Staphylococcal superantigen-like protein 3 triggers murine mast cell adhesion by binding to CD43 and augments mast cell activation.

Staphylococcus aureus is a noteworthy pathogen in allergic diseases, as four staphylococcal exotoxins activate mast cells, a significant contributor to inflammation, in an IgE-independent manner. Although the adhesion of mast cells is an essential process for their immune responses, only a small number of exotoxins have been reported to affect the process. Here, we demonstrated that staphylococcal superantigen-like (SSL) 3, previously identified as a toll-like receptor 2 agonist, induced the adhesion of murine bone marrow-derived mast cells to culture substratum. SSL3-induced adhesion was mediated by fibronectin in an Arg-Gly-Asp (RGD) sequence-dependent manner, suggesting the integrins were involved in the process. Additionally, SSL3 was found to bind to an anti-adhesive surface protein CD43. SSL3 induced the adhesion of HEK293 cells expressing exogenous CD43, suggesting that CD43 is the target molecule for adhesion induced by SSL3. Evaluation of SSL3-derived mutants showed that the C-terminal region (253-326), specifically T285 and H307, are necessary to induce adhesion. SSL3 augmented the IL-13 production of mast cells in response to immunocomplex and SSL12. These findings reveal a novel function of SSL3, triggering cell adhesion and enhancing mast cell activation. This study would clarify the correlation between S. aureus and allergic diseases such as atopic dermatitis.

Krüppel-like Factor-4-Mediated Macrophage Polarization and Phenotypic Transitions Drive Intestinal Fibrosis in THP-1 Monocyte Models In Vitro.

Background and Objectives: Despite the fact that biologic drugs have transformed inflammatory bowel disease (IBD) treatment, addressing fibrosis-related strictures remains a research gap. This study explored the roles of cytokines, macrophages, and Krüppel-like factors (KLFs), specifically KLF4, in intestinal fibrosis, as well as the interplay of KLF4 with various gut components. Materials and Methods: This study examined macrophage subtypes, their KLF4 expression, and the effects of KLF4 knockdown on macrophage polarization and cytokine expression using THP-1 monocyte models. Co-culture experiments with stromal myofibroblasts and a conditioned medium from macrophage subtype cultures were conducted to study the role of these cells in intestinal fibrosis. Human-induced pluripotent stem cell-derived small intestinal organoids were used to confirm inflammatory and fibrotic responses in the human small intestinal epithelium. Results: Each macrophage subtype exhibited distinct phenotypes and KLF4 expression. Knockdown of KLF4 induced inflammatory cytokine expression in M0, M2a, and M2c cells. M2b exerted anti-fibrotic effects via interleukin (IL)-10. M0 and M2b cells showed a high migratory capacity toward activated stromal myofibroblasts. M0 cells interacting with activated stromal myofibroblasts transformed into inflammatory macrophages, thereby increasing pro-inflammatory cytokine expression. The expression of IL-36α, linked to fibrosis, was upregulated. Conclusions: This study elucidated the role of KLF4 in macrophage polarization and the intricate interactions between macrophages, stromal myofibroblasts, and cytokines in experimental in vitro models of intestinal fibrosis. The obtained results may suggest the mechanism of fibrosis formation in clinical IBD.

Simultaneous Evaluation of Membrane Permeability and UDP-Glucuronosyltransferase-Mediated Metabolism of Food-Derived Compounds Using Human Induced Pluripotent Stem Cell-Derived Small Intestinal Epithelial Cells.

Pharmacokinetic prediction after oral ingestion is important for quantitative risk assessment of food-derived compounds. To evaluate the utility of human intestinal absorption prediction, we compared the membrane permeability and metabolic activities of human induced pluripotent stem cell-derived small intestinal epithelial cells (hiPSC-SIECs) with Caco-2 cells or human primary enterocytes (hPECs). We found that membrane permeability in hiPSC-SIECs had better predictivity than that in Caco-2 cells against 21 drugs with known human intestinal availability (r = 0.830 and 0.401, respectively). Membrane permeability in hiPSC-SIECs was only 0.019-0.25-fold as compared with that in Caco-2 cells for 7 in 15 food-derived compounds, primarily those that were reported to undergo glucuronidation metabolism. The metabolic rates of the glucuronide conjugate were similar or higher in hiPSC-SIECs as compared with hPECs but lower in Caco-2 cells. Expression levels of UDP-glucuronosyltransferase (UGT) isoform mRNA in hiPSC-SIECs were similar or higher as compared with hPECs. Therefore, hiPSC-SIECs could be a useful tool for predicting human intestinal absorption to simultaneously evaluate membrane permeability and UGT-mediated metabolism. SIGNIFICANCE STATEMENT: Gastrointestinal absorption is an important step for predicting the internal exposure of food-derived compounds. This research revealed that human induced pluripotent stem cell-derived small intestinal cells (hiPSC-SIECs) had better predictivity of intestinal availability than Caco-2 cells; furthermore, the metabolic rates of UDP-glucuronosyltransferase (UGT) substrates of hiPSC-SIECs were closer to those of human primary enterocytes than those of Caco-2 cells. Therefore, hiPSC-SIECs could be a useful tool for predicting human intestinal absorption to simultaneously evaluate membrane permeability and UGT-mediated metabolism.

Protective Effect of Irsogladine against Aspirin-Induced Mucosal Injury in Human Induced Pluripotent Stem Cell-Derived Small Intestine.

Background and Objectives: Acetylsalicylic acid (ASA) is widely used for preventing cerebrovascular and cardiovascular diseases. Gastrointestinal (GI) tract injury is one of the major complications of aspirin use, potentially leading to severe GI bleeding. However, no drugs for preventing aspirin-induced small intestinal injury have been developed. The aim of this study was to establish a human experimental model for investigating aspirin-induced small intestinal mucosal injury. In addition, we evaluated the protective effect of Irsogladine against aspirin-induced small intestinal mucosal injury using human induced pluripotent stem cell-derived 2D monolayer crypt-villus structural small intestine (2D-hiPSC-SI). Materials and Methods: Human iPS cell-derived intestinal organoids were seeded and cultured in Air-liquid interface. The permeability of 2D-hiPSC-SI was evaluated using Lucifer yellow. Changes in structure and mucosal permeability of 2D-hiPSC-SI after addition of aspirin were confirmed over time, and changes in intestinal epithelium-related markers were evaluated by real-time qPCR and Immunofluorescence staining. The effect of Irsogladine on prevention of aspirin mucosal injury was examined by adding Irsogladine to the culture medium. Results: Cultured 2D-hiPSC-SI showed multi-lineage differentiation into small intestinal epithelium comprised of absorptive cells, goblet cells, enteroendocrine cells, and Paneth cells, which express CD10, MUC2, chromogranin A, and lysozyme, respectively. RNA in situ hybridization revealed intestinal stem cells that express Lgr5. ASA administration induced an increase in the mucosal permeability of 2D-hiPSC-SI. ASA-injured 2D-hiPSC-SI showed decreased mRNA expression of multi-lineage small intestinal cell markers as well as intestinal stem cell marker Lgr5. Administration of Irsogladine on the basal side of the 2D-hiPSC-SI resulted in significant increases in Mki67 and Muc2 mRNA expression by 2D-hiPSCs at 48 h compared with the control group. Administration of 400 µg/mL Irsogladine to the ASA-induced small intestinal injury model resulting in significantly decreased mucosal permeability of 2D-hiPSC-SI. In immunofluorescence staining, Irsogladine significantly increased the fluorescence intensity of MUC2 under normal conditions and administration of 400 µg/mL ASA. Conclusions: we established a novel ASA-induced small intestinal injury model using human iPSC-derived small intestine. Irsogladine maintains mucosal permeability and goblet cell differentiation against ASA-induced small intestinal injury.

Application of Human Induced Pluripotent Stem Cell-Derived Intestinal Organoids as a Model of Epithelial Damage and Fibrosis in Inflammatory Bowel Disease.

Inflammatory bowel disease, which typically manifests as Crohn's disease and ulcerative colitis, is caused by the abnormal production of cytokines such as tumor necrosis factor (TNF)-α and transforming growth factor (TGF)-ß. These cytokines damage intestinal epithelial cells and trigger fibrosis, respectively, for which the current in vitro models have many limitations. Therefore, we tested whether human induced pluripotent stem cell-derived intestinal organoids (HiOs) can mimic inflammatory bowel disease (IBD), and whether such a model is suitable for drug screening. HiOs were treated with TNF-α and TGF-ß to construct mucosal damage and fibrosis models. TNF-α diminished the mRNA expression of intestinal epithelial cell and goblet cell markers in HiOs. TNF-α also induced epithelial cell damage and degradation of tight junctions but not in the presence of infliximab, an antibody used in the clinic to deplete TNF-α. Furthermore, permeation of the non-absorbable marker FD-4 was observed in HiOs treated with TNF-α or ethylene glycol tetraacetic acid (EGTA), but not in the presence of infliximab. In contrast, TNF-α and TGF-ß induced mRNA expression of mesenchymal and fibrosis markers, as well as epithelial-mesenchymal transition. SB431542, a TGF-ß inhibitor, significantly reversed these events. The data indicate that HiOs mimic mucosal damage and fibrosis due to IBD and are thus suitable models for drug screening.

Generation of Intestinal Organoids Suitable for Pharmacokinetic Studies from Human Induced Pluripotent Stem Cells.

Intestinal organoids morphologically resemble intestinal tissues and are expected to be used in both regenerative medicine and drug development studies, including pharmacokinetic studies. However, the pharmacokinetic properties of these organoids remain poorly characterized. In this study, we aimed to generate pharmacokinetically functional intestinal organoids from human induced pluripotent stem (iPS) cells. Human iPS cells were induced to differentiate into the midgut and then seeded on EZSPHERE plates (AGC Techno Glass Inc., Shizuoka, Japan) to generate uniform spheroids, and the floating spheroids were subsequently differentiated into intestinal organoids using small-molecule compounds. Exposure to the small-molecule compounds potently increased the expression of intestinal markers and pharmacokinetic-related genes in the organoids, and the organoids also included various intestinal cells such as enterocytes, intestinal stem cells, goblet cells, enteroendocrine cells, Paneth cells, smooth muscle cells, and fibroblasts. Moreover, microvilli and tight junctions were observed in the organoids. Furthermore, we detected not only the expression of drug transporters but also efflux transport activity through ABCB1/MDR1 and the induction of the drug-metabolizing enzyme CYP3A4 by ligands of nuclear receptors. Our results demonstrated the successful generation of pharmacokinetically functional intestinal organoids from human iPS cells. Thus, these intestinal organoids could be used as a pharmacokinetic evaluation system in drug development studies.

Observation of dynamic interactions between fundamental and second-harmonic modes in a high-power sub-terahertz gyrotron operating in regimes of soft and hard self-excitation.

Dynamic mode interaction between fundamental and second-harmonic modes has been observed in high-power sub-terahertz gyrotrons [T. Notake et al., Phys. Rev. Lett. 103, 225002 (2009); T. Saito et al. Phys. Plasmas 19, 063106 (2012)]. Interaction takes place between a parasitic fundamental or first-harmonic (FH) mode and an operating second-harmonic (SH) mode, as well as among SH modes. In particular, nonlinear excitation of the parasitic FH mode in the hard self-excitation regime with assistance of a SH mode in the soft self-excitation regime was clearly observed. Moreover, both cases of stable two-mode oscillation and oscillation of the FH mode only were observed. These observations and theoretical analyses of the dynamic behavior of the mode interaction verify the nonlinear hard self-excitation of the FH mode.

Suspension culture of human induced pluripotent stem cell-derived intestinal organoids using natural polysaccharides.

Currently, there are many challenges in the culture of human induced pluripotent stem (iPS) cell-derived intestinal organoids (HIOs) for use in drug discovery, disease research, and regenerative medicine. For example, the main culture method, embedding culture, makes industrial large-scale culture difficult, and Matrigel, which is used for almost all HIO cultures, is not respected for its application in regenerative medicine. To overcome these challenges, we herein propose a new culture method using low concentrations of natural polysaccharides in a suspension culture. In the present study, five natural polysaccharides free from heterologous animal-derived components were used, and HIOs were successfully cultured in suspension with FP001 and FP003, which are microbial exopolysaccharide analogs of gellan gum. The fabricated HIOs were similar to living intestinal tracts with respect to their gene expression, microstructure, and protein expression. The observed activities of the drug metabolizing enzymes and drug transporters in the generated HIOs suggested that they have pharmacokinetic functions. We believe that suspension culture of HIOs using FP001 or FP003 can be widely applied to not only drug discovery research but also disease research and regenerative medicine.

Generation of Budding-Like Intestinal Organoids from Human Induced Pluripotent Stem Cells.

Human induced pluripotent stem (iPS) cell-derived intestinal organoids have low invasiveness; however, the current differentiation method does not reflect the crypt-villus-like structure due to structural immaturity. Here, we generated budding-like organoids that formed epithelial tissue-like structures and had the characteristics of the mature small intestine from human iPS cells. They showed a high expression of drug transporters and induced the expression of cytochrome P450 3A4 and P-glycoprotein. When treated with tumor necrosis factor-α and/or transforming growth factor-ß, the budding-like organoids replicated the pathogenesis of mucosal damage or intestinal fibrosis. Upon dissociation and seeding on cell culture inserts, the organoids retained intestinal characteristics, forming polarized intestinal folds with approximately 400 Ω × cm2 transepithelial electrical resistance. This novel method has great potential for disease modeling and drug screening applications.

Dynamic nuclear polarization experiments at 14.1 T for solid-state NMR.

Instrumentation for high-field dynamic nuclear polarization (DNP) at 14.1 T was developed to enhance the nuclear polarization for NMR of solids. The gyrotron generated 394.5 GHz submillimeter (sub-mm) wave with a power of 40 W in the second harmonic TE(0,6) mode. The sub-mm wave with a power of 0.5-3 W was transmitted to the sample in a low-temperature DNP-NMR probe with a smooth-wall circular waveguide system. The (1)H polarization enhancement factor of up to about 10 was observed for a (13)C-labeled compound with nitroxyl biradical TOTAPOL. The DNP enhancement was confirmed by the static magnetic field dependence of the NMR signal amplitude at 90 K. Improvements of the high-field DNP experiments are discussed.

Gellan Gum Promotes the Differentiation of Enterocytes from Human Induced Pluripotent Stem Cells.

The evaluation of drug pharmacokinetics in the small intestine is critical for developing orally administered drugs. Caucasian colon adenocarcinoma (Caco-2) cells are employed to evaluate drug absorption in preclinical trials of drug development. However, the pharmacokinetic characteristics of Caco-2 cells are different from those of the normal human small intestine. Besides this, it is almost impossible to obtain primary human intestinal epithelial cells of the same batch. Therefore, human iPS cell-derived enterocytes (hiPSEs) with pharmacokinetic functions similar to human intestinal epithelial cells are expected to be useful for the evaluation of drug absorption. Previous studies have been limited to the use of cytokines and small molecules to generate hiPSEs. Dietary fibers play a critical role in maintaining intestinal physiology. We used gellan gum (GG), a soluble dietary fiber, to optimize hiPSE differentiation. hiPSEs cocultured with GG had significantly higher expression of small intestine- and pharmacokinetics-related genes and proteins. The activities of drug-metabolizing enzymes, such as cytochrome P450 2C19, and peptide transporter 1 were significantly increased in the GG treatment group compared to the control group. At the end point of differentiation, the percentage of senescent cells increased. Therefore, GG could improve the differentiation efficiency of human iPS cells to enterocytes and increase intestinal maturation by extending the life span of hiPSEs.

Compact radiation module for THz spectroscopy using 300 GHz continuous-wave clinotron.

The results of the development of compact radiation module based on a 300 GHz continuous-wave (CW) clinotron are presented. The clinotron oscillator is proposed as a part of the module designated for high-field dynamic nuclear polarization (DNP) systems for applications in nuclear magnetic resonance (NMR) spectroscopy. The simulation results of clinotron radiation spectra considering the influence of accelerating voltage pulsations are compared with the requirements for THz radiation linewidth for efficient NMR signal enhancement. Based on the simulations, the 300 GHz CW clinotron oscillator was developed and tested together with the high-voltage (HV) power supply, providing the output voltage stability better than 20 ppm. The frequency stability of 33 ppm was observed during the clinotron operation within several hours. The spectral linewidth is better than 8 MHz at 300 GHz that satisfies the requirements for DNP-NMR spectroscopy.

Helium-cooling and -spinning dynamic nuclear polarization for sensitivity-enhanced solid-state NMR at 14 T and 30 K.

We describe a (1)H polarization enhancement via dynamic nuclear polarization (DNP) at very low sample temperature T≈30 K under magic-angle spinning (MAS) conditions for sensitivity-enhanced solid-state NMR measurement. Experiments were conducted at a high external field strength of 14.1 T. For MAS DNP experiments at T<<90 K, a new probe system using cold helium gas for both sample-cooling and -spinning was developed. The novel system can sustain a low sample temperature between 30 and 90K for a period of time >10 h under MAS at ν(R)≈3 kHz with liquid He consumption of ≈6 L/h. As a microwave source, we employed a high-power, continuously frequency-tunable gyrotron. At T≈34 K, (1)H DNP enhancement factors of 47 and 23 were observed with and without MAS, respectively. On the basis of these observations, a discussion on the total NMR sensitivity that takes into account the effect of sample temperature and external field strength used in DNP experiments is presented. It was determined that the use of low sample temperature and high external field is generally rewarding for the total sensitivity, in spite of the slower polarization buildup at lower temperature and lower DNP efficiency at higher field. These findings highlight the potential of the current continuous-wave DNP technique also at very high field conditions suitable to analyze large and complex systems, such as biological macromolecules.