Effects of exposure to the neonicotinoid pesticide clothianidin on α-defensin secretion and gut microbiota in mice.

The mechanism by which the neonicotinoid pesticide clothianidin (CLO) disrupts the intestinal microbiota of experimental animals is unknown. We focused on α-defensins, which are regulators of the intestinal microbiota. Subchronic exposure to CLO induced dysbiosis and reduced short-chain fatty acid-producing bacteria in the intestinal microbiota of mice. Levels of cryptdin-1 (Crp1, a major α-defensin in mice) in feces and cecal contents were lower in the CLO-exposed groups than in control. In Crp1 immunostaining, Paneth cells in the jejunum and ileum of the no-observed-adverse-effect-level CLO-exposed group showed a stronger positive signal than control, likely due to the suppression of Crp1 release. Our results showed that CLO exposure suppresses α-defensin secretion from Paneth cells as part of the mechanism underlying CLO-induced dysbiosis.

A Basic Study of the Effects of Mulberry Leaf Administration to Healthy C57BL/6 Mice on Gut Microbiota and Metabolites.

Mulberry leaves contain α-glucosidase inhibitors, which have hypoglycemic effects and are considered functional foods. However, few reports have covered the effects of mulberry leaf components on normal gut microbiota and gut metabolites. Herein, gut microbiota analysis and NMR-based metabolomics were performed on the feces of mulberry leaf powder (MLP)-treated mice to determine the effects of long-term MLP consumption. Gut microbiota in the mouse were analyzed using 16S-rRNA gene sequencing, and no significant differences were revealed in the diversity and community structure of the gut microbiota in the C57BL/6 mice with or without MLP supplementation. Thirty-nine metabolites were identified via 1H-NMR analysis, and carbohydrates and amino acids were significantly (p < 0.01-0.05) altered upon MLP treatment. In the MLP-treated group, there was a marked increase and decrease in maltose and glucose concentrations, respectively, possibly due to the degradation inhibitory activity of oligosaccharides. After 5 weeks, all amino acid concentrations decreased. Furthermore, despite clear fluctuations in fecal saccharide concentrations, short-chain fatty acid production via intestinal bacterial metabolism was not strongly affected. This study provides the knowledge that MLP administration can alter the gut metabolites without affecting the normal gut microbiota, which is useful for considering MLP as a healthy food source.

Antimicrobial Properties and Mode of Action of Cryptdin-4, a Mouse α-Defensin Regulated by Peptide Redox Structures and Bacterial Cultivation Conditions.

Cryptdin-4 (crp4) is an enteric α-defensin derived from mice, and is a main mediator of immunity to oral infections and a determinant of the composition of the intestinal microbiota. Structurally, crp4 exists in two states: the oxidized form (crp4oxi), constrained by three invariant disulfide bonds, and the reduced form (crp4red) with six free thiol groups, both of which exist in the intestinal tract. In this study, the antibacterial mechanisms of crp4 in both forms under aerobic and anaerobic conditions were investigated using Escherichia coli (E. coli), an anaerobic facultative bacterium, as a model. Fluorescent dye studies revealed that both crp4oxi and crp4red exhibited antimicrobial activity against cells cultured under aerobic conditions via rapid membrane depolarization. Furthermore, the antioxidant treatment experiments suggested that only crp4oxi exhibited antimicrobial activity by the induction and accumulation of reactive oxygen species (ROS). However, under anaerobic culture conditions, the ability of both forms to disrupt the function of bacterial membranes decreased and activity was greatly reduced, but crp4red maintained some antimicrobial activity. This activity may be due to the inhibition of intracellular functions by DNA binding. Altogether, these data indicate that, according to its redox structure and the environmental redox conditions, crp4 could perform different antimicrobial activities via different mechanisms.

Application of Benchtop NMR for Metabolomics Study Using Feces of Mice with DSS-Induced Colitis.

Nuclear magnetic resonance (NMR)-based metabolomics, which comprehensively measures metabolites in biological systems and investigates their response to various perturbations, is widely used in research to identify biomarkers and investigate the pathogenesis of underlying diseases. However, further applications of high-field superconducting NMR for medical purposes and field research are restricted by its high cost and low accessibility. In this study, we applied a low-field, benchtop NMR spectrometer (60 MHz) employing a permanent magnet to characterize the alterations in the metabolic profile of fecal extracts obtained from dextran sodium sulfate (DSS)-induced ulcerative colitis model mice and compared them with the data acquired from high-field NMR (800 MHz). Nineteen metabolites were assigned to the 60 MHz 1H NMR spectra. Non-targeted multivariate analysis successfully discriminated the DSS-induced group from the healthy control group and showed high comparability with high-field NMR. In addition, the concentration of acetate, identified as a metabolite with characteristic behavior, could be accurately quantified using a generalized Lorentzian curve fitting method based on the 60 MHz NMR spectra.

Decreased Paneth cell α-defensins promote fibrosis in a choline-deficient L-amino acid-defined high-fat diet-induced mouse model of nonalcoholic steatohepatitis via disrupting intestinal microbiota.

Nonalcoholic steatohepatitis (NASH) is a chronic liver disease characterized by fibrosis that develops from fatty liver. Disruption of intestinal microbiota homeostasis, dysbiosis, is associated with fibrosis development in NASH. An antimicrobial peptide α-defensin secreted by Paneth cells in the small intestine is known to regulate composition of the intestinal microbiota. However, involvement of α-defensin in NASH remains unknown. Here, we show that in diet-induced NASH model mice, decrease of fecal α-defensin along with dysbiosis occurs before NASH onset. When α-defensin levels in the intestinal lumen are restored by intravenous administration of R-Spondin1 to induce Paneth cell regeneration or by oral administration of α-defensins, liver fibrosis is ameliorated with dissolving dysbiosis. Furthermore, R-Spondin1 and α-defensin improved liver pathologies together with different features in the intestinal microbiota. These results indicate that decreased α-defensin secretion induces liver fibrosis through dysbiosis, further suggesting Paneth cell α-defensin as a potential therapeutic target for NASH.

Shorter sleep time relates to lower human defensin 5 secretion and compositional disturbance of the intestinal microbiota accompanied by decreased short-chain fatty acid production.

Sleep is essential for our health. Short sleep is known to increase disease risks via imbalance of intestinal microbiota, dysbiosis. However, mechanisms by which short sleep induces dysbiosis remain unknown. Small intestinal Paneth cell regulates the intestinal microbiota by secreting antimicrobial peptides including α-defensin, human defensin 5 (HD5). Disruption of circadian rhythm mediating sleep-wake cycle induces Paneth cell failure. We aim to clarify effects of short sleep on HD5 secretion and the intestinal microbiota. Fecal samples and self-reported sleep time were obtained from 35 healthy middle-aged Japanese (41 to 60-year-old). Shorter sleep time was associated with lower fecal HD5 concentration (r = 0.354, p = 0.037), lower centered log ratio (CLR)-transformed abundance of short-chain fatty acid (SCFA) producers in the intestinal microbiota such as [Ruminococcus] gnavus group (r = 0.504, p = 0.002) and Butyricicoccus (r = 0.484, p = 0.003), and lower fecal SCFA concentration. Furthermore, fecal HD5 positively correlated with the abundance of these genera and SCFA concentration. These findings suggest that short sleep relates to disturbance of the intestinal microbiota via decreased HD5 secretion.

Efficient and simple genetic engineering of enteroids using mouse isolated crypts for investigating intestinal functions.

Intestinal epithelial cells separate subepithelial tissues from luminal environment formed with food, incoming pathogens, and resident intestinal microbiota, etc., and elicit various intestinal function. Enteroid, a three-dimensional culture system of small intestinal epithelial cells, has been widely used for analyzing the intestinal function, further a transgenic enteroid was developed to investigate the molecular mechanisms. However, conventional transgenic enteroid production method, which transfer gene into single stem cells, has limitations including low efficiency and time-consuming. Here we show that by gene transfer into small intestinal isolated crypts maintaining stem cell niche, a transgenic enteroid was obtained quickly and efficiently. Isolated crypts were transfected by lentiviral vector without separating into single cells, and transgenic enteroid composed of all lineages of intestinal epithelial cells was generated at day 7 with yield of 56%, maintaining the intestinal function in drug transport and innate immunity. Our efficient and simple transgenic enteroid generation method enables high-throughput investigation of intestinal epithelial cells and contributes to understanding intestinal function.

Hemodynamic response to intestinal pH stimulation measured with spectroscopic video imaging.

To investigate the relationship between the gut and skin (gut-skin axis), head skin hemodynamic responses to gut stimulation including the injection of acetic acid in nude mice were measured by spectroscopic video imaging, which was calculated using a modified Beer-Lambert formula. The relationship with blood proteins was also analyzed. The blood volume changes in three mice injected with acetic acid were highly reproducible in the mathematical model equation. Four proteins correlated with blood volume changes were all related to immunity. These results suggest that intestinal pH can alter the blood volume in the skin and induce immune-related responses.

Intestinal commensal microbiota and cytokines regulate Fut2+ Paneth cells for gut defense.

Paneth cells are intestinal epithelial cells that release antimicrobial peptides, such as α-defensin as part of host defense. Together with mesenchymal cells, Paneth cells provide niche factors for epithelial stem cell homeostasis. Here, we report two subtypes of murine Paneth cells, differentiated by their production and utilization of fucosyltransferase 2 (Fut2), which regulates α(1,2)fucosylation to create cohabitation niches for commensal bacteria and prevent invasion of the intestine by pathogenic bacteria. The majority of Fut2- Paneth cells were localized in the duodenum, whereas the majority of Fut2+ Paneth cells were in the ileum. Fut2+ Paneth cells showed higher granularity and structural complexity than did Fut2- Paneth cells, suggesting that Fut2+ Paneth cells are involved in host defense. Signaling by the commensal bacteria, together with interleukin 22 (IL-22), induced the development of Fut2+ Paneth cells. IL-22 was found to affect the α-defensin secretion system via modulation of Fut2 expression, and IL-17a was found to increase the production of α-defensin in the intestinal tract. Thus, these intestinal cytokines regulate the development and function of Fut2+ Paneth cells as part of gut defense.

Effect of early intensive rehabilitation on the clinical outcomes of patients with acute stroke.

AIM: Intensive rehabilitation effectively improves physical functions in patients with acute stroke, but the frequency of intervention and its cost-effectiveness are poorly studied. This study aimed to examine the effect of early high-frequency rehabilitation intervention on inpatient outcomes and medical expenses of patients with stroke. METHODS: The study retrospectively included 1759 patients with acute stroke admitted to the Kobe City Medical Center General Hospital between 2013 and 2016. Patients with a transient ischemic attack, subarachnoid hemorrhage, and those who underwent urgent surgery were excluded. Patients were divided into two groups according to the frequency of rehabilitation intervention: the high-frequency intervention group (>2 times/day, n = 1105) and normal-frequency intervention group (<2 times/day, n = 654). A modified Rankin scale score ≤2 at discharge, immobility-related complications and medical expenses were compared between the groups. RESULTS: The high-frequency intervention group had a significantly shorter time to first rehabilitation (median [interquartile range], 19.0 h [13.1-38.4] vs. 24.7 h [16.1-49.4], P < 0.001) and time to first mobilization (23.3 h [8.7-47.2] vs. 22.8 h [5.7-62.3], P = 0.65) than the normal-frequency intervention group. Despite higher disease severity, the high-frequency intervention group exhibited favorable outcomes at discharge (modified Rankin scale, ≤2; adjusted odds ratio, 1.89; 95% confidence interval, 1.25-2.85; P = 0.002). No significant differences were observed between the two groups concerning the rate of immobility-related complications and total medical expenses during hospitalization. CONCLUSIONS: High-frequency intervention was associated with improved outcomes and decreased medical expenses in patients with stroke. Our results may contribute to reducing medical expenses by increasing the efficiency of care delivery. Geriatr Gerontol Int 2021; 21: 623-628.

Decrease of α-defensin impairs intestinal metabolite homeostasis via dysbiosis in mouse chronic social defeat stress model.

Psychological stress has been reported to relate to dysbiosis, imbalance of the intestinal microbiota composition, and contribute to the onset and exacerbation of depression, though, underlying mechanisms of psychological stress-related dysbiosis have been unknown. It has been previously established that α-defensins, which are effector peptides of innate enteric immunity produced by Paneth cells in the small intestine, play an important role in regulation of the intestinal microbiota. However, the relationship between disruption of intestinal ecosystem and α-defensin under psychological stress is yet to be determined. Here we show using chronic social defeat stress (CSDS), a mouse depression model that (1) the exposure to CSDS significantly reduces α-defensin secretion by Paneth cells and (2) induces dysbiosis and significant composition changes in the intestinal metabolites. Furthermore, (3) they are recovered by administration of α-defensin. These results indicate that α-defensin plays an important role in maintaining homeostasis of the intestinal ecosystem under psychological stress, providing novel insights into the onset mechanism of stress-induced depression, and may further contribute to discovery of treatment targets for depression.

Simultaneous real-time analysis of Paneth cell and intestinal stem cell response to interferon-γ by a novel stem cell niche tracking method.

Paneth cells and Lgr5+ intestinal stem cells (Lgr5+ ISCs) constitute the stem cell niche and maintain small intestinal epithelial integrity by recognizing various niche factors derived from subepithelial cells and external antigens. Although it has been known that interferon-γ (IFN-γ), a Th1 cytokine, is associated with intestinal epithelial disruption during inflammation as a niche factor, dynamics of Paneth cells and Lgr5+ ISCs in response to IFN-γ remain to be understood. Here we show that CAG-tdTomato;Lgr5-EGFP (CT-LE) mice generated in this study enable to identify Paneth cells and Lgr5+ ISCs separately by fluorescence signals. Lgr5+ ISCs underwent cell death a little earlier than Paneth cells in response to IFN-γ by simultaneous tracking using CT-LE mice. In addition, the timing of cell death in most Paneth cells overlapped with Lgr5+ ISCs, suggesting that Paneth cell depletion is induced directly by IFN-γ. Taken together, we established a novel simultaneous stem cell niche tracking method and clarified the involvement of both Paneth cells and Lgr5+ ISCs in stem cell niche damage induced by IFN-γ, further contribute to understanding the mechanism for maintaining intestinal homeostasis by stem cell niche.

Optimal phase analysis of electrocardiogram-gated computed tomography angiography in patients with Stanford type A acute aortic dissection.

OBJECTIVE: To determine the phase that facilitates flap observation of the ascending aorta in Stanford type A acute aortic dissection with perfused false lumen. METHODS: We reconstructed retrospective Electrocardiogram-gated Computed Tomography Angiography images of the ascending aorta of all 20 patients to 20 phases of curved-multiplanar reconstruction in 5% increment. One radiologist created and randomized 10 cross-sectional images of each phase for every patient and two radiologists scored these images on a 5-point scale depending on the degree of flap stoppage. We calculated the average score for each phase of each case and compared them among the three groups. RESULTS: Image scores were significantly better in the 65 %-100 % R-R interval group than those in the 5%-30 % (p < 2e-16) and 35 %-60 % R-R interval groups(p = 7.2e-10). Similar scores were observed in the Heart Rate > 70 group (p = 0.00039, 2.2e-14). Moreover a similar tendency was observed in the arrhythmia group (p = 0.0035, 0.294). No difference was found in the degree of flap stoppage in the 65 %-100 % R-R interval group between the Heart Rate > 70 and Heart Rate ≤ 70 groups (p = 0.466) and between the arrhythmia and non-arrhythmia groups (p = 0.1240). CONCLUSION: In observing the ascending aorta, We obtained a good image at 65 %-100 % R-R interval and similar tendency was observed in the patients with arrhythmia.

Expression and Localization of Paneth Cells and Their α-Defensins in the Small Intestine of Adult Mouse.

Paneth cells contribute to intestinal innate immunity by sensing bacteria and secreting α-defensin. In Institute of Cancer Research (ICR) mice, α-defensin termed cryptdin (Crp) in Paneth cells consists of six major isoforms, Crp1 to 6. Despite accumulating evidences that α-defensin functions in controlling the intestinal microbiota, topographical localization of Paneth cells in the small intestine in relation to functions of α-defensin remains to be determined. In this study, we examined the expression level of messenger RNA (mRNA) encoding six Crp-isoforms and Crp immunoreactivities using singly isolated crypts together with bactericidal activities of Paneth cell secretions from isolated crypts of duodenum, jejunum, and ileum. Here we showed that levels of Crp mRNAs in the single crypt ranged from 5 x 103 to 1 x 106 copies per 5 ng RNA. For each Crp isoform, the expression level in ileum was 4 to 50 times higher than that in duodenum and jejunum. Furthermore, immunohistochemical analysis of isolated crypts revealed that the average number of Paneth cell per crypt in the small intestine increased from proximal to distal, three to seven-fold, respectively. Both Crp1 and 4 expressed greater in ileal Paneth cells than those in duodenum or jejunum. Bactericidal activities in secretions of ileal Paneth cell exposed to bacteria were significantly higher than those of duodenum or jejunum. In germ-free mice, Crp expression in each site of the small intestine was attenuated and bactericidal activities released by ileal Paneth cells were decreased compared to those in conventional mice. Taken together, Paneth cells and their α-defensin in adult mouse appeared to be regulated topographically in innate immunity to control intestinal integrity.

Mycotoxin Deoxynivalenol Has Different Impacts on Intestinal Barrier and Stem Cells by Its Route of Exposure.

The different effects of deoxynivalenol (DON) on intestinal barrier and stem cells by its route of exposure remain less known. We explored the toxic effects of DON on intestinal barrier functions and stem cells after DON microinjection (luminal exposure) or addition to a culture medium (basolateral exposure) using three-dimensional mouse intestinal organoids (enteroids). The influx test using fluorescein-labeled dextran showed that basolateral DON exposure (1 micromolar (µM) disrupted intestinal barrier functions in enteroids compared with luminal DON exposure at the same concentration. Moreover, an immunofluorescence experiment of intestinal epithelial proteins, such as E-cadherin, claudin, zonula occludens-1 (ZO-1), and occludin, exhibited that only basolateral DON exposure broke down intestinal epithelial integrity. A time-lapse analysis using enteroids from leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5)-enhanced green fluorescence protein (EGFP) transgenic mice and 5-ethynyl-2-deoxyuridine (EdU) assay indicated that only the basolateral DON exposure, but not luminal DON exposure, suppressed Lgr5+ stem cell count and proliferative cell ratio, respectively. These results revealed that basolateral DON exposure has larger impacts on intestinal barrier function and stem cells than luminal DON exposure. This is the first report that DON had different impacts on intestinal stem cells depending on the administration route. In addition, RNA sequencing analysis showed different expression of genes among enteroids after basolateral and luminal DON exposure.

Paneth cell α-defensin misfolding correlates with dysbiosis and ileitis in Crohn's disease model mice.

Crohn's disease (CD) is an intractable inflammatory bowel disease, and dysbiosis, disruption of the intestinal microbiota, is associated with CD pathophysiology. ER stress, disruption of ER homeostasis in Paneth cells of the small intestine, and α-defensin misfolding have been reported in CD patients. Because α-defensins regulate the composition of the intestinal microbiota, their misfolding may cause dysbiosis. However, whether ER stress, α-defensin misfolding, and dysbiosis contribute to the pathophysiology of CD remains unknown. Here, we show that abnormal Paneth cells with markers of ER stress appear in SAMP1/YitFc, a mouse model of CD, along with disease progression. Those mice secrete reduced-form α-defensins that lack disulfide bonds into the intestinal lumen, a condition not found in normal mice, and reduced-form α-defensins correlate with dysbiosis during disease progression. Moreover, administration of reduced-form α-defensins to wild-type mice induces the dysbiosis. These data provide novel insights into CD pathogenesis induced by dysbiosis resulting from Paneth cell α-defensin misfolding and they suggest further that Paneth cells may be potential therapeutic targets.

Butyric Acid and Leucine Induce α-Defensin Secretion from Small Intestinal Paneth Cells.

The intestine not only plays a role in fundamental processes in digestion and nutrient absorption, but it also has a role in eliminating ingested pathogenic bacteria and viruses. Paneth cells, which reside at the base of small intestinal crypts, secrete α-defensins and contribute to enteric innate immunity through potent microbicidal activities. However, the relationship between food factors and the innate immune functions of Paneth cells remains unknown. Here, we examined whether short-chain fatty acids and amino acids induce α-defensin secretion from Paneth cells in the isolated crypts of small intestine. Butyric acid and leucine elicit α-defensin secretion by Paneth cells, which kills Salmonella typhimurium. We further measured Paneth cell secretion in response to butyric acid and leucine using enteroids, a three-dimensional ex vivo culture system of small intestinal epithelial cells. Paneth cells expressed short-chain fatty acid receptors, Gpr41, Gpr43, and Gpr109a mRNAs for butyric acid, and amino acid transporter Slc7a8 mRNA for leucine. Antagonists of Gpr41 and Slc7a8 inhibited granule secretion by Paneth cells, indicating that these receptor and transporter on Paneth cells induce granule secretion. Our findings suggest that Paneth cells may contribute to intestinal homeostasis by secreting α-defensins in response to certain nutrients or metabolites.

A multicenter phase I study of inebilizumab, a humanized anti-CD19 monoclonal antibody, in Japanese patients with relapsed or refractory B-cell lymphoma and multiple myeloma.

This multicenter, phase I, open-label dose escalation study evaluated safety, tolerability, pharmacokinetics, and preliminary anti-tumor activity of inebilizumab in Japanese patients with relapsed/refractory diffuse large B-cell lymphoma (DLBCL), chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), or multiple myeloma (MM) who were ineligible for hematopoietic stem cell transplantation. Patients received inebilizumab 2, 4, or 8 mg/kg intravenously on days 1 and 8 of the first 28-day cycle, and once every 28 days thereafter, with a 12 mg/kg cohort added. Twenty patients (11 FL, six DLBCL, two CLL, and one MM) received inebilizumab at four dose levels (2 mg/kg cohort, n = 3; 4 mg/kg cohort, n = 7; 8 mg/kg cohort, n = 4; 12 mg/kg cohort, n = 6). Three patients experienced dose-limiting toxicities: grade 4 neutropenia/grade 3 leukopenia (n = 1, 12 mg/kg) and grade 3 infusion reaction (n = 1 each, 4 mg/kg and 12 mg/kg); the maximum tolerated dose was 8 mg/kg. Four (three FL and one DLBCL) patients achieved complete response; eight (six FL and two DLBCL) achieved partial response. Overall response rate was 60%. Over the dose ranges evaluated, the pharmacokinetic profile of inebilizumab in Japanese patients was generally dose proportional. This phase I study showed acceptable toxicity and preliminary and promising efficacy of inebilizumab in patients with relapsed/refractory FL and DLBCL.