Protective and aggressive bacterial subsets and metabolites modify hepatobiliary inflammation and fibrosis in a murine model of PSC.

OBJECTIVE: Conflicting microbiota data exist for primary sclerosing cholangitis (PSC) and experimental models. GOAL: define the function of complex resident microbes and their association relevant to PSC patients by studying germ-free (GF) and antibiotic-treated specific pathogen-free (SPF) multidrug-resistant 2 deficient (mdr2-/- ) mice and microbial profiles in PSC patient cohorts. DESIGN: We measured weights, liver enzymes, RNA expression, histological, immunohistochemical and fibrotic biochemical parameters, faecal 16S rRNA gene profiling and metabolomic endpoints in gnotobiotic and antibiotic-treated SPF mdr2-/- mice and targeted metagenomic analysis in PSC patients. RESULTS: GF mdr2-/- mice had 100% mortality by 8 weeks with increasing hepatic bile acid (BA) accumulation and cholestasis. Early SPF autologous stool transplantation rescued liver-related mortality. Inhibition of ileal BA transport attenuated antibiotic-accelerated liver disease and decreased total serum and hepatic BAs. Depletion of vancomycin-sensitive microbiota exaggerated hepatobiliary disease. Vancomycin selectively decreased Lachnospiraceae and short-chain fatty acids (SCFAs) but expanded Enterococcus and Enterobacteriaceae. Antibiotics increased Enterococcus faecalis and Escherichia coli liver translocation. Colonisation of GF mdr2-/- mice with translocated E. faecalis and E. coli strains accelerated hepatobiliary inflammation and mortality. Lachnospiraceae colonisation of antibiotic pretreated mdr2-/- mice reduced liver fibrosis, inflammation and translocation of pathobionts, and SCFA-producing Lachnospiraceae and purified SCFA decreased fibrosis. Faecal Lachnospiraceae negatively associated, and E. faecalis/ Enterobacteriaceae positively associated, with PSC patients' clinical severity by Mayo risk scores. CONCLUSIONS: We identified novel functionally protective and detrimental resident bacterial species in mdr2-/- mice and PSC patients with associated clinical risk score. These insights may guide personalised targeted therapeutic interventions in PSC patients.

Dynamic ultrasonography for optimizing treatment position in superior mesenteric artery syndrome: Two case reports and review of literature.

BACKGROUND: Superior mesenteric artery (SMA) syndrome is a rare cause of duodenal obstruction by extrinsic compression between the SMA and the aorta (SMA-Ao). Although the left lateral recumbent position is considered effective in the treatment of SMA syndrome, individual variations in the optimal patient position have been noted. In this report, we present two elderly cases of SMA syndrome that exhibited rapid recovery due to ultrasonographic dynamic evaluation of the optimal position for each patient. CASE SUMMARY: Case 1: A 90-year-old man with nausea and vomiting. Following diagnosis of SMA syndrome by computed tomography (CT), ultrasonography (US) revealed the SMA-Ao distance in the supine position (4 mm), which slightly improved in the lateral position (5.7-7.0 mm) without the passage of duodenal contents. However, in the sitting position, the SMA-Ao distance was increased to 15 mm accompanied by improved content passage. Additionally, US indicated enhanced passage upon abdominal massage on the right side. By day 2, the patient could eat comfortably with the optimal position and massage. Case 2: An 87-year-old woman with vomiting. After the diagnosis of SMA syndrome and aspiration pneumonia by CT, dynamic US confirmed the optimal position (SMA-Ao distance was improved to 7 mm in forward-bent position, whereas it remained at 5 mm in the supine position). By day 7 when her pneumonia recovered, she could eat with the optimal position. CONCLUSION: The optimal position for SMA syndrome varies among individuals. Dynamic US appears to be a valuable tool in improving patient outcomes.

Validation of rapid fecal calprotectin assay using particle enhanced turbidimetric immunoassay for inflammatory bowel disease.

Fecal calprotectin (FC) is a promising biomarker for diagnosis and treatment of inflammatory bowel disease, ulcerative colitis (UC), and Crohn's disease. An enzyme immunoassay (EIA) is widely used for FC detection, though the considerable lag time, up to several days, causes clinical management delay. This study was performed to examine the new rapid kit fCAL-turbo, which is based on a particle-enhanced turbidimetric immunoassay (15 min), by comparing FC values with other EIAs (EliA, PhiCal, Bühlmann) and endoscopic scores. Using 94 samples, fCAL-turbo showed strong significant positive correlations with the other kits (Spearman's r = 0.9178-0.9886). Of 74 UC patients, 69 underwent an endoscopy and fCAL-turbo reflected endoscopic activity with a moderate correlation with Mayo endoscopic subscore (MES) (r = 0.6945, others r = 0.6682-0.7013). Receiver operating characteristic analyses based on MES 0 versus 1-3 showed a similar efficacy as compared to the other kits (cut-off and area under the curve: 89.70 µg/g and 0.8592, respectively, others 62.35-138.4 µg/g and 0.8280-0.8611, respectively). Furthermore, multiple regression analysis confirmed that fCAL-turbo results significantly contributed to prediction of MES 0 with a higher t-value as compared to the other biomarkers. fCAL-turbo showed strong correlations with the other kits and also demonstrated excellent performance for predicting endoscopic remission of UC.

Giardia Antagonizes Beneficial Functions of Indigenous and Therapeutic Intestinal Bacteria during Malnutrition.

Undernutrition in children commonly disrupts the structure and function of the small intestinal microbial community, leading to enteropathies, compromised metabolic health, and impaired growth and development. The mechanisms by which diet and microbes mediate the balance between commensal and pathogenic intestinal flora remain elusive. In a murine model of undernutrition, we investigated the direct interactions Giardia lamblia, a prevalent small intestinal pathogen, on indigenous microbiota and specifically on Lactobacillus strains known for their mucosal and growth homeostatic properties. Our research reveals that Giardia colonization shifts the balance of lactic acid bacteria, causing a relative decrease in Lactobacillus spp . and an increase in Bifidobacterium spp . This alteration corresponds with a decrease in multiple indicators of mucosal and nutritional homeostasis. Additionally, protein-deficient conditions coupled with Giardia infection exacerbate the rise of primary bile acids and susceptibility to bile acid-induced intestinal barrier damage. In epithelial cell monolayers, Lactobacillus spp . mitigated bile acid-induced permeability, showing strain-dependent protective effects. In vivo, L. plantarum, either alone or within a Lactobacillus spp consortium, facilitated growth in protein-deficient mice, an effect attenuated by Giardia , despite not inhibiting Lactobacillus colonization. These results highlight Giardia's potential role as a disruptor of probiotic functional activity, underscoring the imperative for further research into the complex interactions between parasites and bacteria under conditions of nutritional deficiency.

Superior mesenteric artery syndrome: Diagnosis and management.

Superior mesenteric artery (SMA) syndrome (also known as Wilkie's syndrome, cast syndrome, or aorto-mesenteric compass syndrome) is an obstruction of the duodenum caused by extrinsic compression between the SMA and the aorta. The median age of patients is 23 years old (range 0-91 years old) and predominant in females over males with a ratio of 3:2. The symptoms are variable, consisting of postprandial abdominal pain, nausea and vomiting, early satiety, anorexia, and weight loss and can mimic anorexia nervosa or functional dyspepsia. Because recurrent vomiting leads to aspiration pneumonia or respiratory depression via metabolic alkalosis, early diagnosis is required. The useful diagnostic modalities are computed tomography as a standard tool and ultrasonography, which has advantages in safety and capability of real-time assessments of SMA mobility and duodenum passage. The initial treatment is usually conservative, including postural change, gastroduodenal decompression, and nutrient management (success rates: 70%-80%). If conservative therapy fails, surgical treatment (i.e., laparoscopic duodenojejunostomy) is recommended (success rates: 80%-100%).

Giardia hinders growth by disrupting nutrient metabolism independent of inflammatory enteropathy.

Giardia lamblia (Giardia) is among the most common intestinal pathogens in children in low- and middle-income countries (LMICs). Although Giardia associates with early-life linear growth restriction, mechanistic explanations for Giardia-associated growth impairments remain elusive. Unlike other intestinal pathogens associated with constrained linear growth that cause intestinal or systemic inflammation or both, Giardia seldom associates with chronic inflammation in these children. Here we leverage the MAL-ED longitudinal birth cohort and a model of Giardia mono-association in gnotobiotic and immunodeficient mice to propose an alternative pathogenesis of this parasite. In children, Giardia results in linear growth deficits and gut permeability that are dose-dependent and independent of intestinal markers of inflammation. The estimates of these findings vary between children in different MAL-ED sites. In a representative site, where Giardia associates with growth restriction, infected children demonstrate broad amino acid deficiencies, and overproduction of specific phenolic acids, byproducts of intestinal bacterial amino acid metabolism. Gnotobiotic mice require specific nutritional and environmental conditions to recapitulate these findings, and immunodeficient mice confirm a pathway independent of chronic T/B cell inflammation. Taken together, we propose a new paradigm that Giardia-mediated growth faltering is contingent upon a convergence of this intestinal protozoa with nutritional and intestinal bacterial factors.

Rationally designed bacterial consortia to treat chronic immune-mediated colitis and restore intestinal homeostasis.

Environmental factors, mucosal permeability and defective immunoregulation drive overactive immunity to a subset of resident intestinal bacteria that mediate multiple inflammatory conditions. GUT-103 and GUT-108, live biotherapeutic products rationally designed to complement missing or underrepresented functions in the dysbiotic microbiome of IBD patients, address upstream targets, rather than targeting a single cytokine to block downstream inflammation responses. GUT-103, composed of 17 strains that synergistically provide protective and sustained engraftment in the IBD inflammatory environment, prevented and treated chronic immune-mediated colitis. Therapeutic application of GUT-108 reversed established colitis in a humanized chronic T cell-mediated mouse model. It decreased pathobionts while expanding resident protective bacteria; produced metabolites promoting mucosal healing and immunoregulatory responses; decreased inflammatory cytokines and Th-1 and Th-17 cells; and induced interleukin-10-producing colonic regulatory cells, and IL-10-independent homeostatic pathways. We propose GUT-108 for treating and preventing relapse for IBD and other inflammatory conditions characterized by unbalanced microbiota and mucosal permeability.

Homeostatic regulation of Salmonella-induced mucosal inflammation and injury by IL-23.

IL-12 and IL-23 regulate innate and adaptive immunity to microbial pathogens through influencing the expression of IFN-γ, IL-17, and IL-22. Herein we define the roles of IL-12 and IL-23 in regulating host resistance and intestinal inflammation during acute Salmonella infection. We find that IL-23 alone is dispensable for protection against systemic spread of bacteria, but synergizes with IL-12 for optimal protection. IL-12 promotes the production of IFN-γ by NK cells, which is required for resistance against Salmonella and also for induction of intestinal inflammation and epithelial injury. In contrast, IL-23 controls the severity of inflammation by inhibiting IL-12A expression, reducing IFN-γ and preventing excessive mucosal injury. Our studies demonstrate that IL-23 is a homeostatic regulator of IL-12-dependent, IFN-γ-mediated intestinal inflammation.

Caveolae mediate growth factor-induced disassembly of adherens junctions to support tumor cell dissociation.

Remodeling of cell-cell contacts through the internalization of adherens junction proteins is an important event during both normal development and the process of tumor cell metastasis. Here we show that the integrity of tumor cell-cell contacts is disrupted after epidermal growth factor (EGF) stimulation through caveolae-mediated endocytosis of the adherens junction protein E-cadherin. Caveolin-1 and E-cadherin closely associated at cell borders and in internalized structures upon stimulation with EGF. Furthermore, preventing caveolae assembly through reduction of caveolin-1 protein or expression of a caveolin-1 tyrosine phospho-mutant resulted in the accumulation of E-cadherin at cell borders and the formation of tightly adherent cells. Most striking was the fact that exogenous expression of caveolin-1 in tumor cells that contain tight, well-defined, borders resulted in a dramatic dispersal of these cells. Together, these findings provide new insights into how cells might disassemble cell-cell contacts to help mediate the remodeling of adherens junctions, and tumor cell metastasis and invasion.

Dynamin 2 mediates fluid-phase micropinocytosis in epithelial cells.

It is well-known that dynamin 2 (Dyn2) participates in clathrin- and caveolae-mediated endocytosis; however, the role of Dyn2 in coat-independent endocytic processes remains controversial. Here we demonstrate a role for specific spliced variants of Dyn2 in the micropinocytosis of fluid in epithelial cells, independent of coat-mediated endocytic pathways. A general inhibition of Dyn2 was first performed using either microinjection of anti-dynamin antibodies or Dyn2-siRNA treatment. Both of these methods resulted in reduced uptake of transferrin, a marker for clathrin-mediated endocytosis, and, under unstimulated conditions, reduced the uptake of the fluid-phase markers dextran and horseradish peroxidase (HRP). By contrast, cells treated similarly but stimulated with serum or EGF internalized substantial amounts of dextran or HRP, indicating that Dyn2 is not required for stimulated fluid uptake via macropinocytosis. We next tested whether a specific spliced variant might selectively affect fluid-phase endocytosis. Mutation of specific Dyn2 spliced variants resulted in a differential attenuation of transferrin and dextran internalization. Furthermore, the reduction in fluid uptake in Dyn2-siRNA-treated cells was only rescued upon re-expression of select spliced variants. These findings suggest that Dyn2 function is required for the coat-independent internalization of fluid through endocytic pathways distinct from macropinocytosis and, in addition, implicate different Dyn2 spliced variants in specific endocytic functions.

Leukocyte immunoglobulin-like receptors: novel innate receptors for human basophil activation and inhibition.

Basophils, recruited from the blood to tissues, have been implicated by their presence in diverse allergic disorders including bronchial asthma, allergic rhinitis, and cutaneous contact hypersensitivity. We hypothesized that like other leukocytes involved in inflammatory responses, basophils would express members of the leukocyte immunoglobulin-like receptor (LIR) family of immuno-regulatory molecules on their cell surface. We identified LIR7, an activating member coupled to the common Fc receptor gamma chain, and LIR3, an inhibitory member containing cytoplasmic immunoreceptor tyrosine-based inhibitory motifs, on these cells from human peripheral blood. Cross-linking of LIR7 resulted in the concentration-dependent net release of histamine (29.8 +/- 10.8%) and cysteinyl leukotrienes (cysLTs) (31.4 +/- 8.7 ng/10(6) basophils) that were maximal at 30 minutes, and of interleukin-4 (IL-4) (410.2 +/- 61.6 pg/10(6) basophils) that was maximal at 4 hours and comparable with the response initiated by cross-linking of the high-affinity receptor for immunoglobulin E (FcepsilonRI). Coligation of LIR3 to LIR7 or to FcepsilonRI by means of a second monoclonal antibody significantly inhibited net histamine release, cysLT production, and IL-4 generation. That LIR3 is profoundly counter-regulatory for both adaptive and innate receptors suggests a broad role in containment of the inflammatory response.