Clinical outcomes of patients with remitting ulcerative colitis after discontinuation of indigo naturalis.

Indigo naturalis is an effective treatment for ulcerative colitis. However, long-term use of indigo naturalis causes adverse events, such as pulmonary hypertension. The natural history of patients with ulcerative colitis who discontinued indigo naturalis after induction therapy is unknown. Moreover, the clinical features of patients who relapsed within 52 weeks after the discontinuation of indigo naturalis are unclear. This study aimed to assess the clinical outcomes of patients with ulcerative colitis after discontinuation of indigo naturalis and to identify potential markers responsible for relapse. This single-center retrospective study investigated the follow-up of 72 patients who achieved a clinical response 8 weeks after indigo naturalis treatment. We observed relapse in patients with ulcerative colitis after the discontinuation of indigo naturalis. We analyzed the factors predicting long-term outcomes after discontinuation of indigo naturalis. Relapse was observed in 24%, 57%, and 71% of patients at 8, 26, and 52 weeks, respectively. There were no predictive markers in patients who relapsed within 52 weeks after the discontinuation of indigo naturalis. The ulcerative colitis relapse rate after indigo naturalis discontinuation was high. Follow-up treatment is required after the discontinuation of indigo naturalis in patients with ulcerative colitis.

Neural circuits for retrospective and prospective introspection for the past, present and future in macaque monkeys and humans.

For animals, including humans, to have self-awareness, the ability to reflect on one's own perceptions and cognitions, which is known as metacognition, and an understanding of consistency of the self from the past to the present and into the future based on metacognition is essential. Through the mediation of self-consciousness, animals are thought to be able to proactively act to change their environment rather than passively responding to changes in their environment. However, it has not been known whether animals have self-awareness, and, if so, how it is implemented neurobiologically. In this review article, I introduce our studies examining the neural basis of metacognitive abilities for past, present, and future actions in macaque monkeys and humans, and explore the evolutionary origins of self-awareness.

Double-bundle anterior cruciate ligament reconstruction in patients aged 60 years and older.

Background: This study aimed to examine the clinical outcomes of double-bundle (DB) anterior cruciate ligament (ACL) reconstruction in patients aged ≥60 years. Methods: Anatomical DB-ACL reconstruction using hamstring tendon autografts was performed in 13 patients aged ≥60 years at our institution between June 2012 and May 2018. The patients included seven men and six women, and the mean age at surgery was 65.0 years (range, 60-73 years). The mean time from injury to surgery was 80.5 months (range, 1-480 months), and the mean follow-up time was 26.2 months (range, 24-42 months). All patients were assessed based on physical examination findings, clinical scores, Kellgren-Lawrence grades preoperatively and at the final postoperative follow-up, intraoperative meniscal or chondral lesions, and perioperative complications. Status of returning to sports for all patients was assessed at the final follow-up. Results: The mean side-to-side differences by arthrometer improved from 4.3 mm (range, 2-8 mm) to 0.9 mm (range, 0-2 mm), and the positive pivot-shift test decreased from 100% to 8%. The mean extensor muscle strength was 93.3% (range, 74-116%) postoperatively. The mean Lysholm score improved from 71.1 (range, 27-85) to 95.2 (range, 89-100). Ten of the 13 patients (77%) returned to their pre-injury level of sports performance, and one patient (8%) returned to sports with less intensity. Intraoperatively, meniscal tears were observed in 10 patients (77%), and chondral lesions >grade 2 were observed in 11 (85%). One patient developed perioperative complications. At the final follow-up, the Kellgren-Lawrence grade worsened in only one patient. No re-injury or infection was observed, and revision surgery was not required for any patients. Conclusions: Anatomical DB-ACL reconstruction could provide satisfactory clinical outcomes and knee function restoration in patients aged ≥60 years. Level of evidence: A retrospective study, case series (IV).

Arthroscopic All-Suture Anchor Technique for Unstable Ramp Lesions with Medial Meniscal Defects.

A ramp lesion is a specific type of tear in the meniscocapsular junction of the posterior horn of the medial meniscus, usually associated with anterior cruciate ligament (ACL) injury. Biomechanical cadaveric studies have shown that ACL injury combined with ramp lesions significantly increases anterior tibial translation and external rotation, which ACL reconstruction alone cannot completely control. Additionally, ramp lesions are sometimes associated with medial meniscal defects, especially in cases of chronic ACL deficiency after repetitive traumatic events, in which the anatomical repair of the meniscocapsular junction is infeasible. This report describes a new arthroscopic repair technique using an all-suture anchor through a posteromedial portal for unstable ramp lesions with medial meniscal defects.

Downregulation of chemokine receptor 9 facilitates CD4+CD8αα+ intraepithelial lymphocyte development.

Intestinal intraepithelial lymphocytes (IELs) reside in the gut epithelial layer, where they help in maintaining intestinal homeostasis. Peripheral CD4+ T cells can develop into CD4+CD8αα+ IELs upon arrival at the gut epithelium via the lamina propria (LP). Although this specific differentiation of T cells is well established, the mechanisms preventing it from occurring in the LP remain unclear. Here, we show that chemokine receptor 9 (CCR9) expression is low in epithelial CD4+CD8αα+ IELs, but CCR9 deficiency results in CD4+CD8αα+ over-differentiation in both the epithelium and the LP. Single-cell RNA sequencing shows an enriched precursor cell cluster for CD4+CD8αα+ IELs in Ccr9-/- mice. CD4+ T cells isolated from the epithelium of Ccr9-/- mice also display increased expression of Cbfß2, and the genomic occupancy modification of Cbfß2 expression reveals its important function in CD4+CD8αα+ differentiation. These results implicate a link between CCR9 downregulation and Cbfb2 splicing upregulation to enhance CD4+CD8αα+ IEL differentiation.

The gut microbiota-induced kynurenic acid recruits GPR35-positive macrophages to promote experimental encephalitis.

The intricate interplay between gut microbes and the onset of experimental autoimmune encephalomyelitis (EAE) remains poorly understood. Here, we uncover remarkable similarities between CD4+ T cells in the spinal cord and their counterparts in the small intestine. Furthermore, we unveil a synergistic relationship between the microbiota, particularly enriched with the tryptophan metabolism gene EC:1.13.11.11, and intestinal cells. This symbiotic collaboration results in the biosynthesis of kynurenic acid (KYNA), which modulates the recruitment and aggregation of GPR35-positive macrophages. Subsequently, a robust T helper 17 (Th17) immune response is activated, ultimately triggering the onset of EAE. Conversely, modulating the KYNA-mediated GPR35 signaling in Cx3cr1+ macrophages leads to a remarkable amelioration of EAE. These findings shed light on the crucial role of microbial-derived tryptophan metabolites in regulating immune responses within extraintestinal tissues.

D-amino Acids Ameliorate Experimental Colitis and Cholangitis by Inhibiting Growth of Proteobacteria: Potential Therapeutic Role in Inflammatory Bowel Disease.

BACKGROUND & AIMS: D-amino acids, the chiral counterparts of protein L-amino acids, were primarily produced and utilized by microbes, including those in the human gut. However, little was known about how orally administered or microbe-derived D-amino acids affected the gut microbial community or gut disease progression. METHODS: The ratio of D- to L-amino acids was analyzed in feces and blood from patients with ulcerative colitis (UC) and healthy controls. Also, composition of microbe was analyzed from patients with UC. Mice were treated with D-amino acid in dextran sulfate sodium colitis model and liver cholangitis model. RESULTS: The ratio of D- to L-amino acids was lower in the feces of patients with UC than that of healthy controls. Supplementation of D-amino acids ameliorated UC-related experimental colitis and liver cholangitis by inhibiting growth of Proteobacteria. Addition of D-alanine, a major building block for bacterial cell wall formation, to culture medium inhibited expression of the ftsZ gene required for cell fission in the Proteobacteria Escherichia coli and Klebsiella pneumoniae, thereby inhibiting growth. Overexpression of ftsZ restored growth of E. coli even when D-alanine was present. We found that D-alanine not only inhibited invasion of pathological K. pneumoniae into the host via pore formation in intestinal epithelial cells but also inhibited growth of E. coli and generation of antibiotic-resistant strains. CONCLUSIONS: D-amino acids might have potential for use in novel therapeutic approaches targeting Proteobacteria-associated dysbiosis and antibiotic-resistant bacterial diseases by means of their effects on the intestinal microbiota community.

Bacteriophage therapy against pathological Klebsiella pneumoniae ameliorates the course of primary sclerosing cholangitis.

Primary sclerosing cholangitis (PSC) is characterized by progressive biliary inflammation and fibrosis. Although gut commensals are associated with PSC, their causative roles and therapeutic strategies remain elusive. Here we detect abundant Klebsiella pneumoniae (Kp) and Enterococcus gallinarum in fecal samples from 45 PSC patients, regardless of intestinal complications. Carriers of both pathogens exhibit high disease activity and poor clinical outcomes. Colonization of PSC-derived Kp in specific pathogen-free (SPF) hepatobiliary injury-prone mice enhances hepatic Th17 cell responses and exacerbates liver injury through bacterial translocation to mesenteric lymph nodes. We developed a lytic phage cocktail that targets PSC-derived Kp with a sustained suppressive effect in vitro. Oral administration of the phage cocktail lowers Kp levels in Kp-colonized germ-free mice and SPF mice, without off-target dysbiosis. Furthermore, we demonstrate that oral and intravenous phage administration successfully suppresses Kp levels and attenuates liver inflammation and disease severity in hepatobiliary injury-prone SPF mice. These results collectively suggest that using a lytic phage cocktail shows promise for targeting Kp in PSC.

Myeloid TLR4 signaling promotes post-injury withdrawal resolution of murine liver fibrosis.

The fate of resolution of liver fibrosis after withdrawal of liver injury is still incompletely elucidated. Toll-like receptor 4 (TLR4) in tissue fibroblasts is pro-fibrogenic. After withdrawal of liver injury, we unexpectedly observed a significant delay of fibrosis resolution as TLR4 signaling was pharmacologically inhibited in vivo in two murine models. Single-cell transcriptome analysis of hepatic CD11b+ cells, main producers of matrix metalloproteinases (MMPs), revealed a prominent cluster of restorative Tlr4-expressing Ly6c2-low myeloid cells. Delayed resolution after gut sterilization suggested its microbiome-dependent nature. Enrichment of a metabolic pathway linking to a significant increase of bile salt hydrolase-possessing family Erysipelotrichaceae during resolution. Farnesoid X receptor-stimulating secondary bile acids including 7-oxo-lithocholic acids upregulated MMP12 and TLR4 in myeloid cells in vitro. Fecal material transplant in germ-free mice confirmed phenotypical correlations in vivo. These findings highlight a pro-fibrolytic role of myeloid TLR4 signaling after injury withdrawal and may provide targets for anti-fibrotic therapy.

Imagining the future self through thought experiments.

The ability of the mind to conceptualize what is not present is essential. It allows us to reason counterfactually about what might have happened had events unfolded differently or had another course of action been taken. It allows us to think about what might happen - to perform 'Gedankenexperimente' (thought experiments) - before we act. However, the cognitive and neural mechanisms mediating this ability are poorly understood. We suggest that the frontopolar cortex (FPC) keeps track of and evaluates alternative choices (what we might have done), whereas the anterior lateral prefrontal cortex (alPFC) compares simulations of possible future scenarios (what we might do) and evaluates their reward values. Together, these brain regions support the construction of suppositional scenarios.

Single-cell transcriptomics of human gut T cells identifies cytotoxic CD4+CD8A+ T cells related to mouse CD4 cytotoxic T cells.

Cytotoxic CD4+ T cells (CD4-CTLs) show the presence of cytolytic granules, which include the enzymes granzyme and perforin. The cells have a pathogenic and protective role in various diseases, including cancer, viral infection, and autoimmune disease. In mice, cytotoxic CD4+ T cells express CD8αα+ and reside in the intestine (mouse CD4+CTLs; mCD4-CTLs). The population of cytotoxic CD4+ T cells in the human intestine is currently unknown. Moreover, it is unclear how cytotoxic CD4 T cells change in patients with inflammatory bowel disease (IBD). Here, we aimed to identify cytotoxic CD4+ T cells in the human intestine and analyze the characteristics of the population in patients with IBD using single-cell RNA-seq (scRNA-seq). In CD4+ T cells, granzyme and perforin expression was high in humanMAIT (hMAIT) cells and hCD4+CD8A+ T cell cluster. Both CD4 and CD8A were expressed in hTreg, hMAIT, and hCD4+CD8A+ T cell clusters. Next we performed fast gene set enrichment analysis to identify cell populations that showed homology to mCD4CTLs. The analysis identified the hCD4+CD8A+ T cell cluster (hCTL-like population; hCD4-CTL) similar to mouse CTLs. The percentage of CD4+CD8A+ T cells among the total CD4+ T cells in the inflamed intestine of the patients with Crohn's disease was significantly reduced compared with that in the noninflamed intestine of the patients. In summary, we identified cytotoxic CD4+CD8+ T cells in the small intestine of humans. The integration of the mouse and human sc-RNA-seq data analysis highlight an approach to identify human cell populations related to mouse cell populations, which may help determine the functional properties of several human cell populations in mice.

Comparison of clinical severity, genotype and toxin gene expression of binary toxin-producing Clostridioides difficile clinical isolates in Japan.

The emerging Clostridioides difficile strain BI/NAP1/027 has been reported to be associated with more severe clinical symptoms and higher mortality rates, thought in part due to production of a novel binary toxin alongside conventional A and B toxins. However, recent studies suggest that this may not always be the case. Therefore, the purpose of this report was to investigate the correlation between clinical severity and microbiological characteristics of CDT-producing C. difficile isolates in Japan. Eight Japanese isolates of CDT producing C. difficile were investigated using genotyping, cytotoxic activity assays and toxin gene expression. Correlation with clinical severity was performed retrospectively using the patient record. Three of eight patients were assessed as having severe C. difficile infection (CDI). PCR ribotyping resolved six ribotypes including ribotype 027. No specific genes were identified determining severe compared with non-severe cases. Positive correlation of expression levels of tcdA, tcdB and cdtB were observed although these expression levels were not correlated with cytotoxicity. CDI severity index neither correlated with toxin gene expression level nor cytotoxicity. These data indicate that the possession of the CDT gene and toxin gene expression levels may not relate to C. difficile cytotoxicity or clinical severity.

Localization and movement of Tregs in gastrointestinal tract: a systematic review.

BACKGROUND: The intestine is rich in food-derived and microbe-derived antigens. Regulatory T cells (Tregs) are an essential T-cell population that prevents systemic autoimmune diseases and inhibits inflammation by encountering antigens. Previously, it was reported that the functional loss of Tregs induces systemic inflammation, including inflammatory bowel disease and graft-versus-host disease in human and murine models. However, there is a dearth of information about how Tregs localize in different tissues and suppress effector cells. MAIN BODY: The development of Tregs and their molecular mechanism in the digestive tract have been elucidated earlier using murine genetic models, infectious models, and human samples. Tregs suppress immune and other nonimmune cells through direct effect and cytokine production. The recent development of in vivo imaging technology allows us to visualize how Tregs localize and move in the settings of inflammation and homeostasis. This is important because, according to a recent report, Treg characterization and function are regulated by their location. Tregs located in the proximal intestine and its draining lymph nodes induce tolerance against food antigens, and those located in the distal intestine suppress the inflammation induced by microbial antigens. Taken together, various Tregs are induced in a location-specific manner in the gastrointestinal tract and influence the homeostasis of the gut. CONCLUSION: In this review, we summarize how Tregs are induced in the digestive tract and the application of in vivo Treg imaging to elucidate immune homeostasis in the digestive tract.

Protocol to isolate and enrich mouse splenic naive CD4+ T cells for in vitro CD4+CD8αα+ cell induction.

Recent studies have shown that CD4+CD8αα+ T cells are induced in the hypoxic environment of the small intestinal epithelium. Herein, we describe a protocol for CD4+CD8αα+ T cell induction from freshly isolated naive CD4+ T cells, including procedures for the isolation and enrichment of mouse splenic T cells. In addition, we present an approach that can induce more CD4+CD8αα+ T cells by artificially creating a hypoxic environment in vitro. For complete details on the use and execution of this protocol, please refer to Harada et al. (2022).

CCR9 axis inhibition enhances hepatic migration of plasmacytoid DCs and protects against liver injury.

Plasmacytoid dendritic cells (pDCs) perform dual proinflammatory and immunosuppressive roles. We recently reported the potential of pDC therapy for treatment of intractable acute liver failure. However, establishment of efficient methods to deliver pDCs to the liver is essential for future clinical therapeutic applications. The present study demonstrates a higher abundance of liver and peripheral blood pDCs in mice lacking C-C motif chemokine receptor 9 (CCR9), a pDC gut-homing receptor, than in WT mice. Adoptive transfer of Ccr9-/- pDCs resulted in a higher efficiency of migration to the liver than WT pDCs did, while WT pDCs migrated efficiently to the original target organ, the small intestine. Further, Ccr9-/- pDCs consistently migrated efficiently to livers with concanavalin A-induced inflammation, and exerted a more effective immunosuppressive effect, resulting in better protection against acute liver inflammation than that demonstrated by WT pDCs. These findings highlight the therapeutic potential of the manipulation of the CCR9 axis as an approach to improve migration of immunosuppressive pDCs to the liver in order to exploit their beneficial effects in acute liver disease.

Aryl hydrocarbon receptor signals in epithelial cells govern the recruitment and location of Helios+ Tregs in the gut.

CD4+Foxp3+ regulatory T cells (Tregs) are essential for homeostasis in the colon, but the mechanism by which local environmental cues determine the localization of colonic Tregs is unclear. Here, we administer indigo naturalis (IN), a nontoxic phytochemical aryl hydrocarbon receptor (AhR) agonist used for treating patients with ulcerative colitis (UC) in Asia, and we show that IN increases Helios+ Tregs and MHC class II+ epithelial cells (ECs) in the colon. Interactions between Tregs and MHC class II+ ECs occur mainly near the crypt bottom in the steady state, whereas Tregs dramatically increase and shift toward the crypt top following IN treatment. Moreover, the number of CD25+ T cells is increased near the surface of ECs in IN-treated UC patients compared with that in patients treated with other therapies. We also highlight additional AhR-signaling mechanisms in intestinal ECs that determine the accumulation and localization of Helios+ Tregs in the colon.

Intracellular metabolic adaptation of intraepithelial CD4+CD8αα+ T lymphocytes.

Intestinal intraepithelial lymphocytes (IELs), the first line of defense against microbial and dietary antigens, are classified as natural or induced based on their origin and receptor expression. Induced CD4+CD8αα+TCRß+ T cells (double positive, DPIELs) originated from CD4+CD8α-TCRß+ T cells (single positive, SPIELs) increase with aging. However, the metabolic requirements and the metabolic-related genes in IEL development remain unclear. We determined that the intraepithelial compartment is hypoxic in the presence of microbes and DPIELs increased more than natural IELs in this location. Moreover, DPIELs consumed less oxygen and glucose and exhibited unique alterations in mitochondria. Using inhibitors and genetically modified mice, we revealed that DPIELs adapt to their surrounding oxygen-deprived environment in peripheral tissues by modulating specific genes, including hypoxia-inducible factor, mammalian target of rapamycin complexes (mTORC), phosphorylated ribosomal protein S6 (pS6), and other glycolytic factors. Our findings provide valuable insight into the metabolic properties of IELs.

Conversion of concept-specific decision confidence into integrative introspection in primates.

Introspection based on the integration of uncertain evidence is critical for acting upon abstract thinking and imagining future scenarios. However, it is unknown how confidence read-outs from multiple sources of different concepts are integrated, especially considering the relationships among the concepts. In this study, monkeys performed wagering based on an estimation of their performance in a preceding mnemonic decision. We found that the longer the response times for post-decision wagering, the more relieved the impairments having been caused by frontal disruption. This suggests the existence of a time-consuming compensatory metacognitive process. We found posterior inferior parietal lobe (pIPL) as its candidate, which was not coding the wagering per se (i.e., just high bet or low bet), but became more active when monkeys successfully chose the optimal bet option based on mnemonic decision performance. Thereafter, the pIPL prompts dorsal anterior cingulate cortex to carry the chosen wagering option. Our findings suggest a role for the pIPL in metacognitive concept integration.

Promethazine Downregulates Wnt/ß-Catenin Signaling and Increases the Biomechanical Forces of the Injured Achilles Tendon in the Early Stage of Healing.

BACKGROUND: Wnt/ß-catenin signaling suppresses the differentiation of cultured tenocytes, but its roles in tendon repair remain mostly elusive. No chemical compounds are currently available to treat tendon injury. HYPOTHESIS: We hypothesized that the inhibition of Wnt/ß-catenin signaling would accelerate tendon healing. STUDY DESIGN: Controlled laboratory study. METHODS: Tendon-derived cells (TDCs) were isolated from rat Achilles tendons. The right Achilles tendon was injured via a dermal punch, while the left tendon was sham operated. A Wnt/ß-catenin inhibitor, IWR-1, and an antihistamine agent, promethazine (PH), were locally and intramuscularly injected, respectively, for 2 weeks after surgery. The healing tendons were histologically and biomechanically evaluated. RESULTS: The amount of ß-catenin protein was increased in the injured tendons from postoperative weeks 0.5 to 2. Inhibition of Wnt/ß-catenin signaling by IWR-1 in healing tendons improved the histological abnormalities and decreased ß-catenin, but it compromised the biomechanical properties. As we previously reported that antihistamine agents suppressed Wnt/ß-catenin signaling in human chondrosarcoma cells, we examined the effects of antihistamines on TDCs. We found that a first-generation antihistamine agent, PH, increased the expression of the tendon marker genes Mkx and Tnmd in TDCs. Intramuscular injection of PH did not improve histological abnormalities, but it decreased ß-catenin in healing tendons and increased the peak force and stiffness of the healing tendons on postoperative week 2. On postoperative week 8, however, the biomechanical properties of vehicle-treated tendons became similar to those of PH-treated tendons. CONCLUSION: IWR-1 and PH suppressed Wnt/ß-catenin signaling and improved the histological abnormalities of healing tendons. IWR-1, however, compromised the biomechanical properties of healing tendons, whereas PH improved them. CLINICAL RELEVANCE: PH is a candidate repositioned drug that potentially accelerates tendon repair.

Reassessing associations between white matter and behaviour with multimodal microstructural imaging.

Several studies have established specific relationships between White Matter (WM) and behaviour. However, these studies have typically focussed on fractional anisotropy (FA), a neuroimaging metric that is sensitive to multiple tissue properties, making it difficult to identify what biological aspects of WM may drive such relationships. Here, we carry out a pre-registered assessment of WM-behaviour relationships in 50 healthy individuals across multiple behavioural and anatomical domains, and complementing FA with myelin-sensitive quantitative MR modalities (MT, R1, R2∗). Surprisingly, we only find support for predicted relationships between FA and behaviour in one of three pre-registered tests. For one behavioural domain, where we failed to detect an FA-behaviour correlation, we instead find evidence for a correlation between behaviour and R1. This hints that multimodal approaches are able to identify a wider range of WM-behaviour relationships than focusing on FA alone. To test whether a common biological substrate such as myelin underlies WM-behaviour relationships, we then ran joint multimodal analyses, combining across all MRI parameters considered. No significant multimodal signatures were found and power analyses suggested that sample sizes of 40-200 may be required to detect such joint multimodal effects, depending on the task being considered. These results demonstrate that FA-behaviour relationships from the literature can be replicated, but may not be easily generalisable across domains. Instead, multimodal microstructural imaging may be best placed to detect a wider range of WM-behaviour relationships, as different MRI modalities provide distinct biological sensitivities. Our findings highlight a broad heterogeneity in WM's relationship with behaviour, suggesting that variable biological effects may be shaping their interaction.