The adaptor TRAF5 limits the differentiation of inflammatory CD4(+) T cells by antagonizing signaling via the receptor for IL-6.

The physiological functions of members of the tumor-necrosis factor (TNF) receptor (TNFR)-associated factor (TRAF) family in T cell immunity are not well understood. We found that in the presence of interleukin 6 (IL-6), naive TRAF5-deficient CD4(+) T cells showed an enhanced ability to differentiate into the TH17 subset of helper T cells. Accordingly, TH17 cell-associated experimental autoimmune encephalomyelitis (EAE) was greatly exaggerated in Traf5(-/-) mice. Although it is normally linked with TNFR signaling pathways, TRAF5 constitutively associated with a cytoplasmic region in the signal-transducing receptor gp130 that overlaps with the binding site for the transcription activator STAT3 and suppressed the recruitment and activation of STAT3 in response to IL-6. Our results identify TRAF5 as a negative regulator of the IL-6 receptor signaling pathway that limits the induction of proinflammatory CD4(+) T cells that require IL-6 for their development.

Metabolome-based discrimination of chrysanthemum cultivars for the efficient generation of flower color variations in mutation breeding.

INTRODUCTION: The color variations of ornamental flowers are often generated by ion-beam and gamma irradiation mutagenesis. However, mutation rates differ significantly even among cultivars of the same species, resulting in high cost and intensive labor for flower color breeding. OBJECTIVES: We aimed to establish a metabolome-based strategy to identify biomarkers and select promising parental lines with high mutation rates using Chrysanthemum as the case study. METHODS: The mutation rates associated with flower color were measured in 10 chrysanthemum cultivars with pink, yellow, or white flowers after soft X-ray irradiation at the floret-formation stage. The metabolic profiles of the petals of these cultivars were clarified by widely targeted metabolomics and targeted carotenoid analysis using liquid chromatography-tandem quadrupole mass spectrometry. Metabolome and carotenoid data were subjected to an un-supervised principal component analysis (PCA) and a supervised logistic regression with least absolute shrinkage and selection operator (LASSO). RESULTS: The PCA of the metabolic profile data separated chrysanthemum cultivars according to flower color rather than mutation rates. By contrast, logistic regression with LASSO generated a discrimination model to separate cultivars into two groups with high or low mutation rates, and selected 11 metabolites associated with mutation rates that can be biomarkers candidates for selecting parental lines for mutagenesis. CONCLUSION: This metabolome-based strategy to identify metabolite markers for mutation rates associated with flower color might be applied to other ornamental flowers to accelerate mutation breeding for generating new cultivars with a wider range of flower colors.

Treatment of classic-type Hirschsprung's disease: rectoplasty with posterior triangular colonic flap versus transanal endorectal pull-through with rectoanal myotomy.

PURPOSE: Our institution employs rectoplasty with a posterior triangular colonic flap (RPTCF) for classic-type Hirschsprung's disease. Recently, we employed a modified Soave procedure: transanal endorectal pull-through with rectoanal myotomy (TEPTRAM). In both procedures, the internal sphincter muscle is completely divided vertically at the 6 o'clock position. Unlike RPTCF, TEPTRAM does not require abdominal manipulation. We aimed to verify the usefulness of TEPTRAM. METHODS: 64 patients with classic-type Hirschsprung's disease who underwent surgery between 1970 and 2017 were divided into group R (RPTCF, 47 cases) and group T (TEPTRAM, 17 cases). We compared the defecation function of the groups. RESULTS: No patient showed fecal incontinence (R: 0/41, T: 0/10, ns). Three patients (6%) in group R and four (24%) in group T developed postoperative enterocolitis (P = 0.16). Three patients (10%) in group R and one (20%) in group T needed an enema (P = 0.36). CONCLUSION: There was no adverse effect of rectoanal myotomy; incontinence was not observed in either procedure. Although there was no significant difference, the incidences of enterocolitis and constipation were slightly higher in group T, perhaps because of the residual muscle cuff; therefore, it is necessary to provide best care with attention to constipation immediately after surgery.

Non-lysosomal Degradation of Singly Phosphorylated Oligosaccharides Initiated by the Action of a Cytosolic Endo-ß-N-acetylglucosaminidase.

Phosphorylated oligosaccharides (POSs) are produced by the degradation of dolichol-linked oligosaccharides (DLOs) by an unclarified mechanism in mammalian cells. Although POSs are exclusively found in the cytosol, their intracellular fates remain unclear. Our findings indicate that POSs are catabolized via a non-lysosomal glycan degradation pathway that involves a cytosolic endo-ß-N-acetylglucosaminidase (ENGase). Quantitative and structural analyses of POSs revealed that ablation of the ENGase results in the significant accumulation of POSs with a hexasaccharide structure composed of Manα1,2Manα1,3(Manα1,6)Manß1,4GlcNAcß1,4GlcNAc.In vitroENGase assays revealed that the presence of an α1,2-linked mannose residue facilitates the hydrolysis of POSs by the ENGase. Liquid chromatography-mass spectrometric analyses and fluorescent labeling experiments show that such POSs contain one phosphate group at the reducing end. These results indicate that ENGase efficiently hydrolyzes POSs that are larger than Man4GlcNAc2-P, generating GlcNAc-1-P and neutral Gn1-type free oligosaccharides. These results provide insight into important aspects of the generation and degradation of POSs.

Mesenteric lymph nodes contribute to proinflammatory Th17-cell generation during inflammation of the small intestine in mice.

T cells of the small intestine, including Th17 cells, are critically involved in host protection from microbial infection, and also contribute to the pathogenesis of small bowel inflammatory disorders. Accumulating evidence suggests that mesenteric lymph nodes (MLNs) play important roles in gut-tropic T-cell generation, although it is still unclear if MLNs are involved in the pathogenesis of small intestine inflammation. To address this issue, we analyzed the roles of both MLNs and Peyer's patches (PPs) by evaluating MLN- or PP-deficient mice in an experimental model of small intestine inflammation, induced by CD3-specific mAb injection. Interestingly, MLNs, but not PPs, were essential for the pathogenesis of intestinal inflammation, in particular the accumulation and infiltration of CD4(+) T-cell populations, including Th17 cells, from the blood. In addition, CD4(+) T-cell accumulation was dependent on the function of the α4 ß7 integrin. Furthermore, MLN removal led to a significantly reduced number of peripheral α4 ß7 (+) CD4(+) effector memory T cells under normal conditions, suggesting that MLNs may play a role in maintaining the number of gut-tropic CD4(+) effector memory T cells circulating in the blood. Taken together, the present study highlights the important role of MLNs in contributing to the pathogenesis of small intestine inflammation.

Predictive factor for intraoperative tumor rupture of Wilms tumor.

PURPOSE: For Wilms tumor, intraoperative tumor rupture with wide tumor spillage during surgical manipulation raises the classification to stage 3. Then, postoperative chemotherapy must be more intensive, and abdominal radiotherapy is added. Therefore, intraoperative tumor rupture should be avoided if possible. However, predictive factors for intraoperative tumor rupture have not been sufficiently described. Here we examined the risk factors for intraoperative tumor rupture. METHODS: Patients with Wilms tumor who underwent treatment according to the National Wilms Tumor Study or the Japanese Wilms Tumor Study protocol at our institution were reviewed retrospectively. Collected cases were categorized into two groups: the ruptured group and the non-ruptured group. Risk factors for intraoperative tumor rupture, including the ratio of the tumor area to the abdominal area in a preoperative single horizontal computed tomography slice (T/A ratio), were investigated in both groups. RESULTS: The two groups were not different in age, body weight, tumor laterality, sex, or histological distribution. The T/A ratio in the ruptured group was significantly higher than that in the non-ruptured group. Receiver operating characteristic curve analysis identified a discriminative value for a T/A ratio >0.5. CONCLUSION: The T/A ratio can be a predictive factor for intraoperative tumor rupture of Wilms tumor.

OX40 and IL-7 play synergistic roles in the homeostatic proliferation of effector memory CD4⁺ T cells.

T-cell homeostasis preserves the numbers, the diversity and functional competence of different T-cell subsets that are required for adaptive immunity. Naïve CD4(+) T (TN ) cells are maintained in the periphery via the common γ-chain family cytokine IL-7 and weak antigenic signals. However, it is not clear how memory CD4(+) T-cell subsets are maintained in the periphery and which factors are responsible for the maintenance. To examine the homeostatic mechanisms, CFSE-labeled CD4(+) CD44(high) CD62L(low) effector memory T (TEM ) cells were transferred into sublethally-irradiated syngeneic C57BL/6 mice, and the systemic cell proliferative responses, which can be divided distinctively into fast and slow proliferations, were assessed by CFSE dye dilution. We found that the fast homeostatic proliferation of TEM cells was strictly regulated by both antigen and OX40 costimulatory signals and that the slow proliferation was dependent on IL-7. The simultaneous blockade of both OX40 and IL-7 signaling completely inhibited the both fast and slow proliferation. The antigen- and OX40-dependent fast proliferation preferentially expanded IL-17-producing helper T cells (Th17 cells). Thus, OX40 and IL-7 play synergistic, but distinct roles in the homeostatic proliferation of CD4(+) TEM cells.

Homeostatic proliferation of naive CD4+ T cells in mesenteric lymph nodes generates gut-tropic Th17 cells.

Homeostatic proliferation of naive T cells in the spleen and cutaneous lymph nodes supplies memory-phenotype T cells. The "systemic" proliferative responses divide distinctly into fast or slow cell division rates. The fast proliferation is critical for generation of effector memory T cells. Because effector memory T cells are abundant in the lamina propria of the intestinal tissue, "gut-specific" homeostatic proliferation of naive T cells may be important for generation of intestinal effector memory T cells. However, such organ-specific homeostatic proliferation of naive T cells has not yet been addressed. In this study, we examined the gut-specific homeostatic proliferation by transferring CFSE-labeled naive CD4(+) T cells into sublethally irradiated mice and separately evaluating donor cell division and differentiation in the intestine, mesenteric lymph nodes (MLNs), and other lymphoid organs. We found that the fast-proliferating cell population in the intestine and MLNs had a gut-tropic α4ß7(+) Th17 phenotype and that their production was dependent on the presence of commensal bacteria and OX40 costimulation. Mesenteric lymphadenectomy significantly reduced the Th17 cell population in the host intestine. Furthermore, FTY720 treatment induced the accumulation of α4ß7(+)IL-17A(+) fast-dividing cells in MLNs and eliminated donor cells in the intestine, suggesting that MLNs rather than intestinal tissues are essential for generating intestinal Th17 cells. These results reveal that MLNs play a central role in inducing gut-tropic Th17 cells and in maintaining CD4(+) T cell homeostasis in the small intestine.

A small molecule inhibitor to plasminogen activator inhibitor 1 inhibits macrophage migration.

OBJECTIVE: Macrophage (Mϕ) migration rests on the adhesion/detachment between Mϕ surface components and extracellular matrixes, and the contribution of numerous inflammatory disorders. Plasminogen activator inhibitor (PAI)-1, a serine protease inhibitor, influences Mϕ motility through an action distinct from its classical modulation of the plasmin-based fibrinolytic process. We rely here on a small molecule PAI-1 inhibitor (TM5275) to investigate the role of PAI-1 in Mϕ migration in the pathogenesis of renal injury. APPROACH AND RESULTS: Mϕ migration was inhibited both in vitro and in vivo by TM5275. It was also reduced in T-cell-deficient nude mice, but not in PAI-1-deficient mice. Mϕ migration hinged on the interaction of PAI-1 with low-density lipoprotein receptor-related protein, an interaction prevented by TM5275, but not with vitronectin, urokinase-type plasminogen activator, or tissue-type plasminogen activator. Fed to rats with anti-Thy-1-induced nephritis, TM5275 significantly decreased Mϕ accumulation and ameliorated the progression of renal injury. CONCLUSIONS: These findings suggest that a small molecule PAI-1 inhibitor represents a novel class of anti-inflammatory agents targeting Mϕ migration by the inhibition of the interaction of PAI-1 with low-density lipoprotein receptor-related protein.

Life-threatening risk factors and the role of intestinal transplantation in patients with intestinal failure.

PURPOSE: We elucidated the life-threatening risk factors for intestinal failure (IF) and characterized the role of intestinal transplantation (ITx) in affected patients. METHODS: We conducted a retrospective review of 38 patients with short bowel (SB) and 19 with motility disorders (MD). The SB patients were divided into three categories according to the length of their residual small bowel and the presence of the ileocecal valve. The four disease subcategories were grouped into two categories: low-risk category (mild and moderated SB) and high-risk category (extensive SB and MD). The age at the introduction of parenteral nutrition (PN) was <1 year in 50 patients (infant group, IG) and 1-15 years in 7 patients (pediatric group, PG). RESULTS: Enteral autonomy was rarely achieved in the high-risk category (p < 0.0001). IG was associated with a higher incidence of developing intestinal failure-associated liver disease (IFALD) (p = 0.004). Eight patients died, due to IFALD in four, sepsis in three and acute heart failure in one. Twenty-eight patients (49 %) are currently alive without PN, including four after ITx. CONCLUSION: The treatment of high-risk IF is still challenging. Inclusion of ITx in appropriate timing, along with aggressive medical, nutritional and surgical management, may reduce advanced morbidity and mortality of high-risk IF.

Usefulness of lysophosphatidylcholine measurement in the cerebrospinal fluid for differential diagnosis of neuropathic pain: Possible introduction into clinical laboratory testing.

BACKGROUND: The differential diagnosis of neuropathic pain, especially discrimination between neuropathic pain caused by spinal canal stenosis (SCS) and neuropathic pain associated with causes other than SCS, is sometimes difficult; however, it is important for surgical application. METHODS: We established a reliable method for measuring lysophosphatidylcholine (LPC), a precursor of lysophosphatidic acids which are known as being pain initiators, using a liquid chromatography-tandem mass spectrometry method, and measured the LPC concentrations in the cerebrospinal fluid (CSF) in patients with SCS (SCS group; n = 76), patients with neuropathic pain caused by non-SCS diseases (Others group; n = 49), and control subjects without pain (control group; n = 92). RESULTS: Both within-run and between-run CV(%) were almost < 10 %, suggesting an enough performance for clinical introduction. The CSF concentrations of LPC (16:0) and LPC (18:0) were higher in the SCS group than those in the Control or Others group; the concentrations of LPC (18:1), LPC (18:2), LPC (20:4), LPC (22:6) levels were higher in the SCS group than those in the control or others group, but they were also higher in the Others group than those in the control group. The areas under the curve in the ROC curve analyses of LPC (18:1) for discriminating between the SCS and control groups, others and control groups, and SCS and others groups were 0.994, 0.860, and 0.869, respectively. CONCLUSIONS: LPC measurement in the CSF is useful for the differential diagnosis of neuropathic pain, especially for surgical decision-making, which is expected for clinical introduction.

Intestinal Transplantation at a Single Institution in Japan.

BACKGROUND: This study aimed to compare the clinical outcomes of intestinal transplantation (ITX) between 2 groups by using medications for induction treatment and assess the utility of the current protocol. METHODS: From 2003 to 2020, 11 patients underwent ITX. Recipients were classified into 2 groups: group IL-2Ra (interleukin-2 receptor antagonist therapy, n = 6) and ATG (rabbit antithymocyte globulin therapy, n = 5). We conducted a retrospective review of patient and graft survival rates and the postoperative course. RESULTS: The 1-, 5-, and 10-year patient and graft survival rates of the 11 primary grafts in the 11 recipients were 100%, 88.9%, 62.2% and 90.0%, 78.8%, 56.3%, respectively. The median duration of follow-up for the IL-2Ra and ATG groups was 197.3 and 87.3 months, respectively. The 1-, 5-, and 10-year patient survival rates were 100%, 83.3%, 50% and 100%, 100%, 100% for the IL-2Ra and ATG groups, respectively (P = .25) and 83.3%, 66.7%, 33.3% and 100%, 100%, 100% for graft survival in the IL-2Ra and ATG groups, respectively (P = .08). The incidence of moderate and severe acute rejection was 100% and 20% in the IL-2Ra and ATG groups, respectively (P = .02). The 1- and 5-year moderate and severe rejection-free survival rates were 33.3%, 0% and 80%, 80% in the IL-2Ra and ATG groups, respectively (P = .04). CONCLUSIONS: ATG significantly suppressed moderate and severe acute rejection compared with IL-2Ra, thereby showing better short- and mid-term rejection-free survival rates. Additional clinical experience is needed to determine the optimal regimen for the management of ITX recipients.

Fast determination of isomeric triterpenic acids in Osmanthus fragrans (Thunb.) Lour. fruits by UHPLC coupled with triple quadrupole mass spectrometry.

Triterpenic acids possess rich biological activity. Due to slight differences in structure and polarity, the simultaneous determination of isomeric triterpenic acids is challenging. In the present work, a simple and effective approach to chromatographic separation of such compounds based on conventional C18 stationary phase with gradient elution was developed, which allowed the simultaneous separation of eleven analytes including euscaphic, arjunic, tormentic, arjunolic, asiatic, pomolic, maslinic, corosolic, oleanolic, ursolic and 2-Epi tormentic acid (internal standard). This approach with mass spectrometric detection and ultrasonic extraction was fast, sensitive and accurate for analyzing isomeric triterpenic acids in O. fragrans fruits with a toal duration of the analytical cycle (including pretreatment) within one hour. The LODs lie in ranges of 0.8-12 ng/mL (30 ng/mL for asiatic and corosolic acid). The developed method was validated and successfully applied in ten batches of O. fragrans fruits, which could reflect the detail content difference of triterpenic acid components.

Determination of vitamin D in oily drops using a column-switching system with an on-line clean-up by supercritical fluid chromatography.

A column-switching system, composed of supercritical fluid chromatography (SFC) and reverse phase liquid chromatography/mass spectrometry (RPLC/MS) was developed for the analysis of vitamin D in oily and fatty matrices. The SFC with the similar retention behavior as normal-phase liquid chromatography (NPLC), was applied for an on-line clean-up of oily and fatty samples, then followed by separation and detection using a reverse-phase LC-MS/MS. Three SFC columns packed with materials of different functional groups (Silica, NH2, Diol) were compared and the column with diol groups, on which the retention time of vitamin D was the longest, was finally selected for purification of the samples. 100% methanol was chosen to carry vitamin D from the clean-up column to the pre-treatment column. It was also used as the mobile phase for the separation of vitamin D on a reverse phase C18 column. Vitamin D2 and D3 were baseline separated by using this system. The linearity was calculated with a value of coefficient of determination (r2) ≥ 0.998. The linear range is from 20 ng/mL to 200 ng/mL. Two kinds of liquid vitamin D3 supplements (Baby Ddrops and Vitamin AD drops) were directly analyzed using this system without any fussy preparation procedure. The limit of detection (LOD) for vitamin D3 in the two oily samples was estimated to be 10 ng/mL. The relative standard deviations (RSD) of intra- and inter-day precision, repeatability were 1.47%, 2.43% and 1.59% for Baby Ddrops and 5.76%, 8.24% and 5.86% for Vitamin AD drops. The recoveries vary between 84.3% and 102.8% with 7.1% RSD for Baby Ddrops and 90.8-109.6% with 5.83% RSD for Vitamin AD drops, respectively. These results suggest that the method based on the SFC-RPLC/MS column-switching system is simple and suitable for analysis of vitamin D in oily and fatty samples.

Structural analysis of quazepam metabolites in bile by ion trap time-of-flight mass spectrometry.

Quazepam (QZP) is a long-acting benzodiazepine-type hypnotic. We searched for novel QZP metabolites in bile and determined their structures by liquid chromatography-ion trap time-of-flight mass spectrometry (LC-IT-TOF MS). The metabolites were extracted with ethyl acetate after ß-glucuronidase treatment. First, a single MS spectrum was acquired. Second, MS(n) spectra were acquired for peaks that consisted of ions with the isotope pattern corresponding to molecules bearing one chlorine atom. The novel QZP metabolites found in this study were hydroxyquazepam, hydroxy-methoxyquazepam, hydroxy-oxoquazepam, and hydroxy-methoxy-oxoquazepam, which have the hydroxy and methoxy groups on the fluorophenyl group, and dihydroxy-oxoquazepam and dihydroxy-methoxy-oxoquazepam, which have one hydroxy group at the 3-position of the seven-membered ring and the other hydroxy group and the methoxy group on the fluorophenyl group. We demonstrated that LC-IT-TOF MS was a useful tool for determining the structure of the metabolites. However, the exact locations of the hydroxy and methoxy groups on the fluorophenyl group could not be identified.

Accurate determination of the structural elasticity of human hair by a small-scale bending test.

This paper reports on a small-scale bending method for human hair. The test sample, which is elliptical in cross-section, is fixed to a hollow steel needle using resin to form a cantilever. A loading probe is used to subject this to a lateral load, where the load is applied parallel to either the long or short axis of the elliptical cross-section. From these tests, load-displacement relationships for the hair were obtained. From the experimental data and analysis, we found that the structural elasticity determined is independent of the direction of bending, and precise measurements of the structural elasticity of human hair with scattering of less than 5% were realized using this test scheme. Finally, changes in the structural elasticity of hair due to hair treatments were detected and the changes are discussed based on a theoretical model of the multi-layered structure.