There is limited information regarding responses by slow cycling stem cells during T. spiralis-induced T-cell mediated intestinal inflammation and how such responses may relate to those of Paneth cells. Transgenic mice, in which doxycycline induces expression of histone 2B (H2B)-green fluorescent protein (GFP), were used. Following discontinuation of doxycycline (“chase” period), retention of H2B-GFP enabled the identification of slow cycling stem cells and long-lived Paneth cells. Inflammation in the small intestine (SI) was induced by oral administration of T. spiralis muscle larvae. Epithelial retention of H2B-GFP per crypt cell position (cp) was studied following immunohistochemistry and using the Score and Wincrypts program. Compared to non-infected controls, there was significant reduction in the number of H2B-GFP-retaining stem cells in T. spiralis-infected small intestines. H2B-GFP-retaining stem cells peaked at around cp 4 in control sections, but smaller peaks at higher cell positions (>10) were seen in sections of inflamed small intestines. In the latter, there was a significant increase in the total number of Paneth cells, with significant reduction in H2B-GFP-retaining Paneth cells, but a marked increase in unlabelled (H2B-GFP-negative) Paneth cells. In conclusion, following T. spiralis-infection, putative slow cycling stem cell numbers were reduced. A marked increase in newly generated Paneth cells at the crypt base led to higher cell positions of the remaining slow cycling stem cells.

DEF6, also known as SLAT and IBP, is critical for the development of autoimmune disease and cancer. In T cells, DEF6 participates in TCR-mediated signalling determining T helper cell-mediated immune responses. In addition, DEF6 acts as a guanine nucleotide exchange factor for Rho GTPases facilitating F-actin assembly and stabilisation of the immunological synapse. However, DEF6 is also a component of mRNA processing bodies (P-bodies) linking it to mRNA metabolism. DEF6 can adopt multiple conformations that result in different cellular localisations and functions. Post translational modifications such as phosphorylation result in conformational change liberating functional domains that are masked in the native stage of DEF6. ITK phosphorylation of Try210/222 liberates the N-terminal end and to a certain extend also the C-terminal coiled coil domain of DEF6 resulting in P-body colocalisation. In fact, the N-terminal 45 amino acids of DEF6 that encode a Ca2+-binding EF hand are sufficient to target P-bodies. Mutant proteins that unleashed the C-terminal coiled coil domain of DEF6 spontaneously aggregated forming large vesicle-like, cytoplasmic structures. These aggregates trapped proteins such as the P-body component DCP1 altering its cytoplasmic localisation. However, cellular stress reversed aggregate formation in mutant DEF6 proteins that contained ITAM and PH domain in conjunction with the coiled coil domain resulting in colocalisation with DCP1. Furthermore, coiled coil-mediated aggregates appeared to function like prions enforcing conformational change onto wild type DEF6 protein.

DEF6 (Differentially Expressed in FDCP 6, also known as IBP and/or SLAT) is critical for the development of autoimmune disease and cancer. In T cells, DEF6 participates in TCR-mediated signalling determining T helper cell-mediated immune responses. In addition, DEF6 acts as a guanine nucleotide exchange factor (GEF) for Rho GTPases facilitating F-actin assembly and stabilisation of the immunological synapse (IS). However, DEF6 is also a component of mRNA processing bodies (P-bodies) linking it to mRNA metabolism. Including DEF6, more than 34 proteins have been shown to localise in P-bodies many of which contain a coiled coil domain, a super-secondary structure likely to facilitate interaction between these proteins. Accordingly, we suggested that the coiled coil domain in the C-terminal end of DEF6 was mediating P-body localisation of DEF6 under cellular stress conditions. However, a comprehensive analysis of wild type and mutant DEF6 proteins expressed in COS7 cells revealed that the coiled coil domain is dispensable for P-body colocalisation. Instead, we show here that the N-terminal 45 amino acids of DEF6 that contain one Ca2+-binding EF hand motif are sufficient to target DEF6 to P-bodies whereas the N-terminal 30 amino acids containing a disrupted EF hand motif are insufficient.

Swap70 functions as a guanine nucleotide exchange factor for Rac and RhoA regulating F-actin cytoskeletal rearrangements and playing a crucial role in mammalian cell activation, migration, adhesion and invasion. Here we show that the zebrafish orthologue, Swap70b, is required for convergent and extension cell movement during gastrulation. Swap70b morphants exhibited broader and shorter body axis but cell fate specification appeared normal. While ectopic Swap70b expression robustly rescued Wnt11 morphants, RhoA overexpression was sufficient to rescue Swap70b morphants, establishing Swap70b as a novel member of the non-canonical Wnt/PCP pathway downstream of Wnt11 and upstream of RhoA. This is distinct from the related Def6a protein that acts downstream of Wnt5b. Def6a/ Swap70b morphants resemble Ppt/Slb double mutant embryos suggesting that Swap70b and Def6a delineate Wnt11 and Wnt5b signalling pathways. & 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Spermatogenesis is a classic model of cycling cell lineages that depend on a balance between stem cell self-renewal for continuity and the formation of progenitors as the initial step in the production of differentiated cells. The mechanisms that guide the continuum of spermatogonial stem cell (SSC) to progenitor spermatogonial transition and precise identifiers of subtypes in the process are undefined. Here we used an Id4-eGfp reporter mouse to discover that EGFP intensity is predictive of the subsets, with the ID4-EGFPBright population being mostly, if not purely, SSCs, whereas the ID4-EGFPDim population is in transition to the progenitor state. These subsets are also distinguishable by transcriptome signatures. Moreover, using a conditional overexpression mouse model, we found that transition from the stem cell to the immediate progenitor state requires downregulation of Id4 coincident with a major change in the transcriptome. Collectively, our results demonstrate that the level of ID4 is predictive of stem cell or progenitor capacity in spermatogonia and dictates the interface of transition between the different functional states.

greatly diminished growth of the pathogen was shown in these organs of CAC-mice treated with CCL1 antisense ODN.Conclusion: M1Mϕ were induced in CAC-mice by CCL1 antisense ODN and these mice were survived after E.faecalis infection.These results indicate the sepsis stemming from E.faecalis translocation in CAC-mice is controllable by CCL1 antisense ODN gene therapy. Mo1832 In VivoAnimal Model to Study Antimicrobial Therapy of C. difficile Infection Zhi-Dong Jiang, Shi Ke Recommended therapy of bacterial infection is generally based on in vitro susceptibility of an organism to an infecting organism.Yet drug bioavailability based on solubility, protein binding and other factors along with host resistance and inflammatory-immune response importantly alter response to treatment.The objective of the study was to establish in vivo animal model to study antimicrobial therapy of C. difficile infection.C. difficile isolate with hyper-virulent factors (toxin A/B plus binary toxin and 18 base pair deletion in tcdC gene) was used in the study.The cultured C. difficile was stained with near infrared (NIR) dye.Stained C. difficile were determined by using fluorescent microscope and confocal microscope.To monitor stained bacterial trafficking in the gut animals were imaged in real-time.The intestines from treated animals were dissected at the end of imaging study.The bacterial signal intensity and migration pattern were recorded and compared.C. difficile toxin secretion in the different segment of GI system was analyzed.Results showed the C. difficile can be stained by NIR dye.Stained bacteria can be imaged by NIR camera system to track the C. difficile traffic in the gut in real-time.Untreated control animal has different bacteria migration pattern than the animal with C. difficile infection treated with ciprofloxacin.The NIR florescent probe can be used to visualize C. difficile trafficking in the gastrointentinal tract through culturing, staining and imaging.Fig. 1.Confocal images of C. difficile.Viable bacterial stained with NIR dye (A.Red) and dead bacterial determined with Sytox green (B.Green).Stain efficiency was determined by co-localized all cells (C.Green) and NIR (C.Red).Confocal microscope analyzed NIR dye binding at subcellular level (D).Red and green represent NIR and cell nuclei, respectively.Results demonstrate C. difficile can be stained by NIR dye.Most bacteria remain viable after the staining procedure.

Swap70 functions as a guanine nucleotide exchange factor for Rac and RhoA regulating F-actin cytoskeletal rearrangements and playing a crucial role in mammalian cell activation, migration, adhesion and invasion. Here we show that the zebrafish orthologue, Swap70b, is required for convergent and extension cell movement during gastrulation. Swap70b morphants exhibited broader and shorter body axis but cell fate specification appeared normal. While ectopic Swap70b expression robustly rescued Wnt11 morphants, RhoA overexpression was sufficient to rescue Swap70b morphants, establishing Swap70b as a novel member of the non-canonical Wnt/PCP pathway downstream of Wnt11 and upstream of RhoA. This is distinct from the related Def6a protein that acts downstream of Wnt5b. Def6a/Swap70b morphants resemble Ppt/Slb double mutant embryos suggesting that Swap70b and Def6a delineate Wnt11 and Wnt5b signalling pathways. (C) 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Swap70 functions as a guanine nucleotide exchange factor for Rac and RhoA regulating F-actin cytoskeletal rearrangements and playing a crucial role in mammalian cell activation, migration, adhesion and invasion. Here we show that the zebrafish orthologue, Swap70b, is required for convergent and extension cell movement during gastrulation. Swap70b morphants exhibited broader and shorter body axis but cell fate specification appeared normal. While ectopic Swap70b expression robustly rescued Wnt11 morphants, RhoA overexpression was sufficient to rescue Swap70b morphants, establishing Swap70b as a novel member of the non-canonical Wnt/PCP pathway downstream of Wnt11 and upstream of RhoA. This is distinct from the related Def6a protein that acts downstream of Wnt5b. Def6a/Swap70b morphants resemble Ppt/Slb double mutant embryos suggesting that Swap70b and Def6a delineate Wnt11 and Wnt5b signalling pathways.

cultured crypts/organoids.The COX-2 inhibitor NS-398 (10 uM) caused a significant decrease in the budding and survival of mouse colonic organoids (n=4, p<0.05) and also caused a reduction in proliferation of human colonic crypts.Furthermore, PGE2 (1 uM) caused a 1.5 fold increase in proliferation at the base of human colonic crypts, and also rescued Cox-2 suppression of crypt cell proliferation (n>5, p<0.05).PGE2 increased the number of LGR5/ OLFM4+ cells along the human crypt axis and inhibition of COX-2 by NS-398 reduced the number of LGR5/OLFM4+ cells along the crypt axis, which was rescued by PGE2 (n>5, p<0.05).Use of EP1-4 receptor antagonists showed differential effects on mouse organoid formation and survival.Inhibition of EP2 and 4 had no effect (n=2), however inhibition of EP1 and EP3 showed a significant reduction in organoid survival and budding (n=3, P<0.05).Of note, tissue obtained from IBD sufferers was characterised by internalisation of EP1-4 receptors and an increased number of OLFM4+ stem/progenitor cells.CONCLUSIONS: PGE2 supports the proliferation, growth and survival of cultured human and mouse crypt/ organoids and maintains intestinal stem/progenitor cells.This effect is mediated in part by activation of EP1 and EP3 receptor subtypes.Ongoing work is investigating the effects of EP receptor inhibition on crypt cell dynamics in IBD.

Localization of DEF6 (SLAT/IBP), a Rho-family guanine nucleotide exchange factor, to the center of the immune synapse is dependent upon ITK, a Tec-family kinase that regulates the spatiotemporal organization of components of T cell signaling pathways and Cdc42-dependent actin polymerization. Here we demonstrate that ITK both interacts with DEF6 and phosphorylates DEF6 at tyrosine residues Tyr(210) and Tyr(222). Expression of a GFP-tagged Y210E-Y222E phosphomimic resulted in the formation of DEF6 cytoplasmic granules that co-localized with decapping enzyme 1 (DCP1), a marker of P-bodies; sites of mRNA degradation. Similarly treatment of cells with puromycin or sodium arsenite, reagents that arrest translation, also resulted in the accumulation of DEF6 in cytoplasmic granules. Bioinformatics analysis identified a glutamine-rich, heptad-repeat region; a feature of aggregating proteins, within the C-terminal region of DEF6 with the potential to promote granule formation through a phosphorylation-dependent unmasking of this region. These data suggest that in addition to its role as a GEF, DEF6 may also function in regulating mRNA translation.

Stem cells have been identified in two locations in small intestinal crypts; those intercalated between Paneth cells and another population (which retains DNA label) are located above the Paneth cell zone, at cell position 4. Because of disadvantages associated with the use of DNA label, doxycycline-induced transient transgenic expression of histone 2B (H2B)-green fluorescent protein (GFP) was investigated. H2B-GFP-retaining putative stem cells were consistently seen, with a peak at cell position 4, over chase periods of up to 112 days. After a 28-day chase, a subpopulation of the H2B-GFP-retaining cells was cycling, but the slow cycling status of the majority was illustrated by lack of expression of pHistone H3 and Ki67. Although some H2B-GFP-retaining cells were sensitive to low-dose radiation, the majority was resistant to low- and high-dose radiation-induced cell death, and a proportion of the surviving cells proliferated during subsequent epithelial regeneration. Long-term retention of H2B-GFP in a subpopulation of small intestinal Paneth cells was also seen, implying that they are long lived. In contrast to the small intestine, H2B-GFP-retaining epithelial cells were not seen in the colon from 28-day chase onward. This implies important differences in stem cell function between these two regions of the gastrointestinal tract, which may have implications for region-specific susceptibility to diseases (such as cancer and ulcerative colitis), in which epithelial stem cells and their progeny are involved.

The ID family of helix-loop-helix proteins regulates cell proliferation and differentiation in many different developmental pathways, but the functions of ID4 in mammary development are unknown. We report that mouse Id4 is expressed in cap cells, basal cells and in a subset of luminal epithelial cells, and that its targeted deletion impairs ductal expansion and branching morphogenesis as well as cell proliferation induced by estrogen and/or progesterone. We discover that p38MAPK is activated in Id4-null mammary cells. p38MAPK is also activated following siRNA-mediated Id4 knockdown in transformed mammary cells. This p38MAPK activation is required for the reduced proliferation and increased apoptosis in Id4-ablated mammary glands. Therefore, ID4 promotes mammary gland development by suppressing p38MAPK activity.

Objective. LYL-1 is a transcription factor containing a basic helix-loop-helix motif closely related to SCL/TAL-1, a regulator of erythroid differentiation. Because LYL-1 is expressed in erythroid cell populations, we addressed its role in erythropoiesis using knockin mice.Materials and Methods. Erythropoiesis of LYL-1(-/-) mice was studied by progenitor assays, flow cytometry, reconstitution assays, and functional tests. Expression of LYL-1, SCL, and GATA-1 was assessed at messenger RNA level by quantitative reverse transcription polymerase chain reaction.Results. LYL-1(-/-) mice displayed decreased erythropoiesis with a partial arrest in differentiation, and enhanced apoptosis associated with decreased Bcl-x(L) expression in the bone marrow (BM). In addition, LYL-1(-/-) BM cells were severely impaired in their abilities to reconstitute the erythroid lineage in competitive assays, suggesting a cell autonomous abnormality of erythropoiesis. In parallel, erythroid progenitor and precursor cells were significantly increased in the spleen of LYL-1(-/-) mice. Expression of LYL-1 was differentially regulated during maturation of erythroblasts and strikingly different between spleen- and BM-derived erythroblasts. Expression of LYL-1 decreased during erythroid differentiation in the spleen whereas it increased in the BM to reach the same level in mature erythroblasts as in the soleen. Loss of Lyl-1 expression was accompanied with an increase of SCL/TAL-1 and GATA-1 transcripts in spleen but not in BM-derived erythroblasts. Furthermore, phenylhydrazine-induced stress erythropoiesis was elevated in LYL-1(-/-) mice and mutant BM and spleen erythroid progenitors were hypersensitive to erythropoietin.Conclusions. Taken together, these results suggest that LYL-1 plays a definite role in erythropoiesis, albeit with different effects in BM specifically regulating basal erythropoiesis, and spleen, controlling stress-induced erythropoiesis. (C) 2011 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc.

Background: Wnt signaling regulates intestinal epithelial stem cell function. Wnt ligands bind Frizzled (Fz) receptors and low-density lipoprotein-receptor-related protein (LRP) 5 and 6. Secreted Frizzled-related protein (SFRP) and Dickkopf families inhibit Wnt signaling. Our aim was to study expression of Wnt family of genes in isolated intestinal myofibroblasts and crypt epithelial cells.Methods: Myofibroblasts were isolated from normal colonic and small intestinal mucosal samples and those affected by ulcerative colitis (UC) and Crohn's disease. Expression of the Wnt family of genes was studied by polymerase chain reaction (PCR) array. Epithelial proliferation was studied using IEC-6 cells.Results: Most of the myofibroblast isolates expressed Wnt2, Wnt5A, Wnt5B, Fzd1, Fzd2, Fzd4, Fzd6, Fzd7, Fzd8, LRP6, Dick-kopf1, and SFRP1. Compared to myofibroblasts isolated from normal colonic mucosal samples, real-time reverse transcription-PCR studies (using additional isolates) showed significantly reduced expression of SFRP1 in UC myofibroblasts (3.34-fold reduction, P < 0.01). Recombinant SFRP1 inhibited proliferation of IEC-6 epithelial cells. In colonic crypt epithelial cells, expression of Wnt ligands and their inhibitors was generally either absent or very weak. By contrast, all the crypt epithelial preparations expressed Fzd1, Fzd5, Fzd7, Fzd8, and LRP6.Conclusions: Human intestinal myofibroblasts expressed a number of Wnt ligands, their receptors, and inhibitors. In contrast, colonic crypt epithelial cells predominantly expressed Wnt receptors. Compared to myofibroblasts isolated from normal colonic mucosa, those affected by UC showed significantly reduced expression of SFRP1. Since reduced SFRP1 expression has been associated with malignancy, low myofibroblast expression of this Wnt inhibitor may be implicated in increased risk of cancer in UC.

During gastrulation, convergent extension (CE) cell movements are regulated through the non-canonical Wnt signaling pathway. Wnt signaling results in downstream activation of Rho GTPases that in turn regulate actin cytoskeleton rearrangements essential for co-ordinated CE cell movement. Rho GTPases are bi-molecular switches that are inactive in their GDP-bound stage but can be activated to bind GTP through guanine nucleotide exchange factors (GEFs). Here we show that def6, a novel GEF, regulates CE cell movement during zebrafish gastrulation. Def6 morphants exhibit broadened and shortened body axis with normal cell fate specification, reminiscent of the zebrafish mutants silberblick and pipetail that lack Wnt11 or Wnt5b, respectively. Indeed, def6 morphants phenocopy Wnt5b mutants and ectopic overexpression of def6 essentially rescues Wnt5b morphants, indicating a novel role for def6 as a central GEF downstream of Wnt5b signaling. In addition, by knocking down both def6 and Wnt11, we show that def6 synergises with the Wnt11 signaling pathway.

Inhibitor of DNA binding protein 4 (ID4) is a member of the dominant-negative basic helix-loop-helix transcription factor family that lacks DNA binding activity and has tumor suppressor function. ID4 promoter methylation has been reported in acute myeloid leukemia and chronic lymphocytic leukemia (CLL), although the expression, function, and clinical relevance of this gene have not been characterized in either disease. We demonstrate that the promoter of ID4 is consistently methylated to various degrees in CLL cells, and increased promoter methylation in a univariable analysis correlates with shortened patient survival. However, ID4 mRNA and protein expression is uniformly silenced in CLL cells irrespective of the degree of promoter methylation. The crossing of ID4(+/-) mice with Eμ-TCL1 mice triggers a more aggressive murine CLL as measured by lymphocyte count and inferior survival. Hemizygous loss of ID4 in nontransformed TCL1-positive B cells enhances cell proliferation triggered by CpG oligonucleotides and decreases sensitivity to dexamethasone-mediated apoptosis. Collectively, this study confirms the importance of the silencing of ID4 in murine and human CLL pathogenesis.