Isolation of a Novel β4 Integrin-binding Protein (p27BBP) Highly Expressed in Epithelial Cells

The integrin beta4 has a long cytodomain necessary for hemidesmosome formation. A yeast two-hybrid screen using beta4 cytodomain uncovered a protein called p27(BBP) that represents a beta4 interactor. Both in yeast and in vitro, p27(BBP) binds the two NH2-terminal fibronectin type III modules of beta4, a region required for signaling and hemidesmosome formation. Sequence analysis of p27(BBP) revealed that p27(BBP) was not previously known and has no homology with any isolated mammalian protein, but 85% identical to a yeast gene product of unknown function. Expression studies by Northern analysis and in situ hybridization showed that, in vivo, p27(BBP) mRNA is highly expressed in epithelia and proliferating embryonic epithelial cells. An antibody raised against p27(BBP) COOH-terminal domain showed that all beta4-containing epithelial cell lines expressed p27(BBP). The p27(BBP) protein is insoluble and present in the intermediate filament pool. Furthermore, subcellular fractionation indicated the presence of p27(BBP) both in the cytoplasm and in the nucleus. Confocal analysis of cultured cells showed that part of p27(BBP) immunoreactivity was both nuclear and in the membrane closely apposed to beta4. These results suggest that the p27(BBP) is an in vivo interactor of beta4, possibly linking beta4 to the intermediate filament cytoskeleton

The High Mobility Group (Hmg) Boxes of the Nuclear Protein Hmg1 Induce Chemotaxis and Cytoskeleton Reorganization in Rat Smooth Muscle Cells

HMG1 (high mobility group 1) is a ubiquitous and abundant chromatin component. However, HMG1 can be secreted by activated macrophages and monocytes, and can act as a mediator of inflammation and endotoxic lethality. Here we document a role of extracellular HMG1 in cell migration. HMG1 (and its individual DNA-binding domains) stimulated migration of rat smooth muscle cells in chemotaxis, chemokinesis, and wound healing assays. HMG1 induced rapid and transient changes of cell shape, and actin cytoskeleton reorganization leading to an elongated polarized morphology typical of motile cells. These effects were inhibited by antibodies directed against the receptor of advanced glycation endproducts, indicating that the receptor of advanced glycation endproducts is the receptor mediating the HMG1-dependent migratory responses. Pertussis toxin and the mitogen-activated protein kinase kinase inhibitor PD98059 also blocked HMG1-induced rat smooth muscle cell migration, suggesting that a Gi/o protein and mitogen-activated protein kinases are required for the HMG1 signaling pathway. We also show that HMG1 can be released by damage or necrosis of a variety of cell types, including endothelial cells. Thus, HMG1 has all the hallmarks of a molecule that can promote atherosclerosis and restenosis after vascular damage

Absence of Rac1 and Rac3 GTPases in the nervous system hinders thymic, splenic and immune-competence development

The nervous system influences organ development by direct innervation and the action of hormones. We recently showed that the specific absence of Rac1 in neurons (Rac1N) in a Rac3-deficient (Rac3KO) background causes motor behavioural defects, epilepsy, and premature mouse death around postnatal day 13. We report here that Rac1N/Rac3KO mice display a progressive loss of immune-competence. Comparative longitudinal analysis of lymphoid organs from control, single Rac1N or Rac3KO, and double Rac1N/Rac3KO mutant animals showed that thymus development is preserved up to postnatal day 9 in all animals, but is impaired in Rac1N/Rac3KO mice at later times. This is evidenced by a drastic reduction in thymic cell numbers. Cell numbers were also reduced in the spleen, leading to splenic tissue disarray. Organ involution occurs in spite of unaltered thymocyte and lymphocyte subset composition, and proper mature T-cell responses to polyclonal stimuli in vitro. Suboptimal thymus innervation by tau-positive neuronal terminals possibly explains the suboptimal thymic output and arrested thymic development, which is accompanied by higher apoptotic rates. Our results support a role for neuronal Rac1 and Rac3 in dictating proper lymphoid organ development, and suggest the existence of lymphoid-extrinsic mechanisms linking neural defects to the loss of immune-competence

Cutting edge: IgE plays an active role in tumor immunosurveillance in mice

Exogenous IgE acts as an adjuvant in tumor vaccination in mice, and therefore a direct role of endogenous IgE in tumor immunosurveillance was investigated. By using genetically engineered mice, we found that IgE ablation rendered mice more susceptible to the growth of transplantable tumors. Conversely, a strengthened IgE response provided mice with partial or complete resistance to tumor growth, depending on the tumor type. By genetic crosses, we showed that IgE-mediated tumor protection was mostly lost in mice lacking FceRI. Tumor protection was also lost after depletion of CD8+ T cells, highlighting a cross-Talk between IgE and T cell- mediated tumor immunosurveillance. Our findings provide the rationale for clinical observations that relate atopy with a lower risk for developing cancer and open new avenues for the design of immunotherapeutics relevant for clinical oncology. The Journal of Immunology, 2016, 197: 2583-2588

Selective preservation of bone marrow mature recirculating but not marginal zone B cells in murine models of chronic inflammation

Inflammation promotes granulopoiesis over B lymphopoiesis in the bone marrow (BM). We studied B cell homeostasis in two murine models of T cell mediated chronic inflammation, namely calreticulin-deficient fetal liver chimeras (FLC), which develop severe blepharitis and alopecia due to T cell hyper responsiveness, and inflammatory bowel disease (IBD) caused by injection of CD4+ naïve T cells into lymphopenic mice. We show herein that despite the severe depletion of B cell progenitors during chronic, peripheral T cell-mediated inflammation, the population of BM mature recirculating B cells is unaffected. These B cells are poised to differentiate to plasma cells in response to blood borne pathogens, in an analogous fashion to non-recirculating marginal zone (MZ) B cells in the spleen. MZ B cells nevertheless differentiate more efficiently to plasma cells upon polyclonal stimulation by Toll-like receptor (TLR) ligands, and are depleted during chronic T cell mediated inflammation in vivo. The preservation of mature B cells in the BM is associated with increased concentration of macrophage migration inhibitory factor (MIF) in serum and BM plasma. MIF produced by perivascular dendritic cells (DC) in the BM provides a crucial survival signal for recirculating B cells, and mice treated with a MIF inhibitor during inflammation showed significantly reduced mature B cells in the BM. These data indicate that MIF secretion by perivascular DC may promote the survival of the recirculating B cell pool to ensure responsiveness to blood borne microbes despite loss of the MZ B cell pool that accompanies depressed lymphopoiesis during inflammation

AAV6-mediated Systemic shRNA Delivery Reverses Disease in a Mouse Model of Facioscapulohumeral Muscular Dystrophy

Treatment of dominantly inherited muscle disorders remains a difficult task considering the need to eliminate the pathogenic gene product in a body-wide fashion. We show here that it is possible to reverse dominant muscle disease in a mouse model of facioscapulohumeral muscular dystrophy (FSHD). FSHD is a common form of muscular dystrophy associated with a complex cascade of epigenetic events following reduction in copy number of D4Z4 macrosatellite repeats located on chromosome 4q35. Several 4q35 genes have been examined for their role in disease, including FRG1. Overexpression of FRG1 causes features related to FSHD in transgenic mice and the FRG1 mouse is currently the only available mouse model of FSHD. Here we show that systemic delivery of RNA interference expression cassettes in the FRG1 mouse, after the onset of disease, led to a dose-dependent long-term FRG1 knockdown without signs of toxicity. Histological features including centrally nucleated fibers, fiber size reduction, fibrosis, adipocyte accumulation, and inflammation were all significantly improved. FRG1 mRNA knockdown resulted in a dramatic restoration of muscle function. Through RNA interference (RNAi) expression cassette redesign, our method is amenable to targeting any pathogenic gene offering a viable option for long-term, body-wide treatment of dominant muscle disease in humans

Selective Preservation of Bone Marrow Mature Recirculating but Not Marginal Zone B Cells in Murine Models of Chronic Inflammation

Inflammation promotes granulopoiesis over B lymphopoiesis in the bone marrow (BM). We studied B cell homeostasis in two murine models of T cell mediated chronic inflammation, namely calreticulin-deficient fetal liver chimeras (FLC), which develop severe blepharitis and alopecia due to T cell hyper responsiveness, and inflammatory bowel disease (IBD) caused by injection of CD4+ naïve T cells into lymphopenic mice. We show herein that despite the severe depletion of B cell progenitors during chronic, peripheral T cell-mediated inflammation, the population of BM mature recirculating B cells is unaffected. These B cells are poised to differentiate to plasma cells in response to blood borne pathogens, in an analogous fashion to non-recirculating marginal zone (MZ) B cells in the spleen. MZ B cells nevertheless differentiate more efficiently to plasma cells upon polyclonal stimulation by Toll-like receptor (TLR) ligands, and are depleted during chronic T cell mediated inflammation in vivo. The preservation of mature B cells in the BM is associated with increased concentration of macrophage migration inhibitory factor (MIF) in serum and BM plasma. MIF produced by perivascular dendritic cells (DC) in the BM provides a crucial survival signal for recirculating B cells, and mice treated with a MIF inhibitor during inflammation showed significantly reduced mature B cells in the BM. These data indicate that MIF secretion by perivascular DC may promote the survival of the recirculating B cell pool to ensure responsiveness to blood borne microbes despite loss of the MZ B cell pool that accompanies depressed lymphopoiesis during inflammation

Speciazione del ferro nell'acqua di mare: sviluppo, ottimizzazione e caratterizzazione di un nuovo metodo di Competitive Ligand Equilibration - Cathodic Stripping Voltammetry (CLE-CSV)

Iron speciation in seawater is of the utmost importance in understanding the biogeochemical cycle of this element, in view of its central role in regulating the primary productivity of the oceanic environment and its connection to global planetary cycles. The Competitive Ligand Equilibration-Cathodic Stripping Voltammetry (CLS-CSV) methods, often with the catalytic enhancement of the signal, fit for this purpose, as they allow to study the organic speciation of the metal by determining the organic iron binding ligands concentration and their stability constant for iron. Moreover, they avoid any sample separation or pretreatment step showing acceptable detection capabilities and robustness towards the saline matrix. Aim of the present PhD project was the development, optimization and characterization of a new method based on this technique characterized by optimal detection capabilities (sensitivity and limit of detection) and by an easy to use instrumental configuration. In fact, several methods experienced a development during the last 20 years, but each of them was characterized by the presence of important drawbacks as poor sensitivity, too high time analysis requirement and also, in some cases, by the use of a suspected carcinogenic species as catalytic enhancer. In particular, 2,3-dihydroxynaphthalene and atmospheric oxygen were employed as iron chelator and catalytic enhancer, respectively, as they ensure the best analytical performances. A new hardware configuration was firstly developed, leading to an overall simplification of the system: a silver wire pseudoreference was installed replacing the traditional Ag/AgCl 3 M KCl reference electrode and air was directly introduced into the close cell by an air pump. An extensive optimization procedure was accordingly performed, and the detection capabilities were carefully evaluated: fully satisfactory analytical performances for trace and ultratrace iron determination were reached. A comprehensive approach to the characterization of an adsorptive cathodic stripping voltammetry with catalytic enhancement followed. The focus of this part was on the understanding of the chemical features and processes involved by method and their consecutive effect on the analytical signal to appreciate possible limitations and future directions for improvements. Accordingly, the ligand degradation was study and its consequences on the analytical sensitivity and the speciation procedure evaluated. The stoichiometry of the FeDHN complex was also determined as it plays a major role in the speciation data treatment. Furthermore, the thermodynamics of ligand and complex adsorption onto the electrode surface was considered to evaluate a possible competition between the two species. As the final step of this part of the work, the electron transfer kinetics (k0 ) and catalytic enhancement (k\u2019cat) were study to completely characterize the electrode reaction, i.e., to give full insight into the intimate mechanism responsible for the high sensitivity. The new CLE-CSV method was subsequently developed. Firstly, the side reaction coefficients for the FeDHN complex were calibrated against EDTA and the conditional stability constant calculated. The method was successfully validated in UV digested seawater using diethylenetriaminepentaacetic acid (DTPA) deferoxamine (DFO), and protoporphyrin IX (PPIX). Furthermore, good detection capabilities also for humic acid (HA) were proved. The analysis of six seawater samples from a Ross Sea water column (Antarctica) was then performed, demonstrating the fit for purpose for the detection of trace amounts of iron ligands in seawater. Lastly, as a further development of the technique, the pH buffer usually employed to perform voltammetric analysis in seawater was removed. The latter innovation was enabled by the natural pH buffer capacity of seawater due to the presence of the CO2/HCO3 - /CO3 2- system. The application of the unbuffered method resulted in not statistically different values for both the side reaction coefficients and conditional stability constant with respect to the buffered method. Again, the unbuffered method was successfully validated in UV digested seawater using both an artificial (diethylenetriaminepentaacetic acid) and a natural ligand (deferoxamine), and then applied to the same six seawater samples from the Ross Sea water column. The proposed unbuffered approach may demonstrate relevant to already existing speciation procedures for iron, resulting in a correct determination of complexing capacity and stability constant of organic iron binding ligands. The new method, both in its buffered and unbuffered version, achieved a tenfold sample size reduction and a tenfold increase in the analytical sensitivity compared with other methods employing 2,3- dihydroxynaphthalene. Furthermore, the analysis time was halved with respect to the fastest method reported in the literature as half an hour resulted enough to measure a twelve points titration