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.

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.

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 immunoregulatory 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 (Fc⑀RI). Coligation of LIR3 to LIR7 or to Fc⑀RI by means of a second monoclonal antibody significantly inhibited net histamine release, cysLT production, and IL-4 generation. That LIR3 is profoundly counterregulatory for both adaptive and innate receptors suggests a broad role in containment of the inflammatory response. IntroductionBecause they resemble mast cells in their panel of mediators and cell surface expression of the high-affinity receptor for the Fc portion of immunoglobulin E (Fc⑀RI), peripheral blood basophils have in the past often been studied as mast cell surrogates due to the relative ease with which they can be isolated. However, basophils differ importantly from mast cells in their developmental pathway and limited supply of effector molecules. Developmentally, basophils originate and mature in the bone marrow, circulating as terminally differentiated cells, while mast cells circulate as precursors and complete their development after transendothelial migration to specific tissues. [1][2][3] Indeed, basophils bear a closer developmental relationship to eosinophils than to mast cells. 4,5 A case report documents the absence of both eosinophils and basophils in a single patient, 6 possibly secondary to destruction or dysfunction of a shared progenitor cell. Infection of Ws/Ws rats, which are deficient in mast cells due to a mutation in c-kit, with the parasite Nippostrongylus brasiliensis led to a more than 50-fold increase in peripheral blood basophils, equal to that observed in wild-type rats. 7 Basophils also appear to have a limited capacity to provide inflammatory mediators in comparison with mast cells. 8 Those currently recognized include histamine stored in preformed granules, leukotriene (LT) C 4 generated from arachidonic acid with cell activation, and interleukin (IL) 4 induced also by activation. 9 That basophils can provide IL-4 has led to the suggestion that these cells may reinforce T helper 2 (Th2) cell polarization. [10][11][12] Basop...

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. INTRODUCTIONPolarized epithelial cells such as those in ductular organs, including the pancreas, form and maintain their tubular tissue architecture through regulated associations with adjacent cells (Hogan and Kolodziej, 2002;Lubarsky and Krasnow, 2003;Zegers et al., 2003). The integrity of these lateral interactions is mediated, in part, by adherens junctions (AJs), of which the transmembrane protein E-cadherin (E-cad) is a major component. On the extracellular side, homophilic antiparallel interactions between E-cad molecules present on adjacent cells mediate the assembly and maintenance of AJs, whereas on the intracellular side the cytoplasmic tail of E-cad is associated with an array of actin cytoskeletal proteins as well as signaling molecules such as catenins, small GTPases, and nonreceptor tyrosine kinases (Perez-Moreno et al., 2003;Gumbiner, 2005). Although maintenance of stable junctions is important for tissue integrity and the functional properties of polarized epithelia, AJs are also dynamic structures undergoing cycles of assembly and disassembly. Indeed, reorganization of AJs is a key aspect of tissue morphogenesis both during normal development as well as tumor cell metastasis, when the structural integrity of AJs is compromised as tumor cells lose polarity and subsequently dissociate before migration (Thiery, 2002;Cavallaro and Christofori, 2004).Pancreatic cancer is a particularly deadly disease and is listed as one of the top five most lethal cancers in the United States (Jemal et al., 2006). Although less prevalent than other cancers, its mortality rate is well over 90% within 6 mo of diagnosis. This exceptionally high lethality is due to a lack of early diagnostic tools, the dispersed organization of the pancreas within the abdomen, and a significant propensity of neoplastic cells to disseminate and migrate from the pancreas to nearby organs (Shi et al., 2001;Freelove and Walling, 2006).A reduction ...

ObjectiveConflicting 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.DesignWe 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.ResultsGF 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.ConclusionsWe 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.