Epigenetic Modification as a Regulatory Mechanism for Spatiotemporal Dynamics of ANO1 Expression in Salivary Glands.

Anoctamin1 (ANO1), a calcium activated chloride channel, is known to play a critical role in salivary secretion. In the salivary gland, ANO1 is expressed exclusively in the acinar cells, with no expression in the ductal cells. However, the mechanisms that determine this distinctive cell type-dependent expression pattern of ANO1 remain unknown. In this study, we discovered that the cell-dependent expression of ANO1 during salivary gland organogenesis is regulated by DNA methylation of ANO1 CpG islands. ANO1 CpG islands in e12 embryonic submandibular glands (eSMG) are highly methylated, but those in e14 eSMG or adult SMG are significantly unmethylated. The differential expression pattern of ANO1 in duct and acini is defined at e14. Artificial demethylation by treatment with the demethylating agent 5-aza-2'-deoxycytidine (5-Aza-CdR), induced the expression of ANO1 in both the ductal cell line Human Submandibular Gland (HSG) and in the duct cells of adult mouse SMG. During the trans-differentiation in Matrigel of duct-origin HSG cells into acinar-like phenotype, significant demethylation of ANO1 CpG islands is observed. This may be due to the reduced expression of DNA methyltransferase (DNMT) 3a and 3b. These results suggest that the differential expression of ANO1 in salivary glands during organogenesis and differentiation is mainly regulated by epigenetic demethylation of the ANO1 gene.

Risk of death, relapse or progression, and loss of life expectancy at different progression-free survival milestones in primary central nervous system lymphoma.

In this study, we analyzed the evolution of the prognosis of primary central nervous system lymphoma (PCNSL) patients as they reach selected progression-free survival (PFS) milestones after high-dose methotrexate (HD-MTX)-based therapy. In total, 258 and 146 patients were included from Denmark and British Columbia, respectively. All patients were diagnosed during 2000-2017. The 5-year PFS was 27% (95% CI 23; 32); however, for patients reaching 5 years of PFS, this increased to 71% (95% CI 57; 86). Within the first 5 years after diagnosis, patients lost 2.0 years (95% CI 1.8; 2.2) when compared to a similar background population. This reduced to 0.5 years (95% CI 0.2; 0.9) for patients reaching 5 years of PFS. Treatment with rituximab was associated with improved outcomes. The prognosis of patients with PCNSL treated with HD-MTX-based regimens in this cohort is poor, although it improves as patients survive without progression/relapse. However, survival does not conclusively normalize to that of a similar background population.

Tissue Distribution of Kir7.1 Inwardly Rectifying K+ Channel Probed in a Knock-in Mouse Expressing a Haemagglutinin-Tagged Protein.

Kir7.1 encoded by the Kcnj13 gene in the mouse is an inwardly rectifying K+ channel present in epithelia where it shares membrane localization with the Na+/K+-pump. Further investigations of the localisation and function of Kir7.1 would benefit from the availability of a knockout mouse, but perinatal mortality attributed to cleft palate in the neonate has thwarted this research. To facilitate localisation studies we now use CRISPR/Cas9 technology to generate a knock-in mouse, the Kir7.1-HA that expresses the channel tagged with a haemagglutinin (HA) epitope. The availability of antibodies for the HA epitope allows for application of western blot and immunolocalisation methods using widely available anti-HA antibodies with WT tissues providing unambiguous negative control. We demonstrate that Kir7.1-HA cloned from the choroid plexus of the knock-in mouse has the electrophysiological properties of the native channel, including characteristically large Rb+ currents. These large Kir7.1-mediated currents are accompanied by abundant apical membrane Kir7.1-HA immunoreactivity. WT-controlled western blots demonstrate the presence of Kir7.1-HA in the eye and the choroid plexus, trachea and lung, and intestinal epithelium but exclusively in the ileum. In the kidney, and at variance with previous reports in the rat and guinea-pig, Kir7.1-HA is expressed in the inner medulla but not in the cortex or outer medulla. In isolated tubules immunoreactivity was associated with inner medulla collecting ducts but not thin limbs of the loop of Henle. Kir7.1-HA shows basolateral expression in the respiratory tract epithelium from trachea to bronchioli. The channel also appears basolateral in the epithelium of the nasal cavity and nasopharynx in newborn animals. We show that HA-tagged Kir7.1 channel introduced in the mouse by a knock-in procedure has functional properties similar to the native protein and the animal thus generated has clear advantages in localisation studies. It might therefore become a useful tool to unravel Kir7.1 function in the different organs where it is expressed.

Uterine, but not ovarian, female reproductive organ involvement at presentation by diffuse large B-cell lymphoma is associated with poor outcomes and a high frequency of secondary CNS involvement.

Involvement of the internal female reproductive organs by diffuse large B-cell lymphoma (DLBCL) is uncommon, and there are sparse data describing the outcomes of such cases. In total, 678 female patients with DLBCL staged with positron emission tomography/computed tomography and treated with rituximab-containing chemotherapy were identified from databases in Denmark, Great Britain, Australia, and Canada. Overall, 27/678 (4%) had internal reproductive organ involvement: uterus (n = 14), ovaries (n = 10) or both (n = 3). In multivariate analysis, women with uterine DLBCL experienced inferior progression-free survival and overall survival compared to those without reproductive organ involvement, whereas ovarian DLBCL was not predictive of outcome. Secondary central nervous system (CNS) involvement (SCNS) occurred in 7/17 (41%) women with uterine DLBCL (two patients with concomitant ovarian DLBCL) and 0/10 women with ovarian DLBCL without concomitant uterine involvement. In multivariate analysis adjusted for other risk factors for SCNS, uterine involvement by DLBCL remained strongly associated with SCNS (Hazard ratio 14·13, 95% confidence interval 5·09-39·25, P < 0·001). Because involvement of the uterus by DLBCL appears to be associated with a high risk of SCNS, those patients should be considered for CNS staging and prophylaxis. However, more studies are needed to determine whether the increased risk of secondary CNS involvement also applies to women with localized reproductive organ DLBCL.

Nanomedicine: a pharma perspective.

Nanotechnology as applied to medicine is not a new field. The first nanomedicines approved for use were developed from research dating back to the 1970s. These liposomal formulations of existing drugs showed improved therapeutic activity and reduced toxicity in the nonclinical model systems. However, these benefits proved more subtle and harder to demonstrate in patients. This fact, combined with the technical challenges in commercial-scale production of nanoparticles, led to only limited investment in nanomedicines by the major pharmaceutical companies. Even so, research on nanomedicines has proceeded apace in academic laboratories and smaller biopharmaceutical companies. New materials and drug combinations have been studied, and targeting moieties added with the aim of improving the therapeutic index. Today many of these new designs are in, or are approaching, clinical testing. It will take only one or two to be successful to change the way pharmaceutical companies view this field of innovative research.

Phase 1 dose-escalation study of the PARP inhibitor CEP-9722 as monotherapy or in combination with temozolomide in patients with solid tumors.

PURPOSE: Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme important in DNA repair. PARP-1 activation at points of DNA strand break results in poly(ADP-ribose) polymer formation, opening the DNA structure, and allowing access of other repair enzymes. CEP-9722 inhibits PARP-1 and PARP-2 and is designed to potentiate DNA-damaging chemotherapies. METHODS: This dose-escalating phase 1 study assessed the safety, maximum tolerated dose (MTD), and pharmacokinetics/pharmacodynamics of CEP-9722 plus temozolomide in adults with solid tumors. Tumor response was also assessed. Participants received a 14-day cycle of CEP-9722 (days 1 and 3-5 or days 1-5), followed by 28-day cycles of CEP-9722 plus temozolomide 150 mg/m(2) on days 1-5. The initial CEP-9722 dose (cohort 1) was 150 mg/day; dose escalation followed a modified Fibonnaci sequence. RESULTS: Twenty-six patients received CEP-9722 150-1,000 mg/day combined with temozolomide. Dose-limiting toxicities of asthenia and persistent weight loss at 1,000 mg/day resulted in 750 mg/day being defined as the MTD and recommended dose for further study. Overall, 24 (92 %) patients had treatment-related adverse events (TRAEs), mostly grade 1 or 2, with nausea, vomiting, and diarrhea having the strongest relation to CEP-9722. Four patients had grade 3 TRAEs (asthenia, myositis, diarrhea, and fatigue). Systemic exposure generally increased with dosage, with high inter- and intra-patient variability at all doses. Pharmacodynamic assessment confirmed PARP inhibition although no dose response was apparent. One patient with melanoma achieved a partial response (1,000 mg/day). CONCLUSIONS: CEP-9722 was adequately tolerated with temozolomide; the MTD was 750 mg/day. Only limited clinical activity was observed.

Higher World Health Organization grades of follicular lymphoma correlate with better outcome in two Nordic Lymphoma Group trials of rituximab without chemotherapy.

Abstract A common treatment for follicular lymphoma is rituximab monotherapy. To identify patients for whom this regimen is adequate as first-line therapy, we applied the World Health Organization (WHO) classification for grading follicular lymphoma in a prospective central pathology review of the biopsies of previously untreated patients in two randomized trials of rituximab without chemotherapy. In the first trial (n1 = 53), higher WHO grades correlated with longer time to next treatment, independently of clinical prognostic factors (p = 0.030); the finding was replicated in the second trial (n2 = 221; p = 0.019). Higher grades were associated with better treatment responses (p = 0.018). Furthermore, also grades externally confirmed by independent local pathologists correlated with time to next treatment (p = 0.048). Flow cytometry in a separate patient series showed that the intensity of CD20 increased with the malignant cell size (p < 0.00005). In conclusion, WHO grade 1 follicular lymphoma correlates with inferior outcome after rituximab monotherapy. WHO grading might provide a clinically useful tool for personalized therapy.

Cerebrospinal fluid secretion by the choroid plexus.

The choroid plexus epithelium is a cuboidal cell monolayer, which produces the majority of the cerebrospinal fluid. The concerted action of a variety of integral membrane proteins mediates the transepithelial movement of solutes and water across the epithelium. Secretion by the choroid plexus is characterized by an extremely high rate and by the unusual cellular polarization of well-known epithelial transport proteins. This review focuses on the specific ion and water transport by the choroid plexus cells, and then attempts to integrate the action of specific transport proteins to formulate a model of cerebrospinal fluid secretion. Significant emphasis is placed on the concept of isotonic fluid transport across epithelia, as there is still surprisingly little consensus on the basic biophysics of this phenomenon. The role of the choroid plexus in the regulation of fluid and electrolyte balance in the central nervous system is discussed, and choroid plexus dysfunctions are described in a very diverse set of clinical conditions such as aging, Alzheimer's disease, brain edema, neoplasms, and hydrocephalus. Although the choroid plexus may only have an indirect influence on the pathogenesis of these conditions, the ability to modify epithelial function may be an important component of future therapies.

Effects of octane derivatives on activity of the volume-regulated anion channel in rat pancreatic ß-cells.

BACKGROUND: Saturated free fatty acids (FFAs) have a dual action on pancreatic ß-cells, consisting of an initial enhancement and subsequent suppression of glucose-induced electrical activity and insulin release. These stimulatory and inhibitory effects have been attributed, at least in part, to the activation and inhibition, respectively, of the volume-regulated anion channel (VRAC) by FFAs. Both effects were independent of their metabolism. We have now investigated the effects of related aliphatic compounds in order to further define the determinants of FFA interaction with VRAC. METHODS: ß-Cell VRAC and electrical activity were measured by conventional whole-cell and perforated patch recording, respectively. Cell volume was measured using a video-imaging technique. RESULTS: In common with octanoic acid, addition of methyl octanoate or n-octanol resulted in a rapid, pronounced and reversible inhibition of VRAC activity. Addition of n-octane had no significant effect on VRAC activity. n-Octanol had a biphasic effect on ß-cell membrane potential, namely a small transient depolarization followed by a marked hyperpolarization. n-Octanol was also found to prevent regulatory volume decrease in cells exposed to a hypotonic medium, consistent with VRAC inhibition. CONCLUSION: It is suggested that methyl octanoate and n-octanol can mimic the effects of FFAs on the pancreatic ß-cell via modulation of VRAC activity. The structural requirements for this effect appear to be a medium or long chain aliphatic compound containing at least one oxygen atom.

A novel surface electrocardiogram-based marker of ventricular arrhythmia risk in patients with ischemic cardiomyopathy.

BACKGROUND: Better sudden cardiac death risk markers are needed in ischemic cardiomyopathy (ICM). Increased heterogeneity of electrical restitution is an important mechanism underlying the risk of ventricular arrhythmia (VA). Our aim was to develop and test a novel quantitative surface electrocardiogram-based measure of VA risk in patients with ICM: the Regional Restitution Instability Index (R2I2). METHODS AND RESULTS: R2I2, the mean of the standard deviation of residuals from the mean gradient for each ECG lead at a range of diastolic intervals, was measured retrospectively from high-resolution 12-lead ECGs recorded during an electrophysiology study. Patient groups were as follows: Study group, 26 patients with ICM being assessed for implantable defibrillator; Control group, 29 patients with supraventricular tachycardia undergoing electrophysiology study; and Replication group, 40 further patients with ICM. R2I2 was significantly higher in the Study patients than in Controls (mean ± standard error of the mean: 1.09±0.06 versus 0.63±0.04, P<0.001). Over a median follow-up period of 23 months, 6 of 26 Study group patients had VA or death. R2I2 predicted VA or death independently of demographic factors, electrophysiology study result, left ventricular ejection fraction, or QRS duration (Cox model, P=0.029). R2I2 correlated with peri-infarct zone as assessed by cardiac magnetic resonance imaging (r=0.51, P=0.024). The findings were replicated in the Replication group: R2I2 was significantly higher in 11 of 40 Replication patients experiencing VA (1.18±0.10 versus 0.92±0.05, P=0.019). In combined analysis of ICM cohorts, R2I2 ≥1.03 identified subjects with significantly higher risk of VA or death (43%) compared with R2I2 <1.03 (11%) (P=0.004). CONCLUSIONS: In this pilot study, we have developed a novel VA risk marker, R2I2, and have shown that it correlated with a structural measure of arrhythmic risk and predicted risk of VA or death in patients with ICM. R2I2 may improve risk stratification and merits further evaluation. (J Am Heart Assoc. 2012;1:e001552 doi: 10.1161/JAHA.112.001552.).

The glucokinase activator GKA50 causes an increase in cell volume and activation of volume-regulated anion channels in rat pancreatic ß-cells.

Glucokinase plays a key role in the metabolism of glucose by pancreatic ß-cells. In this study the effects of the glucokinase activator GKA50 on cell volume and electrical activity in rat ß-cells were examined. One micro molar GKA50 caused an increase in ß-cell volume in the presence of 4mM glucose. GKA50 also caused a depolarisation of ß-cell membrane potential and increased electrical activity. These changes were associated with the activation of inward whole-cell currents, and were attenuated by the anion channel inhibitor 5-nitro-2-(3-phenylpropylamino) benzoic acid. In single channel experiments, the open probability of volume-regulated anion channels (VRAC) was increased from 0.03±0.01 to 0.19±0.04 (n=3) by the GKA50. The data suggest that a GKA50-evoked increase in glucose metabolism causes an increase in ß-cell volume. This in turn activates VRAC leading to a depolarisation of the cell membrane potential.

Functional characterization of bestrophin-1 missense mutations associated with autosomal recessive bestrophinopathy.

PURPOSE: Autosomal recessive bestrophinopathy (ARB) is a retinal dystrophy affecting macular and retinal pigmented epithelium function resulting from homozygous or compound heterozygous mutations in BEST1. In this study we characterize the functional implications of missense bestrophin-1 mutations that cause ARB by investigating their effect on bestrophin-1's chloride conductance, cellular localization, and stability. METHODS: The chloride conductance of wild-type bestropin-1 and a series of ARB mutants were determined by whole-cell patch-clamping of transiently transfected HEK cells. The effect of ARB mutations on the cellular localization of bestrophin-1 was determined by confocal immunofluorescence on transiently transfected MDCK II cells that had been polarized on Transwell filters. Protein stability of wild-type and ARB mutant forms of bestrophin-l was determined by the addition of proteasomal or lysosomal inhibitors to transiently transfected MDCK II cells. Lysates were then analyzed by Western blot analysis. RESULTS: All ARB mutants investigated produced significantly smaller chloride currents compared to wild-type bestrophin-1. Additionally, co-transfection of compound heterozygous mutants abolished chloride conductance in contrast to co-transfections of a single mutant with wild-type bestrophin-l, reflecting the recessive nature of the condition. In control experiments, expression of two dominant vitelliform macular dystrophy mutants was shown to inhibit wild-type currents. Cellular localization of ARB mutants demonstrated that the majority did not traffic correctly to the plasma membrane and that five of these seven mutants were rapidly degraded by the proteasome. Two ARB-associated mutants (p.D312N and p.V317M) that were not trafficked correctly nor targeted to the proteasome had a distinctive appearance, possibly indicative of aggresome or aggresome-like inclusion bodies. CONCLUSIONS: Differences in cellular processing mechanisms for different ARB associated mutants lead to the same disease phenotype. The existence of distinct pathogenic disease mechanisms has important ramifications for potential gene replacement therapies since we show that missense mutations associated with an autosomal recessive disease have a pathogenic influence beyond simple loss of function.

Electrical activity in pancreatic islet cells: The VRAC hypothesis.

A major aspect of stimulation of ß-cell function by glucose is the induction of electrical activity. The ionic events that underlie ß-cell electrical activity are understood in some detail. At sub-stimulatory glucose concentrations, the ß-cell is electrically 'silent'. Increasing the glucose concentration to stimulatory levels results in a gradual depolarisation of the membrane potential to a threshold potential where 'spikes' or action potentials are generated. These action potentials represent the gating of voltage-sensitive Ca²(+) channels, leading to Ca²(+) entry into the cell, thus triggering the release of insulin. The stimulatory actions of glucose on the ß-cell depend on the metabolism of the hexose. A major question concerns the molecular mechanism(s) whereby ß-cell plasma membrane potential is regulated by changes in glucose metabolism in the cell. This article provides a brief summary of the evidence suggesting that, in addition to metabolically-regulated K(ATP) channels, ß-cells are equipped with a volume-regulated anion channel that is activated by glucose concentrations within the range effective in modulating electrical activity and insulin release.

Epithelial pathways in choroid plexus electrolyte transport.

A stable intraventricular milieu is crucial for maintaining normal neuronal function. The choroid plexus epithelium produces the cerebrospinal fluid and in doing so influences the chemical composition of the interstitial fluid of the brain. Here, we review the molecular pathways involved in transport of the electrolytes Na+, K+, Cl-, and HCO3(-)across the choroid plexus epithelium.

Regulatory volume increase in epithelial cells isolated from the mouse fourth ventricle choroid plexus involves Na(+)-H(+) exchange but not Na(+)-K(+)-2Cl(-) cotransport.

The aim of this study was to determine the ability of choroid plexus epithelial cells to volume regulate when exposed to hypertonic solutions, and furthermore to identify the ion transporters involved in any volume regulation. Experiments were performed on cells freshly isolated, using the enzyme dispase, from the mouse fourth ventricle choroid plexus. Cell volume was measured using a video-imaging method. Cells used in this study were all of a similar morphology and had a mean volume of 0.71pl. Cells shrank when superfused with hypertonic solutions to a minimum relative cell volume of 0.84+/-0.01 (n=8) in 3min. They then exhibited a regulatory volume increase (RVI) to reach a relative volume of 0.92+/-0.02 over the following 12min. The RVI was HCO(3)(-)-dependent, that is it was not observed in hepes-buffered solutions. A post-regulatory volume decrease RVI (post-RVD RVI) was also observed in cells following exposure to hypotonic solutions. The RVI and post-RVD RVI were inhibited by 10microM 5-(N-ethyl-N-isopropyl) amiloride or 10microM 5-(N-methyl-N-isobutyl) amiloride, both selective inhibitors of Na(+)-H(+) exchange (NHE). They were also inhibited by the anion transport inhibitor 100microM 2,2'-(1,2-ethenediyl) bis (5-isothiocyanatobenzenesulfonic acid). The Na(+)-K(+)-2Cl(-) cotransporter inhibitor, 10microM bumetanide, was without effect on either the RVI or the post-RVD RVI. The data indicate that NHE, probably in combination with Cl(-)-HCO(3)(-) exchangers, contributes to RVI in choroid plexus epithelial cells.

Opposing effects of tenidap on the volume-regulated anion channel and K(ATP) channel activity in rat pancreatic beta-cells.

Tenidap (5-chloro-2-hydroxy-3-(thiophene-2-carbonyl)indole-1-carboxamide) is a non-steroidal anti-inflammatory and anti-rheumatic drug with several cellular actions including inhibition of anion transport processes. Since other anion transport inhibitors have been shown to inhibit activity of the volume-regulated anion channel (VRAC), the present study investigated the effects of tenidap on activity of this channel in pancreatic beta-cells. Membrane potential, VRAC currents and input conductance were recorded from single rat beta-cells in primary culture using perforated patch, conventional whole-cell and cell-attached configurations of the patch-clamp technique. Relative cell volume was measured using a video-imaging method. Tenidap (0.1mM) was found to rapidly hyperpolarise the beta-cell membrane potential and terminate glucose-induced electrical activity. This effect was associated with a pronounced outward current shift at a holding potential of -65mV. Tenidap was found to inhibit activity of the volume-regulated anion channel with IC(50) values of 31 and 43microM for outward and inward currents respectively. Tenidap also markedly increased beta-cell input conductance, representing an activation of the K(ATP) conductance. beta-cell regulatory volume decrease following hypotonically-induced cell swelling was sensitive to inhibition by 50microM tenidap. Tenidap is a potent inhibitor of the volume-regulated anion channel and K(ATP) channel activator in rat pancreatic beta-cells. These actions could at least in part explain the recently reported inhibitory actions of the drug on electrical and secretory activity in the beta-cell, and could also underlie other pharmacological actions of the drug.

Missense mutations in a retinal pigment epithelium protein, bestrophin-1, cause retinitis pigmentosa.

Bestrophin-1 is preferentially expressed at the basolateral membrane of the retinal pigmented epithelium (RPE) of the retina. Mutations in the BEST1 gene cause the retinal dystrophies vitelliform macular dystrophy, autosomal-dominant vitreochoroidopathy, and autosomal-recessive bestrophinopathy. Here, we describe four missense mutations in bestrophin-1, three that we believe are previously unreported, in patients diagnosed with autosomal-dominant and -recessive forms of retinitis pigmentosa (RP). The physiological function of bestrophin-1 remains poorly understood although its heterologous expression induces a Cl--specific current. We tested the effect of RP-causing variants on Cl- channel activity and cellular localization of bestrophin-1. Two (p.L140V and p.I205T) produced significantly decreased chloride-selective whole-cell currents in comparison to those of wild-type protein. In a model system of a polarized epithelium, two of three mutations (p.L140V and p.D228N) caused mislocalization of bestrophin-1 from the basolateral membrane to the cytoplasm. Mutations in bestrophin-1 are increasingly recognized as an important cause of inherited retinal dystrophy.

Studies of the mechanism of activation of the volume-regulated anion channel in rat pancreatic beta-cells.

There is evidence that depolarization of the pancreatic beta cell by glucose involves cell swelling and activation of the volume-regulated anion channel (VRAC). However, it is unclear whether cell swelling per se or accompanying changes in intracellular osmolality and/or ionic strength are responsible for VRAC activation. VRAC activity was measured in rat beta cells by conventional or perforated patch whole-cell recording. Cell volume was measured by video imaging. In conventional whole-cell recordings, VRAC activation was achieved by exposure of the cells to a hyposmotic bath solution, by application of positive pressure to the pipette, or by use of a hyperosmotic pipette solution. Increased concentrations of intracellular CsCl also caused channel activation, but with delayed kinetics. In perforated patch recordings, VRAC activation was induced by isosmotic addition of the permeable osmolytes urea, 3-O-methyl glucose, arginine, and NH4Cl. These effects were all accompanied by beta-cell swelling. It is concluded that increased cell volume, whether accompanied by raised intracellular osmolality or ionic strength, is a major determinant of VRAC activation in the beta cell. However, increased intracellular ionic strength markedly reduced the rate of VRAC activation. These findings are consistent with the hypothesis that the accumulation of glucose metabolites in the beta cell, and the resultant increase in cell volume, provides a signal coupling glucose metabolism with VRAC activation.