The inhalation of medical aerosols is widely used for the treatment of lung disorders such as asthma, chronic obstructive pulmonary disease, cystic fibrosis, respiratory infection and, more recently, lung cancer. Targeted aerosol delivery to the affected lung tissue may improve therapeutic efficiency and minimize unwanted side effects. Despite enormous progress in optimizing aerosol delivery to the lung, targeted aerosol delivery to specific lung regions other than the airways or the lung periphery has not been adequately achieved to date. Here, we show theoretically by computer-aided simulation, and for the first time experimentally in mice, that targeted aerosol delivery to the lung can be achieved with aerosol droplets comprising superparamagnetic iron oxide nanoparticles--so-called nanomagnetosols--in combination with a target-directed magnetic gradient field. We suggest that nanomagnetosols may be useful for treating localized lung disease, by targeting foci of bacterial infection or tumour nodules.

Reversible assembly of the V0V1 holoenzyme from V0 and V1 subcomplexes is a widely used mechanism for regulation of vacuolar-type H ؉ -ATPases (V-ATPases) in animal cells. In the blowfly (Calliphora vicina) salivary gland, V-ATPase is located in the apical membrane of the secretory cells and energizes the secretion of a KCl-rich saliva in response to the hormone serotonin. We have examined whether the cAMP pathway, known to be activated by serotonin, controls V-ATPase assembly and activity. Fluorescence measurements of pH changes at the luminal surface of isolated glands demonstrate that cAMP, Sp-adenosine-3 ,5 -cyclic monophosphorothioate, or forskolin, similar to serotonin, cause VATPase-dependent luminal acidification. In addition, V-ATPasedependent ATP hydrolysis increases upon treatment with these agents. Immunofluorescence microscopy and pelleting assays have demonstrated further that V1 components become translocated from the cytoplasm to the apical membrane and V-ATPase holoenzymes are assembled at the apical membrane during conditions that increase intracellular cAMP. Because these actions occur without a change in cytosolic Ca 2؉ , our findings suggest that the cAMP pathway mediates the reversible assembly and activation of V-ATPase molecules at the apical membrane upon hormonal stimulus.regulation ͉ translocation ͉ secretion T he vacuolar-type H ϩ -ATPases (V-ATPases) are multisubunit heteromeric complexes that are organized into two domains, designated V 0 and V 1 (1-4). V 0 forms a membranespanning proton-translocating complex; in yeast, it is composed of at least five different subunits termed a, c, cЈ, cЉ, and d, of which subunit c binds bafilomycin A 1 , a specific inhibitor of V-ATPases (5-8). The V 1 sector is attached to the cytoplasmic side of the V 0 sector, consists of at least eight different subunits termed A-H, and contains catalytic and noncatalytic ATPbinding sites. V-ATPase is vital for almost every eukaryotic cell and fulfils a variety of functions. On intracellular acidic membrane systems, such as endosomes, lysosomes, and synaptic vesicles, these proton pumps are involved in protein sorting during biosynthetic and endocytotic pathways, zymogen activation, and transmitter uptake, respectively (3, 4). V-ATPase molecules in the plasma membrane of animal cells, especially on the apical plasma membrane of epithelial cells, contribute to intracellular pH homeostasis, extracellular acidification, or alkalinization, or they energize the plasma membrane for secondary transport processes (3, 4).In some cells, V-ATPase requires a considerable amount of energy. For reasons of economy, it is thus favorable if V-ATPase activity is adapted to the physiological needs of the cell. Several regulatory mechanisms have been identified (1,2,4,9). One of these is the reversible dissociation of the V 1 sector from the V 0 sector, as revealed by experiments performed in yeast, midgut epithelial cells of the tobacco hornworm Manduca sexta, mammalian dendritic cells, and renal epithelial cells (10-16). In th...

In the two inflammatory bowel diseases, ulcerative colitis (UC) and Crohn’s disease (CD), altered expression of tight junction (TJ) proteins leads to an impaired epithelial barrier including increased uptake of luminal antigens supporting the inflammation. Here we focused on regulation of tricellulin, a protein of the tricellular TJ essential for the barrier against macromolecules, and hypothesized a role in paracellular antigen uptake. We report that tricellulin is downregulated in UC, but not in CD, and that its reduction increases the passage of macromolecules. Using a novel visualization method, passage sites were identified at TJ regions usually sealed by tricellulin. We show that interleukin-13 (IL-13), beyond its known effect on claudin-2, downregulates tricellulin expression. These two effects of IL-13 are regulated by different signaling pathways: The IL-13 receptor α1 upregulates claudin-2, while IL-13 receptor α2 downregulates tricellulin. We suggest to target the α2 receptor in future developments of therapeutical IL-13-based biologicals.

SUMMARYSecretory activity in blowfly salivary glands is activated by the hormone serotonin. We have investigated the distribution and activity of two cation pumps that are possibly involved with transepithelial ion transport, i.e. Na+/K+-ATPase and vacuolar-type H+-ATPase(V-ATPase). By immunofluorescence labelling of secretory cells,Na+/K+-ATPase was localized on the basolateral plasma membrane and V-ATPase on the highly folded apical membrane. Activities of both ATPases were probed in salivary gland homogenates by applying specific inhibitors for these ion pumps, namely ouabain and bafilomycin A1. In control glands, bafilomycin-A1-sensitive V-ATPase activity and ouabain-sensitive Na+/K+-ATPase activity accounted for 36% and 19%, respectively, of the total ATPase activity. V-ATPase activity increased approximately twofold after stimulation with serotonin, whereas Na+/K+-ATPase activity was not significantly affected. Biochemical assays provided evidence that the serotonin-induced activation of V-ATPase activity was accompanied by a recruitment of peripheral V1subunits from the cytosol to the plasma membrane, indicative of the assembly of V0V1 holoenzymes.These data show that a V-ATPase located in the apical plasma membranes of the secretory cells is a component of the apical `potassium pump' that has been identified previously by physiological approaches. The V-ATPase energizes the apical membrane and provides the primary driving force for fuelling a putative K+/nH+ antiporter and, thus, for fluid secretion. Serotonin-induced assembly of V0V1holoenzymes might constitute a regulatory mechanism for the control of pump activity.

Leptin receptor-deficient db/db mice are a commonly used research model and it is known that the genetic background, on which the mutation is bred, modulates the phenotype. While diabetes-resistant strains sustain near normal glycemia and hyperinsulinemia, susceptible backgrounds develop overt hyperglycemia and islet involution. We hypothesized that genetically-determined differences in the proliferative capacity and the apoptotic frequency of pancreatic beta cells contribute to this phenotypic disparity. We studied C57BLKS/J (BKS; diabetes-susceptible) and C57BL/6 (B6; diabetes-resistant) db/db mice and heterozygous controls from 5 to 12 weeks of age. Body weight, fasting blood glucose, plasma insulin, HOMA-IR, alpha cell mass, beta cell mass, proliferation and apoptosis were measured. Comparable insulin resistance developed in the 2 db/db strains, which was well compensated for on both genetic backgrounds until 7 weeks of age. As expected, the BKS mice became hyperglycemic at 9 weeks. Beta cell proliferation was initially increased in both db/db strains but decreased rapidly in the BKS mice with advancing age. The rate of beta cell apoptosis was already higher in prediabetic BKS mice than in their B6 counterparts. Beta cell mass increased continuously in the B6 strain until 12 weeks of age, but declined from 7 weeks onwards in BKS. An age-dependent decline of beta cell proliferation and an increased rate of beta cell apoptosis already in the prediabetic state probably contribute to the diabetes susceptibility of db/db BKS mice. These factors could also play a role in the genetic predisposition for type 2 diabetes in humans.

Key pointsr Interleukin-13 (IL-13) causes intestinal epithelial barrier dysfunction, and is implicated in the pathogenesis of Th2-driven intestinal inflammation (e.g. ulcerative colitis). However, it is unclear whether the epithelial sodium channel (ENaC) -the main limiting factor for sodium absorption in the distal colon -is also influenced by IL-13 and if so, by what mechanism(s).r We demonstrate in an intestinal cell model as well as in mouse distal colon that IL-13 causes reduced ENaC activity.r We show that IL-13 impairs ENaC-dependent sodium transport by activating the JAK1/2-STAT6 signalling pathway.r These results improve our understanding of the mechanisms through which IL-13 functions as a key effector cytokine in ulcerative colitis, thereby contributing to the distinct pathology of this disease.Abstract Interleukin-13 (IL-13) has been strongly implicated in the pathogenesis of ulcerative colitis, possibly by disrupting epithelial integrity. In the distal colon, the epithelial sodium channel (ENaC) is an important factor in the regulation of sodium absorption, and therefore plays a critical role in minimizing intestinal sodium and water losses. In the present study, we investigated whether IL-13 also acts as a potent modulator of epithelial sodium transport via ENaC, and the signalling components involved. The effect of IL-13 on ENaC was examined in HT-29/B6-GR/MR human colon cells, as well as in mouse distal colon, by measuring amiloride-sensitive short-circuit current (I SC ) in Ussing chambers. The expression levels of ENaC subunits and the cellular components that contribute to ENaC activity were analysed by qRT-PCR and promoter gene assay. We show that IL-13, in both the cell model and in native intestinal tissue, impaired epithelial sodium absorption via ENaC (J Na ) as a result of decreased transcription levels of β-and γ-ENaC subunits and SGK1, a post-translational regulator of ENaC activity, due to impaired promoter activity. The reduction in J Na was prevented by inhibition of JAK1/2-STAT6 signalling. This inhibition also affected the IL-13-induced decrease in p38 MAPK phosphorylation. The contribution of STAT6 to IL-13-mediated ENaC inactivation was confirmed in a STAT6−/− mouse model. In conclusion, these results indicate that IL-13, the levels of which are elevated in ulcerative colitis, contributes to impaired ENaC activity via modulation of the STAT6/p38 MAPK pathways.Please note that Figures 1, 3, 4, 5 and 6 that appear in this final copy-edited version have been updated since the paper was published online as an Accepted Article. The new figures have been supplied by the authors and have been approved by the Editor. The conclusions of the study remain the same. Abbreviations DBA, dexamethasone plus butyrate and aldosterone; ENaC, epithelial sodium channel; GR, glucocorticoid receptor; GRE, glucocorticoid response element; HRE, hormone response element; IL-13, interleukin-13; I SC , amiloride sensitive short-circuit current; J Na , epithelial sodium absorption via the ENaC; MAPK, mitoge...

SUMMARY The paired salivary glands in the cockroach are composed of acini with ion-transporting peripheral P-cells and protein-secreting central C-cells, and a duct system for the modification of the primary saliva. Secretory activity is controlled by serotonergic and dopaminergic neurons, whose axons form a dense plexus on the glands. The spatial relationship of release sites for serotonin and dopamine to the various cell types was determined by anti-synapsin immunofluorescence confocal microscopy and electron microscopy. Every C-cell apparently has only serotonergic synapses on its surface. Serotonergic and dopaminergic fibres on the acini have their release zones at a distance of ∼0.5 μm from the P-cells. Nerves between acinar lobules may serve as neurohaemal organs and contain abundant dopaminergic and few serotonergic release sites. Some dopaminergic and serotonergic release sites reside in the duct epithelium, the former throughout the duct system, the latter only in segments next to acini. These findings are consistent with the view that C-cells respond exclusively to serotonin, P-cells to serotonin and dopamine, and most duct cells only to dopamine. Moreover, the data suggest that C-cells are stimulated by serotonin released close to their surface,whereas P-cells and most duct cells are exposed to serotonin/dopamine liberated at some distance.

BackgroundThe transcription factor Pax6 functions in the specification and maintenance of the differentiated cell lineages in the endocrine pancreas. It has two DNA binding domains, the paired domain and the homeodomain, in addition to a C-terminal transactivation domain. The phenotype of Pax6-/- knockout mice suggests non-redundant functions of the transcription factor in the development of glucagon-expressing α-cells as this cell type is absent in the mutants. We ask the question of how the differentiation of pancreatic endocrine cells, in particular that of α-cells, is affected by selective inactivation of either one of the three major domains of Pax6.ResultsThe Pax6Aey18 mutant mouse line, in which the paired domain is inactivated, showed a phenotype similar to that of Pax6-/- knockout mice with a near complete absence of glucagon-positive α-cells (0-4 cells/section; ≤1% of wt), reduced β-cell area (74% of wt) and disorganized islets. The proportion of ghrelin-positive ε-cells was expanded. In Pax6Sey-Neu mutants, which lack the transactivation domain, α-and β-cells where reduced to 25 and 40% of wt, respectively. We also studied two mouse lines with mutations in the homeodomain, Pax64Neu and Pax6132-14Neu. Neighboring amino acids are affected in the two lines and both point mutations abolish DNA binding of the classical P3 homeodomain target sequence. The pancreatic phenotype of the two mutants however was divergent. While Pax64Neu homozygotes showed a reduction of α- and β-cells to 59 and 61%, respectively, pancreatic endocrine development was unaltered in the Pax6132-14Neu mutant strain.ConclusionsWe show that inactivation of the Pax6 paired domain leads to a more severe phenotype with regards to the differentiation of pancreatic α-cells than the loss of the transactivation domain. The analysis of two different homeodomain mutants suggests that the binding of Pax6 to P3 homeodomain consensus sequences is not required for α-cell development. It rather seems that the homeodomain has a modulating role in Pax6 function, possibly by facilitating a PH0-like binding confirmation on paired domain target genes like proglucagon. This function is differentially affected by the two homeodomain mutations analyzed in this study.