.7%, 2005-2010: 2.1%, 2011-2014: 1.9%, p < 0.001). In-hospital mortality among patients with MCs was 46%. Thirty-three percent of patients with MCs were not able to undergo surgical repair due to advanced age or severe cardiogenic shock. In-hospital mortality among patients who had undergone surgical repair was 29% (VSR: 21%, FWR: 33%, PMR: 60%). In patients with MCs, hazard ratio for in-hospital mortality according to multivariate analysis of without surgical repair was 5.63 (95% CI 3.54-8.95). In patients with surgical repair, the hazard ratios of blow-out-type FWR (5.53, 95% confidence interval (CI) 2.22-13.76), those with renal dysfunction (3.11, 95% CI 1.37-7.05), and those receiving venoarterial extracorporeal membrane oxygenation (VA-ECMO) (3.79, 95% CI 1.81-7.96) were significantly high. Although primary percutaneous coronary intervention (PCI) is associated with decreased incidence of MCs, high in-hospital mortality persisted in patients with MCs that also presented with renal dysfunction and in those requiring VA-ECMO. Early detection and surgical repair of MCs are essential.

Indispensable role of Notch ligand-dependent signaling in the proliferation and stem cell niche maintenance of APC-deficient intestinal tumors.

Ligand-dependent activation of Notch signaling is required to maintain the stem-cell niche of normal intestinal epithelium. However, the precise role of Notch signaling in the maintenance of the intestinal tumor stem cell niche and the importance of the RBPJ-independent non-canonical pathway in intestinal tumors remains unknown. Here we show that Notch signaling was activated in LGR5+ve cells of APC-deficient mice intestinal tumors. Accordingly, Notch ligands, including Jag1, Dll1, and Dll4, were expressed in these tumors. In vitro studies using tumor-derived organoids confirmed the intrinsic Notch activity-dependent growth of tumor cells. Surprisingly, the targeted deletion of Jag1 but not RBPJ in LGR5+ve tumor-initiating cells resulted in the silencing of Hes1 expression, disruption of the tumor stem cell niche, and dramatic reduction in the proliferation activity of APC-deficient intestinal tumors in vivo. Thus, our results highlight the importance of ligand-dependent non-canonical Notch signaling in the proliferation and maintenance of the tumor stem cell niche in APC-deficient intestinal adenomas.

Higher hemoglobin A1c levels are associated with impaired left ventricular diastolic function and higher incidence of adverse cardiac events in patients with nonischemic dilated cardiomyopathy.

We aimed to elucidate the relationship between glycated hemoglobin (HbA1c), cardiac systolic/diastolic function, and heart failure (HF) prognosis during guideline-directed medical therapy in patients with nonischemic dilated cardiomyopathy (NIDCM). We evaluated 283 hospitalized NIDCM patients, who were grouped according to baseline (BL) and 1-year (1Y) levels of HbA1c (<6.0, 6.0-6.9, and ≥7.0 %). The primary endpoint was defined as either readmission for HF worsening or cardiac death. Approximately half of the patients had BL- or 1Y-HbA1c ≥6.0 % (31 % at BL, 34 % at 1Y had 6.0-6.9 %; 12 % at BL, 12 % at 1Y had ≥7.0 %). The absolute value of left ventricular ejection fraction (LVEF) and its improvement during 1 year showed no significant difference among the 1Y-HbA1c groups (p = 0.273), whereas a lower absolute value and a more significant reduction in the early diastolic velocity of the mitral annulus (E a) were seen in the group with 1Y-HbA1c ≥7.0 % (both p < 0.001). In multiple regression analysis, higher 1Y-plasma B-type natriuretic peptide and lower 1Y-Ea were independently associated with higher 1Y-HbA1c (both adjusted p < 0.05). The cumulative incidence of the primary endpoint was highest in the group with 1Y-HbA1c ≥7.0 % (log-rank p = 0.001). Multivariate analysis demonstrated that higher 1Y-HbA1c was independently associated with a higher incidence of the primary endpoint (adjusted p = 0.005). In conclusion, hyperglycemia during clinical follow-up is a risk factor for progression of concomitant LV abnormal relaxation, leading to poor HF prognosis in patients with NIDCM.

Prognostic significance of beta-blocker up-titration in conjunction with cardiac resynchronization therapy in heart failure management.

Clinical practice guidelines emphasize that optimal pharmacotherapy, including beta-blockers (BB), is a prerequisite before receiving cardiac resynchronization therapy (CRT) in eligible patients with heart failure (HF). However, the optimal dose of BB before CRT implantation cannot be tolerated in a number of patients. Sixty-three consecutive patients who underwent CRT in 2006-2013 were retrospectively investigated. Before receiving CRT, BB could not be introduced in 20 patients (32 %); the daily carvedilol-equivalent dose in other 43 patients was 5.6 ± 7.0 mg because of significant HF and bradycardia. After receiving CRT, BB could be introduced in almost all patients (n = 61, 97 %), and the daily BB dose increased from 5.6 ± 7.0 to 13.2 ± 7.8 mg (P < 0.001). Multivariate analysis indicated that the change of BB dose after CRT was independently associated with improved left ventricular end-systolic volume (LVESV) [ß = -0.36; 95 % confidence interval (CI) -2.13 to -0.45; P < 0.01] after 6-months follow-up. Furthermore, Cox proportional hazard analysis also showed that the change in the BB dose (hazard ratio, 0.92; 95 % CI, 0.87-0.98; P < 0.01) as well as the New York Heart Association functional classification was an independent predictor of cardiac events. After initiating CRT, BB therapy can be introduced and up-titrated in intolerant HF patients. The up-titrated dose of BB after CRT was an independent predictor for the improvement of LVESV and HF prognosis.

Clinical significance of endomyocardial biopsy in conjunction with cardiac magnetic resonance imaging to predict left ventricular reverse remodeling in idiopathic dilated cardiomyopathy.

Endomyocardial biopsy (EMB) and cardiac magnetic resonance (CMR) are useful modalities to study the characteristics of myocardial tissue. However, the prognostic impact of both diagnostic tools to predict subsequent left ventricular reverse remodeling (LVRR) has not been well elucidated. A total of 187 consecutive patients with idiopathic dilated cardiomyopathy (IDCM) who were treated by optimal pharmacotherapy (OPT) and underwent EMB of the LV wall were investigated. The myocardial specimens were semiquantitatively evaluated measuring cardiomyocyte degeneration (CD), interstitial fibrosis (IF), and hypertrophy. In addition, late gadolinium enhancement (LGE)-CMR was performed in 78 (48 %) patients. Seventy-eight (48 %) patients developed LVRR, defined as a ≥10 % increase in LV ejection fraction with a ≥10 % decrease in indexed LV end-diastolic dimension at 12 months after OPT. Multivariate regression analysis revealed that CD (P = 0.003), but not IF (P = 0.320), was an independent predictor of LVRR. In the patients with not only EMB but also CMR, the CD score and LGE area were independent predictors of LVRR (odds ratios/P values 0.268/0.010, 0.855/<0.001, respectively). The patients with mild CD and negative LGE had a better achievement rate of LVRR than those with severe CD and positive LGE (74 vs. 19 %). A combination of CD score on EMB and LGE-CMR is useful to predict subsequent LVRR in IDCM patients.

Cardiac fibrosis detected by magnetic resonance imaging on predicting time course diversity of left ventricular reverse remodeling in patients with idiopathic dilated cardiomyopathy.

This study aimed to identify the association between the time course of left ventricular reverse remodeling (LVRR) and late gadolinium enhancement in cardiac magnetic resonance imaging (LGE-cMRI) in patients with idiopathic dilated cardiomyopathy (IDCM). We identified 214 IDCM patients treated by optimal pharmacotherapies. LVRR was defined as ≥10 % increment in LV ejection fraction along with ≥10 % reduction in LV end-diastolic dimension. Findings of LGE-cMRI focusing on presence and extent of LGE were evaluated at baseline. Echocardiographic evaluation for detecting LVRR was performed in all patients for 3 years. The primary endpoint was defined as composite events (CEs) including readmission for heart failure, detection of major ventricular arrhythmia, and all-cause mortality. LVRR was found at <1 year in 59 patients (28 %, early responder), ≥1 year in 56 patients (26 %, late responder), and was absent in 99 patients (46 %, non-responder). Multivariate Cox-proportional hazards analysis revealed that both early responders (P = 0.02) and late responders (P < 0.001) had lower incidence of CEs than non-responders. Among 66 subjects (23 %) with complete cMRI evaluation, LGE was detected more often in late and non- than early responders (65, 83 vs. 23 % P < 0.001, respectively), whereas the LGE area was smaller in both early and late than non-responders (2 ± 3, 4 ± 3 vs. 12 ± 10 %, P < 0.001, respectively). In conclusion, evaluating the presence and the extent of LGE is useful for predicting the clinical differences of LVRR time course and subsequent long-term outcomes.

Time course of left ventricular reverse remodeling in response to pharmacotherapy: clinical implication for heart failure prognosis in patients with idiopathic dilated cardiomyopathy.

The present study aimed to identify the clinical significance of differences in detection timings of left ventricular reverse remodeling (LVRR) on heart failure (HF) prognosis in patients with idiopathic dilated cardiomyopathy (IDCM). We investigated 207 patients with IDCM who underwent pharmacotherapeutic treatment. LVRR was defined as improvements in both LV ejection fraction ≥10 % and indexed LV end-diastolic dimension (LVEDDi) ≥10 %. Patients were stratified into 3 groups by LVRR timing: patients with LVRR <24 months (Early LVRR), those with LVRR ≥24 months (Delayed LVRR), and those without LVRR during the entire follow-up period (No LVRR). The major endpoint was first detection of composite event including readmission for decompensated HF, major ventricular arrhythmias, or all-cause mortality. LVRR was recognized in 108 patients (52 %): Early LVRR in 83 (40 %), Delayed LVRR in 25 (12 %), and No LVRR in 99 (48 %). The survival rate for the major endpoint was significantly higher for Delayed LVRR than for No LVRR (P = 0.001); there was no significant difference between Early and Delayed LVRR. Among patients without LVRR <24 months (Delayed + No LVRR), receiver operating characteristic curve analysis showed that the area under the curve for improvement in LVEDDi during the first 6 months for predicting subsequent LVRR (Delayed LVRR) [0.822 (95 % confidence interval, 0.740-0.916; P = 0.038)] was greater than that for improvement in LVEF. In conclusion, LVRR was a favorable prognostic indicator in patients with IDCM irrespective of its detection timing. Reduced LVEDDi during the first 6 months was predictive for subsequent LVRR in the later phase.

Impact of Doctor Car with Mobile Cloud ECG in reducing door-to- balloon time of Japanese ST-elevation myocardial infarction patients.

Early reperfusion by percutaneous coronary intervention (PCI) is the current standard therapy for ST-elevation myocardial infarction (STEMI). To achieve better prognoses for these patients, reducing the door-to-balloon time is essential. As we reported previously, the Kitasato University Hospital Doctor Car (DC), an ambulance with a physician on board, is equipped with a novel mobile cloud 12-lead ECG system. Between September 2011 and August 2013, there were 260 emergency dispatches of our Doctor Car, of which 55 were for suspected acute myocardial infarction with chest pain and cold sweat. Among these 55 calls, 32 patients received emergent PCI due to STEMI (DC Group). We compared their data with those of 76 STEMI patients who were transported directly to our hospital by ambulance around the same period (Non-DC Group). There were no differences in patient age, gender, underlying diseases, or Killip classification between the two groups. The door-to-balloon time in the DC group was 56.1 ± 13.7 minutes and 74.0 ± 14.1 minutes in the Non-DC Group (P < 0.0001). Maximum levels of CPK were 2899 ± 308 and 2876 ± 269 IU/L (P = 0.703), and those of CK-MB were 292 ± 360 and 295 ± 284 ng/mL (P = 0.423), respectively, in the 2 groups. The Doctor Car system with the Mobile Cloud ECG was useful for reducing the door-to-balloon time.

Hes1 promotes the IL-22-mediated antimicrobial response by enhancing STAT3-dependent transcription in human intestinal epithelial cells.

Notch signaling plays an essential role in the proliferation and differentiation of intestinal epithelial cells (IECs). We have previously shown that Notch signaling is up-regulated in the inflamed mucosa of ulcerative colitis (UC) and thereby plays an indispensable role in tissue regeneration. Here we show that in addition to Notch signaling, STAT3 signaling is highly activated in the inflamed mucosa of UC. Forced expression of the Notch target gene Hes1 dramatically enhanced the IL-22-mediated STAT3-dependent transcription in human IECs. This enhancement of STAT3-dependent transcription was achieved by the extended phosphorylation of STAT3 by Hes1. Microarray analysis revealed that Hes1-mediated enhancement of IL-22-STAT3 signaling significantly increased the induction of genes encoding antimicrobial peptides, such as REG1A, REG3A and REG3G, in human IECs. Conversely, the reduction of Hes1 protein levels with a γ-secretase inhibitor significantly down-regulated the induction of those genes in IECs, resulting in a markedly poor response to IL-22. Our present findings identify a new role for the molecular function of Hes1 in which the protein can interact with cytokine signals and regulate the immune response of IECs.

Fluorescent labelling of intestinal epithelial cells reveals independent long-lived intestinal stem cells in a crypt.

BACKGROUND AND AIMS: The dynamics of intestinal stem cells are crucial for regulation of intestinal function and maintenance. Although crypt stem cells have been identified in the intestine by genetic marking methods, identification of plural crypt stem cells has not yet been achieved as they are visualised in the same colour. METHODS: Intestinal organoids were transferred into Matrigel® mixed with lentivirus encoding mCherry. The dynamics of mCherry-positive cells was analysed using time-lapse imaging, and the localisation of mCherry-positive cells was analysed using 3D immunofluorescence. RESULTS: We established an original method for the introduction of a transgene into an organoid generated from mouse small intestine that resulted in continuous fluorescence of the mCherry protein in a portion of organoid cells. Three-dimensional analysis using confocal microscopy showed a single mCherry-positive cell in an organoid crypt that had been cultured for >1year, which suggested the presence of long-lived mCherry-positive and -negative stem cells in the same crypt. Moreover, a single mCherry-positive stem cell in a crypt gave rise to both crypt base columnar cells and transit amplifying cells. Each mCherry-positive and -negative cell contributed to the generation of organoids. CONCLUSIONS: The use of our original lentiviral transgene system to mark individual organoid crypt stem cells showed that long-lived plural crypt stem cells might independently serve as intestinal epithelial cells, resulting in the formation of a completely functional villus.

[The case of a giant malignant phyllodes tumor of the breast].

A 75-year-old female patient complained of a mass in her left breast 2 years ago. The patient experienced a rapid enlargement of the mass 2 months later and visited our hospital. A computed tomography (CT) scan indicated a 25-cm tumor with infiltration of the left breast skin. Pectoral muscle invasion was considered. Swelling of the axillary lymph node and remote metastases were not found. A needle biopsy indicated a phyllodes tumor. A pectoral muscle-preserving mastectomy was undertaken. The tumor weighed 7.1 kg. Pathological examination indicated hyperplasia of the stroma and part of the epithelium, which had invaded the skin layer and fatty tissue. The pathological diagnosis was a malignant phyllodes tumor. This paper reports the case of a giant malignant phyllodes tumor.

Single cell analysis revealed that two distinct, unique CD4+ T cell subsets were increased in the small intestinal intraepithelial lymphocytes of aged mice.

Recent advances in research suggest that aging has a controllable chronic inflammatory disease aspect. Aging systemic T cells, which secrete pro-inflammatory factors, affect surrounding somatic cells, and accelerate the aging process through chronic inflammation, have attracted attention as potential therapeutic targets in aging. On the other hand, there are few reports on the aging of the intestinal immune system, which differs from the systemic immune system in many ways. In the current study, we investigated the age-related changes in the intestinal immune system, particularly in T cells. The most significant changes were observed in the CD4+ T cells in the small intestinal IEL, with a marked increase in this fraction in old mice and reduced expression of CD27 and CD28, which are characteristic of aging systemic T cells. The proliferative capacity of aging IEL CD4+ T cells was significantly more reduced than that of aging systemic T cells. Transcriptome analysis showed that the expression of inflammatory cytokines was not upregulated, whereas Cd8α, NK receptors, and Granzymes were upregulated in aging IEL CD4+ T cells. Functional analysis showed that aging IEL T cells had a higher cytotoxic function against intestinal tumor organoids in vitro than young IEL T cells. scRNAseq revealed that splenic T cells show a transition from naïve to memory T cells, whereas intestinal T cells show the emergence of a CD8αα+CD4+ T cell fraction in aged mice, which is rarely seen in young cells. Further analysis of the aging IEL CD4+ T cells showed that two unique subsets are increased that are distinct from the systemic CD4+ T cells. Subset 1 has a pro-inflammatory component, with expression of IFNγ and upregulation of NFkB signaling pathways. Subset 2 does not express IFNγ, but upregulates inhibitory molecules and nIEL markers. Expression of granzymes and Cd8a was common to both. These fractions were in opposite positions in the clustering by UMAP and had different TCR repertoires. They may be involved in the suppression of intestinal aging and longevity through anti-tumor immunity, elimination of senescent cells and stressed cells in the aging environment. This finding could be a breakthrough in aging research.