NLRP1B and NLRP3 Control the Host Response following Colonization with the Commensal Protist Tritrichomonas musculis.

Commensal intestinal protozoa, unlike their pathogenic relatives, are neglected members of the mammalian microbiome. These microbes have a significant impact on the host's intestinal immune homeostasis, typically by elevating anti-microbial host defense. Tritrichomonas musculis, a protozoan gut commensal, strengthens the intestinal host defense against enteric Salmonella infections through Asc- and Il1r1-dependent Th1 and Th17 cell activation. However, the underlying inflammasomes mediating this effect remain unknown. In this study, we report that colonization with T. musculis results in an increase in luminal extracellular ATP that is followed by increased caspase activity, higher cell death, elevated levels of IL-1ß, and increased numbers of IL-18 receptor-expressing Th1 and Th17 cells in the colon. Mice deficient in either Nlrp1b or Nlrp3 failed to display these protozoan-driven immune changes and lost resistance to enteric Salmonella infections even in the presence of T. musculis These findings demonstrate that T. musculis-mediated host protection requires sensors of extracellular and intracellular ATP to confer resistance to enteric Salmonella infections.

Lgr5 and stem/progenitor gene expression in gastric/gastroesophageal junction carcinoma - significance of potentially retained stemness.

BACKGROUND: Gastric/gastroesophageal junction (GEJ) adenocarcinomas are heterogeneous, comprising four molecularly distinct subtypes, namely EBV-positive, microsatellite instability (MSI), chromosomal instability (CIN) and genomically stable (GS) subtypes, and a part of this heterogeneity may hypothesized to be different cells-of-origin. Stem/progenitor cell hierarchy in the stomach is complex, which include the Lgr5(+) gastric stem cells (GSCs). METHODS: While previous studies have focused on non-nuclear Lgr5 expression, nuclear Lgr5 expression has been reported in a subset of stem cells, and we examined nuclear Lgr5 expression in a local cohort of 95 cases of gastric/GEJ adenocarcinoma. mRNA levels for LGR5 and other stem cell marker genes were examined in the TCGA cohort. RESULTS: We observed nuclear Lgr5 expression in a 18/95 cases. Near mutual exclusivity was seen between nuclear Lgr5 and strong non-nuclear Lgr5. Both strong non-nuclear and nuclear Lgr5 expression tended to be seen more frequently with the intestinal histotype and approximated CIN molecular subtype. With respect to overall survival (OS), nuclear Lgr5 expression appears to be protective, with the worst survival being seen in the cases lacking nuclear Lgr5 and with low non-nuclear Lgr5 expression. When compared to other stem/progenitor cell markers, LGR5 mRNA expression clusters with other GSC marker genes, including VIL1. Higher expression of these GSC marker genes was associated with better OS. CONCLUSIONS: Our results show that Lgr5 expression is dynamic in gastric/GEJ adenocarcinoma and heterogeneous across the several disease attributes. We postulate that this may reflect "retained stemness" in the form of Lgr5High-GSC signature that appears to be associated with better survival.

Isolated Optic Perineuritis as the Presenting Sign of Sarcoidosis.

A 35-year-old healthy African-American woman presented with a 4-month history of gradual loss of vision in the right eye from an optic neuropathy. MRI of the orbits with gadolinium showed isolated thickening and enhancement of the right optic nerve sheath. Chest x-ray and CT-scan of the chest were performed and showed bilateral hilar and mediastinal lymphadenopathy. This was suggestive of sarcoidosis, and the diagnosis was confirmed with histopathology. The patient promptly recovered vision with high-dose corticosteroids; the thickening of the optic nerve sheath also regressed. Isolated optic nerve sheath thickening from sarcoidosis is rare and may mimic compressive optic neuropathies such as optic nerve sheath meningiomas. A systemic evaluation for systemic inflammatory etiologies should be considered in such cases.

Clinical, endoscopic, and histologic characteristics of lymphocytic esophagitis: a systematic review.

OBJECTIVE: Lymphocytic esophagitis (LyE) is a novel, yet poorly described, clinicopathologic entity. The aim of this systematic review was to characterize the demographic, clinical, endoscopic, and histologic features of LyE in observational studies of adult and pediatric patients. DESIGN: We searched the Embase, MEDLINE, and SCOPUS databases for relevant studies in 2018. Two authors reviewed and extracted data from studies that met the inclusion and exclusion criteria. RESULTS: We identified 20 studies for analysis of demographic, clinical, and endoscopic features of LyE. The mean age ranged from 9 to 67 years. When pooled, there were 231 (52.7%) patients with LyE that were female. The most common presenting symptom was dysphagia reported in 191 (48.8%) patients. On endoscopy, most patients with LyE tended to have abnormal findings (69.0%), which included erosive esophagitis, multiple esophageal rings, linear furrows, and narrow-caliber esophagus. In the 31 studies used to assess the histologic definition, the cut-off number of intraepithelial lymphocytes (IELs) was reported in 16 (51.6%) studies, peripapillary IEL specification in 18 (58.1%) studies, and presence of spongiosis in 6 (19.4%) studies. CONCLUSION: We identified a spectrum of demographic, clinical, and endoscopic findings characteristic of patients with LyE. A consensus on the diagnostic criteria of LyE is required.

NKT Cell-Deficient Mice Harbor an Altered Microbiota That Fuels Intestinal Inflammation during Chemically Induced Colitis.

NKT cells are unconventional T cells that respond to self and microbe-derived lipid and glycolipid Ags presented by the CD1d molecule. Invariant NKT (iNKT) cells influence immune responses in numerous diseases. Although only a few studies have examined their role during intestinal inflammation, it appears that iNKT cells protect from Th1-mediated inflammation but exacerbate Th2-mediated inflammation. Studies using iNKT cell-deficient mice and chemically induced dextran sodium sulfate (DSS) colitis have led to inconsistent results. In this study, we show that CD1d-deficient mice, which lack all NKT cells, harbor an altered intestinal microbiota that is associated with exacerbated intestinal inflammation at steady-state and following DSS treatment. This altered microbiota, characterized by increased abundance of the bacterial phyla Proteobacteria, Deferribacteres, and TM7, among which the mucin-eating Mucispirillum, as well as members of the genus Prevotella and segmented filamentous bacteria, was transmissible upon fecal transplant, along with the procolitogenic phenotype. Our results also demonstrate that this proinflammatory microbiota influences iNKT cell function upon activation during DSS colitis. Collectively, alterations of the microbiota have a major influence on colitis outcome and therefore have to be accounted for in such experimental settings and in studies focusing on iNKT cells.

Mechanisms of Chronic Muscle Wasting and Dysfunction after an Intensive Care Unit Stay. A Pilot Study.

RATIONALE: Critical illness survivors often experience permanent functional disability due to intensive care unit (ICU)-acquired weakness. The mechanisms responsible for long-term weakness persistence versus resolution are unknown. OBJECTIVES: To delineate cellular mechanisms underlying long-term weakness persistence in ICU survivors. METHODS: We conducted a nested, prospective study of critically ill patients mechanically ventilated for 7 days or longer. The patients were recruited from the RECOVER program and serially assessed over 6 months after ICU discharge. Twenty-seven of 82 patients consented to participate; 15 and 11 patients were assessed at 7 days and 6 months after ICU discharge, respectively. MEASUREMENTS AND MAIN RESULTS: We assessed motor functional capacity, quadriceps size, strength, and voluntary contractile capacity and performed electromyography, nerve conduction studies, and vastus lateralis biopsies for histologic, cellular, and molecular analyses. Strength and quadriceps cross-sectional areas were decreased 7 days after ICU discharge. Weakness persisted to 6 months and correlated with decreased function. Quadriceps atrophy resolved in 27% patients at 6 months. Muscle mass reconstitution did not correlate with resolution of weakness, owing to persistent impaired voluntary contractile capacity. Compared with Day 7, increased ubiquitin-proteasome system-mediated muscle proteolysis, inflammation, and decreased mitochondrial content all normalized at 6 months. Autophagy markers were normal at 6 months. Patients with sustained atrophy had decreased muscle progenitor (satellite) cell content. CONCLUSIONS: Long-term weakness in ICU survivors results from heterogeneous muscle pathophysiology with variable combinations of muscle atrophy and impaired contractile capacity. These findings are not explained by ongoing muscle proteolysis, inflammation, or diminished mitochondrial content. Sustained muscle atrophy is associated with decreased satellite cell content and compromised muscle regrowth, suggesting impaired regenerative capacity.

Prognostic significance of human tissue kallikrein-related peptidases 11 and 15 in gastric cancer.

Human tissue kallikrein-related peptidases (KLK) are a group of 15 serine proteases which have been investigated as potential cancer biomarkers. This study determined the prognostic significance of KLK 11 and 15 expression levels in gastric carcinoma specimens. Expression of KLK11 and KLK15 was assessed by immunohistochemistry staining on a tissue microarray constructed from 113 gastrectomy specimens from patients with gastric carcinoma. To minimize inter-observer variability, expression levels were quantified using an automated algorithm. Epithelial and stromal staining were assessed separately. Both KLK11 and KLK15 were expressed in gastric carcinoma. There was no significant correlation between either KLK11 or KLK15 expression and the presence of lymph node metastases or Lauren classification (intestinal vs. diffuse). Higher levels of KLK11 expression in gastric carcinoma were associated with significantly worse overall survival (p = 0.008), and a multivariate analysis showed that it had prognostic value independent of tumor stage and differentiation (p = 0.004). Variations in KLK15 expression were not significantly associated with prognosis. KLK11 shows promise as a potential independent prognostic marker for gastric carcinoma.

Folic Acid Supplementation Adversely Affects Chemosensitivity of Colon Cancer Cells to 5-fluorouracil.

Folic acid (FA) fortification and widespread supplemental use have significantly increased folate status in North America. Furthermore, >50% of colorectal cancer patients use FA supplement. The increased folate status may interfere with cancer chemotherapy. We investigated the effect of FA supplementation on chemosensitivity of human colon cancer cells to 5-fluorouracil (5-FU) using a xenograft model. Mice harboring human HCT116 colon cancer xenografts were randomized to receive the control, or 4× or 12.5× supplemental levels of FA. Within each diet group, mice were randomized to receive 5-FU+leucovorin or saline and xenograft growth and characteristics were determined. The expression of genes involved in folate metabolism and cancer treatment was determined. FA supplementation and 5-FU significantly interacted to influence xenograft growth (P < 0.007). At the control level, 5-FU significantly inhibited the growth of the xenografts (P < 0.0001). However, at the 4× supplemental level, 5-FU-treated xenografts grew faster than untreated xenografts (P = 0.048) while at the 12.5× supplemental level, 5-FU exhibited no effect. Cell proliferation, degree of necrosis, and expression of the selected genes did not significantly differ by the supplemental levels of FA. Our data suggest that FA supplementation may be detrimental to 5-FU chemotherapy of colon cancer and pose public health concern.

HLA-B27, but not HLA-B7, immunodominance to influenza is ERAP dependent.

Endoplasmic reticulum-associated aminopeptidase-1 (ERAP1) plays a critical role in the processing of peptides prior to binding to MHC class I molecules. In this article, we show for the first time, to our knowledge, that the HLA-B27 immunodominant influenza nucleoprotein (NP) 383-391 epitope is made as an N-terminally extended 14-mer before it is trimmed by ERAP. In the absence of ERAP, there is a significant reduction in the CTL response to the B27/NP383-391 epitope in influenza A (flu)-infected B27/ERAP(-/-) mice. With the use of tetramer staining, the number of naive CD8(+) T cells expressing TCR Vß8.1 in B27/ERAP(-/-) transgenic mice is significantly lower than that seen in B27/ERAP(+/+) mice. HLA-B27 surface expression in naive and flu-infected B27/ERAP(-/-) mice is also lower than the expression seen for the same allele in naive and flu-infected B27/ERAP(+/+) mice. In contrast, surface expression of HLA-B7 was unaffected by the absence of ERAP in B7/ERAP(-/-) transgenic mice. The B7-restricted NP418-426 CTL response in flu-infected B7/ERAP(-/-) and B7/ERAP(+/+) mice was also similar. These results provide, to our knowledge, the first in vivo demonstration of ERAP functionally influencing host immune response in an HLA allele-specific manner. This principle has relevance to diseases such as ankylosing spondylitis, in which HLA-B27 and ERAP jointly contribute to disease predisposition.

Inflammatory pseudotumours of the oesophagus--histological and immunohistochemical findings.

AIMS: Inflammatory oesophageal pseudotumours are rare lesions, thought to be reactive. Due to marked atypia of the stromal cells, these can be misdiagnosed as malignancies. The objective of this study was to characterize histological and immunohistochemical features of a series of inflammatory pseudotumours of the oesophagus. METHODS AND RESULTS: We present 12 cases of inflammatory oesophageal pseudotumours, occurring in seven females and five males, with a mean age of 57.3 years. Clinical presentations were variable; dysphagia, abdominal pain and weight loss and upper gastrointestinal bleed. In a majority of the cases, nodules or masses in the distal oesophagus were identified at endoscopy. Microscopically, the lamina propria in all 12 cases contained inflammation and granulation tissue. Ten of 12 cases showed mucosal ulceration and 11 of 12 cases had acutely inflamed epithelium. Markedly atypical pleomorphic stromal cells with prominent nucleoli were identified in all 12 cases. Immunohistochemistry showed uniform positivity for vimentin in 11 of 11 cases, and two of seven cases demonstrated weak focal positivity for smooth muscle actin. The cells were negative for all other markers. CONCLUSIONS: Reactive oesophageal lesions can show marked nuclear atypia in stromal fibroblasts/myofibroblasts, which are easily mistaken for malignancies. Pathologists must consider the diagnosis of an inflammatory pseudotumour if stromal atypia is present in an inflammatory background.

Prognostic significance of human tissue kallikrein-related peptidases 6 and 10 in gastric cancer.

The prognosis of patients following surgery for gastric cancer is often poor and is estimated using traditional clinicopathological parameters, which can be inaccurate predictors of future survival. Kallikreins are a group of serine proteases, which are differentially expressed in many human tumors and are being investigated as potential cancer biomarkers. This study assessed the prognostic utility of human tissue kallikrein-like peptidases 6 and 10 (KLK6 and KLK10) and correlated their expression with histopathological and clinical parameters in gastric cancer. We constructed a gastric tumor tissue microarray from 113 gastrectomy specimens and quantified KLK6 and KLK10 expression using immunohistochemistry. To overcome the problem of inter-observer variability and subjectivity in immunohistochemistry interpretation, a whole-slide scanned image of the tissue microarray was analyzed using an automated algorithm to quantify staining intensity. KLK6 expression was positively correlated with nodal involvement (p=0.002) and was predictive of advanced-stage disease (p<0.05). Kaplan-Meier survival curves revealed that tumors expressing high levels of KLK6 were significantly associated with significantly lower overall survival (p=0.04). KLK10 overexpression was also a predictor of advanced-stage disease (p<0.01), but was not significantly correlated with lymph node involvement or survival period. Our results show the potential ability of KLK6 as a prognostic marker for gastric cancer.

ATG16L1 protects from interferon-γ-induced cell death in the small intestinal crypt.

The breakdown of the intestinal mucosal barrier is thought to underlie the progression to Crohn disease (CD), whereby numerous risk factors contribute. For example, a genetic polymorphism of the autophagy gene ATG16L1, associated with an increased risk of developing CD, contributes to the perturbation of the intestinal epithelium. We examined the role of Atg16l1 in protecting the murine small intestinal epithelium from T-cell-mediated damage using the anti-CD3 model of enteropathy. Our work showed that mice specifically deleted for Atg16l1 in intestinal epithelial cells (IECs) (Atg16l1ΔIEC) had exacerbated intestinal damage, characterized by crypt epithelial cell death, heightened inflammation, and decreased survival. Moreover, Atg16l1 deficiency delayed the recovery of the intestinal epithelium, and Atg16l1-deficient IECs were impaired in their proliferative response. Pathology was largely driven by interferon (IFN)-γ signaling in Atg16l1ΔIEC mice. Mechanistically, although survival was rescued by blocking tumor necrosis factor or IFN-γ independently, only anti-IFN-γ treatment abrogated IEC death in Atg16l1ΔIEC mice, thereby decoupling IEC death and survival. In summary, our findings suggest differential roles for IFN-γ and tumor necrosis factor in acute enteropathy and IEC death in the context of autophagy deficiency and suggest that IFN-γ-targeted therapy may be appropriate for patients with CD with variants in ATG16L1.

Intestinal microRNAs and bacterial taxa in juvenile mice are associated, modifiable by allochthonous lactobacilli, and affect postnatal maturation.

The interplay between the intestinal microbiota and host is critical to intestinal ontogeny and homeostasis. MicroRNAs (miRNAs) may be an underlying link. Intestinal miRNAs are microbiota-dependent and, when shed in the lumen, affect resident microorganisms. Yet, longitudinal relationships between intestinal tissue miRNAs, luminal miRNAs, and luminal microorganisms have not been elucidated, especially in early life. Here, we investigated the postnatal cecal miRNA and microbiota populations, their relationship, and their impact on intestinal maturation in specific pathogen-free mice; we also assessed if they can be modified by intervention with allochthonous probiotic lactobacilli. We report that cecal and cecal content miRNA and microbiota signatures are temporally regulated, correlated, and modifiable by probiotics with implications for intestinal maturation. These findings help understand causal relationships within the gut ecosystem and provide a basis for preventing and managing their alterations in diseases throughout life. IMPORTANCE The gut microbiota affects intestinal microRNA (miRNA) signatures and is modified by host-derived luminal miRNA. This suggests the existence of close miRNA-microbiota relationships that are critical to intestinal homeostasis. However, an integrative analysis of these relationships and their evolution during intestinal postnatal maturation is lacking. We provide a system-level longitudinal analysis of miRNA-microbiota networks in the intestine of mice at the weaning transition, including tissue and luminal miRNA and luminal microbiota. To address causality and move toward translational applications, we used allochthonous probiotic lactobacilli to modify these longitudinal relationships and showed that they are critical for intestinal maturation in early life. These findings contribute to understand mechanisms that underlie the maturation of the intestinal ecosystem and suggest that interventions aiming at maintaining, or restoring, homeostasis cannot prescind from considering relationships among its components.

Microvascular density as an independent predictor of clinical outcome in renal cell carcinoma: an automated image analysis study.

Tumor microvascular density (MVD) has been shown to correlate with the aggressiveness of several cancers. With the introduction of targeted anti-angiogenic therapy, assessment of MVD has the potential not only as a prognostic but also as a therapeutic marker. The significance of tumor vascularity in clear cell renal cell carcinoma (ccRCC) has been debated, with studies showing contradictory results. Previous studies were limited by manual quantification of MVD within a small area of tumor. Since then, the validity of this method has been questioned. To avoid the inaccuracies of manual quantification, we employed a computerized image analysis, which allowed assessment of large areas of tumor and adjacent normal tissue. The latter was used as an internal reference for normalization. MVD and vascular endothelial growth factor (VEGF) were assessed in 57 cases of ccRCC. Sections were immunostained for CD34 and VEGF. Areas of ccRCC and normal kidney medulla were analyzed within scanned images using software that counted CD34-positive vessels and measured the intensity of VEGF staining. We obtained unadjusted values from tumoral areas and calculated adjusted values as tumor/normal ratios. Unadjusted MVD had no association with clinical outcome. However, similarly to tumor stage, higher adjusted MVD was associated with shorter disease-free survival (log-rank P=0.037, Cox P=0.02). This was significant in univariate and multivariate analyses. MVD did not correlate with tumor stage, pointing to its independent prognostic value. As expected due to the known molecular abnormalities in ccRCC, most tumors showed higher VEGF expression than normal tissue. Higher adjusted VEGF was associated with high tumor grade (P=0.049). The finding of increased MVD as an independent marker of tumor aggressiveness may prove useful in the development of new tests for prognostic and therapeutic guidance. Digital techniques can provide more accurate assessment of immunomarkers and may reveal less obvious associations.

Video. "No scar" small bowel resection in a survival porcine model using transcolonic NOTES(®) and transabdominal approach.

BACKGROUND: Fundamental techniques and essential tools for performing "no scar" surgery still need to be developed. Our study was designed to evaluate the feasibility of performing small bowel resection by transcolonic NOTES(®) and transabdominal approach using rigid laparoscopic and flexible endoscopic instruments. METHODS: One non survival and four survival experiments were performed using a porcine model. The endoscope with an overtube was advanced into the peritoneal cavity through the colotomy. Mini-laparoscopic instruments were placed through the abdominal wall under the endoscopic observation. The endoscope was replaced with a rigid linear stapler. The small bowel was identified. The segment of the small bowel was resected by firing the endo stapler, and extracted through the colon. The two limbs of the small bowel were approximated with two stay-sutures. An enterotomy was then created on the antimesenteric sides of each line. A side-to-side anastomosis was performed with another application of the endo stapler. The stapler was withdrawn. The enterotomy was closed by suturing. The colotomy was closed with endoclips and the endoscope was withdrawn. The mini-laparoscopic instruments were removed. RESULTS: Small bowel resection was successfully performed in all animals. The surgery time was 70 minutes. There was no mortality or complications. The animals recovered uneventfully, and survived the 2 weeks postprocedure period. They remained healthy, and gained weight. Necropsy was performed 2 weeks after the surgery. On necropsy, evaluation of the abdominal skin revealed no scars. The peritoneal cavity was examined. No signs of infection, bleeding, perforations, and adhesions were noted. Endoscopic examination of the colotomy and anastomosis revealed complete healing that was confirmed by histopathology. CONCLUSIONS: The study has demonstrated the feasibility of small bowel resection using transcolonic NOTES(®) and transabdominal approach. Simultaneous use of flexible endoscopic and rigid laparoscopic instruments in NOTES(®) is not only feasible but has significant advantages and greatly facilitates the performance of the operation, yet leaves no scars.

Feasibility and safety of adhesiolysis using transgastric NOTES approach: a pilot survival study in a porcine model.

OBJECTIVES: Although adhesions can be removed by adhesiolysis using laparotomy or laparoscopy, they typically recur sometimes with equal severity. It is suggested that minimizing the invasiveness of the operative technique by using natural orifice translumenal endoscopic surgery (NOTES) may reduce adhesion re-formation. The aim of the study was to evaluate the feasibility and safety of adhesiolysis by using a novel transgastric NOTES approach and collect pilot data on adhesion recurrence after transgastric NOTES adhesiolysis. METHODS: One nonsurvival and 5 survival female pigs were used in this experimental survival study. Interventions included (a) induction of adhesions by laparotomy, (b) 2 weeks survival, (c) transgastric NOTES adhesiolysis with endoscopic evaluation and scoring of adhesions before and immediately after adhesiolysis, (d) 2 weeks survival, and (e) necropsy with endoscopic and necroscopic evaluation and scoring of recurrent adhesions. Main outcome measures were (a) survival and complication rates and (b) assessment of adhesion formation and re-formation using the Hopkins Adhesion Formation Score. RESULTS: No mortality and no complications were observed. A total of 11 adhesions formed before the adhesiolysis in 5 survival study animals. All were successfully divided. The frequency of adhesions and median adhesion formation score decreased significantly immediately after adhesiolysis compared with that prior to the procedure (frequency, 11 vs 0, P = .011; the median score = 2.0 [range 1-3] vs 0.0 [range 0-0], P = .004). The treatment gains maintained at 2 weeks after the adhesiolysis. LIMITATIONS: The limitations of this study were the low number of study animals and short-term follow-up data. CONCLUSIONS: Adhesiolysis using NOTES transgastric approach is feasible, safe, and effective with minimal adhesion re-formation.