Tight junction channel regulation by interclaudin interference.

Tight junctions form selectively permeable seals across the paracellular space. Both barrier function and selective permeability have been attributed to members of the claudin protein family, which can be categorized as pore-forming or barrier-forming. Here, we show that claudin-4, a prototypic barrier-forming claudin, reduces paracellular permeability by a previously unrecognized mechanism. Claudin-4 knockout or overexpression has minimal effects on tight junction permeability in the absence of pore-forming claudins. However, claudin-4 selectively inhibits flux across cation channels formed by claudins 2 or 15. Claudin-4-induced loss of claudin channel function is accompanied by reduced anchoring and subsequent endocytosis of pore-forming claudins. Analyses in nonepithelial cells show that claudin-4, which is incapable of independent polymerization, disrupts polymeric strands and higher order meshworks formed by claudins 2, 7, 15, and 19. This process of interclaudin interference, in which one claudin disrupts higher order structures and channels formed by a different claudin, represents a previously unrecognized mechanism of barrier regulation.

In vitro electrochemical measurement of serotonin release in the human jejunum mucosa using a diamond microelectrode.

We report herein on the use of a boron-doped diamond microelectrode (DME) to record oxidation currents in vitro associated with the release of serotonin from enterochromaffin cells in the epithelium of the human intestinal mucosa. Continuous amperometric measurements were made as a function of distance (ln current vs. distance) from the tissue surface in human jejunum specimens. The results demonstrate the capabilities of the DME for the stable and reproducible detection of serotonin in the complex environment of the human tissue. Serotonin release was evoked by the shearing force of a continuously flowing Krebs buffer solution at 36 °C with the tissue pinned down in a flow bath. Reproducible currents with distance were recorded for serotonin oxidation. Increased oxidation currents were observed in the presence of the selective serotonin reuptake inhibitor, fluoxetine, indicating that a significant fraction of the amperometric current recorded is attributable to serotonin oxidation. The nominal reciprocal slope, |slope-1|, of the ln current vs. distance plots increased from 270 ± 25 µm-1 in Krebs buffer (N = 3) to 471 ± 65 µm-1 during fluoxetine addition (N = 3), reflective of a reduced rate of reuptake in the presence of the SERT antagonist. The paper reports on the characterization of the diamond microelectrodes and the in vitro electrochemical measurement data.

Platform for the Delivery of Unformulated RNA In Vivo.

The successful delivery of RNA therapeutics is the gating hurdle to greater clinical translation and utility of this novel class of therapeutics. Delivery strategies today are limited and predominantly rely on lipid nanoparticles or conjugates, which can facilitate hepatic delivery but are poor for achieving uptake outside the liver. The ability to deliver RNA to other organs outside the liver in a formulation-agnostic approach could serve to unlock the broader potential of these therapies and enable their use in a broader set of disease. Here we demonstrate this formulation-agnostic delivery of two model siRNAs using low-frequency ultrasound to the gastrointestinal (GI) tract. Unformulated siRNAs targeting ß-catenin (Ctnnb 1) and Sjögren syndrome antigen B (SSB) genes were successfully delivered to colonic mucosa in mice, achieving robust knockdown of the target mRNA from whole-colon tissue samples. Indeed, the capacity to target and successfully suppress expression from genes underscores the power of this platform to rapidly deliver unformulated and unoptimized sequences against a range of targets in the GI tract.

Ultra-rapid drug delivery in the oral cavity using ultrasound.

The delivery of therapeutics to the gastrointestinal (GI) mucosa remains primarily a function of diffusion and rapid delivery is a significant goal in drug delivery science. However, delivery is hindered by the molecular barrier properties of the mucosa, as well as environmental factors. We hypothesized that low-frequency ultrasound can overcome these barriers, achieving rapid delivery in an engineered, clinically-relevant system for buccal administration. The hand-held system enabled delivery of macromolecules in short, 60-s treatment times ex vivo. Tolerability of the prototype was demonstrated in awake, (unsedated) dogs. Finally, this technology enhanced the efficacy of the anti-inflammatory agent, budesonide, allowing for prophylactic treatment in a hamster model of oral inflammatory lesions in vivo. The capacity to deliver therapeutics in a targeted and rapid manner in a clinically-relevant form-factor presents an intriguing capability to expand the repertoire of therapeutics that can be applied topically in the mouth and beyond.

The mucosal barrier at a glance.

Mucosal barriers separate self from non-self and are essential for life. These barriers, which are the first line of defense against external pathogens, are formed by epithelial cells and the substances they secrete. Rather than an absolute barrier, epithelia at mucosal surfaces must allow selective paracellular flux that discriminates between solutes and water while preventing the passage of bacteria and toxins. In vertebrates, tight junctions seal the paracellular space; flux across the tight junction can occur through two distinct routes that differ in selectivity, capacity, molecular composition and regulation. Dysregulation of either pathway can accompany disease. A third, tight-junction-independent route that reflects epithelial damage can also contribute to barrier loss during disease. In this Cell Science at a Glance article and accompanying poster, we present current knowledge on the molecular components and pathways that establish this selectively permeable barrier and the interactions that lead to barrier dysfunction during disease.

Sex-related differences in small intestinal transit and serotonin dynamics in high-fat-diet-induced obesity in mice.

Obesity alters gastrointestinal (GI) motility and 5-HT signalling. Altered 5-HT signalling disrupts control of GI motility. Levels of extracellular 5-HT depend on enterochromaffin (EC) cell release and serotonin transporter (SERT) uptake. We assessed GI transit and 5-HT signalling in the jejunum of normal and obese mice. Male and female mice were fed a control diet (CD; 10% of kilocalories as fat) or a high-fat diet (HFD; 60% of kilocalories as fat). Gastrointestinal transit was increased in male HFD-fed and female CD-fed compared with male CD-fed mice. The 5-HT3 receptor blocker, alosetron, increased gastric emptying in male CD-fed mice, but decreased transit in female CD-fed mice. The 5-HT-induced jejunal longitudinal muscle contractions in vitro were similar in all mice. In contrast to male CD-fed mice, 5-HT uptake (measured using continuous amperometry in vitro) in male HFD-fed mice was fluoxetine insensitive, yet sensitive to cocaine and the dopamine transporter (DAT) blocker GBR 12909. Immunoreactivity for DAT was present in the mucosa, and protein levels were greater in male HFD-fed compared with CD-fed mice. Extracellular 5-HT and mucosal 5-hydroxyindolacetic acid (5-HT metabolite) were similar in male HFD-fed compared with CD-fed mice. 5-Hydroxytryptamine uptake was fluoxetine sensitive in all females. Greater SERT protein, decreased extracellular 5-HT and greater mucosal 5-hydroxyindolacetic acid were observed in female HFD-fed compared with CD-fed mice. Mucosal 5-HT and EC cell numbers were similar in CD-fed and HFD-fed mice of both sexes; female 5-HT and EC cell numbers were increased compared with males. The HFD did not alter plasma sex hormone levels in any mice. Overall, obesity alters GI transit and 5-HT signalling in a sex-dependent manner.

Nonspecific immunoglobulin replacement in lung transplantation recipients with hypogammaglobulinemia: a cohort study taking into account propensity score and immortal time bias.

BACKGROUND: After lung transplantation (LT), immunoglobulin (Ig) G plasma concentrations<6 g/L are common and correlate with an increased risk of chronic lung allograft dysfunction (CLAD) and a poorer survival. METHODS: We conducted an open substitution intervention with nonspecific intravenous Ig (IVIg), in all patients with IgG plasma less than 6 g/L post-LT in 54 of 84 consecutive recipients since 1998 who survived more than 3 months. Pre-LT and post-LT events were retrospectively analyzed. RESULTS: Both substituted and nonsubstituted groups demonstrated similar donor or recipient characteristics and events over a median follow-up of 2.8 years (Q1-Q3, 1.4-5.7], except for initial diagnosis with more chronic obstructive pulmonary disease patients and less cases of pulmonary arterial hypertension in NS group. Intravenous Ig substitution started 3.5 months (0.5-9.4) after transplantation and lasted 4.5 months after (1.0-17.7), mean cumulative dose was 52.8±47.7 g. In multivariate Cox regression model, hypogammaglobulinemic patients who were substituted with IVIg had actually a 5-year survival (hazard ratio, 0.63; 95% confidence interval, 0.26-1.49; P=0.29) and CLAD-free 5-year survival (hazard ratio, 0.51; 95% confidence interval, 0.15-1.67; P=0.27) really close to nonhypogammaglobulinemic and nonsubstituted patients. Complementary analysis using propensity score and time-dependent analysis showed that survival and CLAD-free survival were not different in both groups. CONCLUSION: Intravenous Ig post-LT achieved similar survival and CLAD-free survival in recipients with hypogammaglobulinemia as compared to those with normal IgG plasmatic rate. A randomized control trial is required to confirm benefic effects of IVIg and disentangle mechanisms, including protection from infections, acute cellular and humoral rejections in patients with hypogammaglobulinemia after LT.

Electrochemical activation of diamond microelectrodes: implications for the in vitro measurement of serotonin in the bowel.

The electrochemical pretreatment of diamond microelectrodes was investigated for the purpose of learning how an anodic, cathodic or a combined anodic + cathodic polarization affects the charge-transfer kinetics for two surface-sensitive redox systems: ferri/ferrocyanide and serotonin (5-hydroxytryptamine, 5-HT). The pretreatments were performed in 0.5 mol L(-1) H2SO4. The anodic pretreatment was performed galvanically for 30 s at 250 mA cm(-2). The cathodic pretreatment was performed for 180 s at -250 mA cm(-2). The combined pretreatment involved application of the anodic step first followed by the cathodic step. The results clearly demonstrate that the best performance for both redox systems is obtained after the cathodic polarization, which presumably activates the electrode by cleaning the surface and removing site-blocking surface carbon-oxygen functionalities. The cathodic pretreatment was found to be effective at activating a fouled microelectrode in situ. This observation has important implication for the measurement of 5-HT in the bowel.

Aberrant striatal dopamine transmitter dynamics in brain-derived neurotrophic factor-deficient mice.

Brain-derived neurotrophic factor (BDNF) modulates the synaptic transmission of several monoaminergic neuronal systems, including forebrain dopamine-containing neurons. Recent evidence shows a strong correlation between neuropsychiatric disorders and BDNF hypofunction. The aim of the present study was to characterize the effect of low endogenous levels of BDNF on dopamine system function in the caudate-putamen using heterozygous BDNF (BDNF(+/-) ) mice. Apparent extracellular dopamine levels in the caudate-putamen, determined by quantitative microdialysis, were significantly elevated in BDNF(+/-) mice compared with wildtype controls (12 vs. 5 nM, respectively). BDNF(+/-) mice also had a potentiated increase in dopamine levels following potassium (120 mM)-stimulation (10-fold) relative to wildtype controls (6-fold). Slice fast-scan cyclic voltammetry revealed that BDNF(+/-) mice had reductions in both electrically evoked dopamine release and dopamine uptake rates in the caudate-putamen. Superfusion of BDNF led to partial recovery of the electrically stimulated dopamine release response in BDNF(+/-) mice. Conversely, tissue accumulation of L-3,4-dihydroxyphenylalanine, extracellular levels of dopamine metabolites, and spontaneous locomotor activity were unaltered. Together, this study indicates that endogenous BDNF influences dopamine system homeostasis by regulating the release and uptake dynamics of pre-synaptic dopamine transmission.