Novel role for pendrin in orchestrating bicarbonate secretion in cystic fibrosis transmembrane conductance regulator (CFTR)-expressing airway serous cells.

In most HCO(3)(-)-secreting epithelial tissues, SLC26 Cl(-)/HCO(3)(-) transporters work in concert with the cystic fibrosis transmembrane conductance regulator (CFTR) to regulate the magnitude and composition of the secreted fluid, a process that is vital for normal tissue function. By contrast, CFTR is regarded as the only exit pathway for HCO(3)(-) in the airways. Here we show that Cl(-)/HCO(3)(-) anion exchange makes a major contribution to transcellular HCO(3)(-) transport in airway serous cells. Real-time measurement of intracellular pH from polarized cultures of human Calu-3 cells demonstrated cAMP/PKA-activated Cl(-)-dependent HCO(3)(-) transport across the luminal membrane via CFTR-dependent coupled Cl(-)/HCO(3)(-) anion exchange. The pharmacological and functional profile of the luminal anion exchanger was consistent with SLC26A4 (pendrin), which was shown to be expressed by quantitative RT-PCR, Western blot, and immunofluorescence. Pendrin-mediated anion exchange activity was confirmed by shRNA pendrin knockdown (KD), which markedly reduced cAMP-activated Cl(-)/HCO(3)(-) exchange. To establish the relative roles of CFTR and pendrin in net HCO(3)(-) secretion, transepithelial liquid secretion rate and liquid pH were measured in wild type, pendrin KD, and CFTR KD cells. cAMP/PKA increased the rate and pH of the secreted fluid. Inhibiting CFTR reduced the rate of liquid secretion but not the pH, whereas decreasing pendrin activity lowered pH with little effect on volume. These results establish that CFTR predominately controls the rate of liquid secretion, whereas pendrin regulates the composition of the secreted fluid and identifies a critical role for this anion exchanger in transcellular HCO(3)(-) secretion in airway serous cells.

Ligation of TLR9 induced on human IL-10-secreting Tregs by 1alpha,25-dihydroxyvitamin D3 abrogates regulatory function.

Signaling through the TLR family of molecular pattern recognition receptors has been implicated in the induction of innate and adaptive immune responses. A role for TLR signaling in the maintenance and/or regulation of Treg function has been proposed, however its functional relevance remains unclear. Here we have shown that TLR9 is highly expressed by human Treg secreting the antiinflammatory cytokine IL-10 induced following stimulation of blood and tissue CD3+ T cells in the presence of 1alpha,25-dihydroxyvitamin D3 (1alpha25VitD3), the active form of Vitamin D, with or without the glucocorticoid dexamethasone. By contrast, TLR9 was not highly expressed by naturally occurring CD4+CD25+ Treg or by Th1 and Th2 effector cells. Induction of TLR9, but not other TLRs, was IL-10 dependent and primarily regulated by 1alpha25VitD3 in vitro. Furthermore, ingestion of calcitriol (1alpha25VitD3) by human volunteers led to an increase of both IL-10 and TLR9 expression by CD3+CD4+ T cells analyzed directly ex vivo. Stimulation of 1alpha25VitD3-induced IL-10-secreting Treg with TLR9 agonists, CpG oligonucleotides, resulted in decreased IL-10 and IFN-gamma synthesis and a concurrent loss of regulatory function, but, unexpectedly, increased IL-4 synthesis. We therefore suggest that TLR9 could be used to monitor and potentially modulate the function of 1alpha25VitD3-induced IL-10-secreting Treg in vivo, and that this has implications in cancer therapy and vaccine design.

The role of p53 and pRB in apoptosis and cancer.

Loss of function of both the p53 pathway and the retinoblastoma protein (pRB) pathway plays a significant role in the development of most human cancers. Loss of pRB results in deregulated cell proliferation and apoptosis, whereas loss of p53 desensitizes cells to checkpoint signals, including apoptosis. In the past two years, mouse genetics and gene expression profiling have led to major advances in our understanding of how the pRB and p53 pathways regulate apoptosis and thus the development of tumours.

PI3Kgamma is the dominant isoform involved in migratory responses of human T lymphocytes: effects of ex vivo maintenance and limitations of non-viral delivery of siRNA.

Use of mice in which individual PI3K isoforms have been deleted or mutated by gene targeting, has determined that PI3Kgamma provides a key migratory signal for T lymphocyte migration. Since PI3Kgamma can be a dispensable signal for directional migration of human T cells, we have adopted a pharmacological and siRNA strategy to assess the contribution of individual PI3K isoforms to chemokine-stimulated migration of human T cells. The broad spectrum PI3K isoform inhibitor Ly294002 inhibits CXCL12-stimulated migration of freshly isolated T lymphocytes. Use of second generation inhibitors that can discriminate between individual PI3K isoforms, revealed that PI3Kgamma was the major contributor to CXCL12-induced migration and PI3K/Akt signaling (as assessed by S6 phosphorylation). Non-viral delivery of siRNA targeting class I (PI3Kgamma), class II (PI3KC2alpha and PI3KC2beta) and class III PI3Ks, followed by 3 days ex vivo culture, reduces the levels of isoform mRNA, but is insufficient to impact on cell migration responses. However, ex vivo maintenance of T cells alone, independently of siRNA treatment, resulted in the migratory response of T cells toward CXCL12 becoming insensitive to Ly294002. Remarkably, random migration remains sensitive to Ly294002. This study therefore, highlights that the migratory response of freshly isolated human T cells is dependent on PI3K signals that are provided predominantly by PI3Kgamma. However, the role of PI3K in cell migration is context-dependent and diminishes during ex vivo maintenance.

Antigen Selection for Enhanced Affinity T-Cell Receptor-Based Cancer Therapies.

Evidence of adaptive immune responses in the prevention of cancer has been accumulating for decades. Spontaneous T-cell responses occur in multiple indications, bringing the study of de novo expressed cancer antigens to the fore and highlighting their potential as targets for cancer immunotherapy. Circumventing the immune-suppressive mechanisms that maintain tumor tolerance and driving an antitumor cytotoxic T-cell response in cancer patients may eradicate the tumor or block disease progression. Multiple strategies are being pursued to harness the cytotoxic potential of T cells clinically. Highly promising results are now emerging. The focus of this review is the target discovery process for cancer immune therapeutics based on affinity-matured T-cell receptors (TCRs). Target cancer antigens in the context of adoptive cell transfer technologies and soluble biologic agents are discussed. To appreciate the impact of TCR-based technology and understand the TCR discovery process, it is necessary to understand key differences between TCR-based therapy and other immunotherapy approaches. The review first summarizes key advances in the cancer immunotherapy field and then discusses the opportunities that TCR technology provides. The nature and breadth of molecular targets that are tractable to this approach are discussed, together with the challenges associated with finding them.

Chronic caffeine produces sexually dimorphic effects on amphetamine-induced behavior, anxiety and depressive-like behavior in adolescent rats.

Caffeine consumption has been increasing rapidly in adolescents; however, most research on the behavioral effects of caffeine has been conducted in adults. Two experiments were conducted in which adolescent male and female rats were treated with a moderate dose of caffeine (0.25 g/l) in their drinking water beginning on P26-28. In the first experiment, animals were maintained on caffeinated drinking water or normal tap water for 14 days and were then tested for behavioral and striatal c-Fos response to amphetamine (1.5 mg/kg). In the second experiment, rats were maintained on caffeinated drinking water or normal tap water beginning on P28 and were tested for novel object recognition, anxiety in the light/dark test (L/D) and elevated plus maze (EPM), and depressive like behavior in the forced swim test (FST) beginning on the 14th day of caffeine exposure. Caffeine decreased amphetamine-induced rearing in males, but had no effect in females; however, this behavioral effect was not accompanied by changes in striatal c-Fos, which was increased by amphetamine but not altered by caffeine. No effects of caffeine were observed on novel object recognition or elevated plus maze behavior. However, in the L/D test, there was a sex by caffeine interaction on time spent in the light driven by a caffeine-induced increase in light time in the males but not the females. On the pretest day of the FST, sex by caffeine interactions were observed for swimming and struggling; caffeine decreased struggling behavior and increased swimming behavior in males and caffeine-treated females demonstrated significantly more struggling and significantly less swimming than caffeine-treated males. A similar pattern was observed on the test day in which caffeine decreased immobility overall and increased swimming. These data reveal sex dependent effects of caffeine on behavior in adolescent rats.

Direct molecular mimicry enables off-target cardiovascular toxicity by an enhanced affinity TCR designed for cancer immunotherapy.

Natural T-cell responses generally lack the potency to eradicate cancer. Enhanced affinity T-cell receptors (TCRs) provide an ideal approach to target cancer cells, with emerging clinical data showing significant promise. Nevertheless, the risk of off target reactivity remains a key concern, as exemplified in a recent clinical report describing fatal cardiac toxicity, following administration of MAGE-A3 specific TCR-engineered T-cells, mediated through cross-reactivity with an unrelated epitope from the Titin protein presented on cardiac tissue. Here, we investigated the structural mechanism enabling TCR cross-recognition of MAGE-A3 and Titin, and applied the resulting data to rationally design mutants with improved antigen discrimination, providing a proof-of-concept strategy for altering the fine specificity of a TCR towards an intended target antigen. This study represents the first example of direct molecular mimicry leading to clinically relevant fatal toxicity, mediated by a modified enhanced affinity TCR designed for cancer immunotherapy. Furthermore, these data demonstrate that self-antigens that are expressed at high levels on healthy tissue should be treated with extreme caution when designing immuno-therapeutics.

Protein phosphatase 1 coordinates CFTR-dependent airway epithelial HCO3- secretion by reciprocal regulation of apical and basolateral membrane Cl(-)-HCO3- exchangers.

BACKGROUND AND PURPOSE: Our recent studies on human airway serous-like Calu-3 cells showed that cAMP agonists stimulated a HCO3(-) rich secretion containing up to 80 mM HCO3(-). This alkaline secretion relied on a coordinated switch in the activity of distinct Cl(-)-HCO3(-) anion exchangers (AE) located at different regions of the cell. At the apical membrane, cAMP agonists activated the electroneutral AE pendrin (SLC26A4), together with cystic fibrosis transmembrane conductance regulator (CFTR), while at the basolateral membrane the agonists inhibited AE2 (SLC4A2). However, the underlying mechanism(s) that orchestrates this cAMP-dependent switch in AE activity has not been elucidated. EXPERIMENTAL APPROACH: Apical and basolateral Cl(-)-HCO3(-) exchange was assessed by measuring Cl(-)-dependent changes in intracellular pH (pH(i)). KEY RESULTS: We show that protein phosphatase 1 (PP1), together with CFTR, play central roles in this reciprocal regulation of AE activity. Activation of pendrin by cAMP agonists, but not inhibition of the basolateral exchanger, was protein kinase A-dependent. Knocking down CFTR expression, or blocking its activity with GlyH-101, led to incomplete inhibition of the basolateral AE by cAMP, supporting a role for CFTR in this process. Addition of the PP1/2A inhibitor, okadaic acid, but not the PP2A specific inhibitor fostreicin, mimicked the effect of cAMP stimulation. Furthermore, okadaic acid-treated Calu-3 monolayers produced a more alkaline fluid than untreated cells, which was comparable with that produced by cAMP stimulation. CONCLUSIONS AND IMPLICATIONS: These results identify PP1 as a novel regulator of AE activity which, in concert with CFTR, coordinates events at both apical and basolateral membranes, crucial for efficient HCO3(-) secretion from Calu-3 cells.

New non-viral method for gene transfer into primary cells.

The availability of genetically altered cells is an essential prerequisite for many scientific and therapeutic applications including functional genomics, drug development, and gene therapy. Unfortunately, the efficient gene transfer into primary cells is still problematic. In contrast to transfections of most cell lines, which can be successfully performed using a variety of methods, the introduction of foreign DNA into primary cells requires a careful selection of gene transfer techniques. Whereas viral strategies are time consuming and involve safety risks, non-viral methods proved to be inefficient for most primary cell types. The Nucleofector technology is a novel gene transfer technique designed for primary cells and hard-to-transfect cell lines. This non-viral gene transfer method is based on a cell type specific combination of electrical parameters and solutions. In this report, we show efficient transfer of DNA expression vectors and siRNA oligonucleotides into a variety of primary cell types from different species utilizing the Nucleofector technology, including human B-CLL cells, human CD34+ cells, human lymphocytes, rat cardiomyocytes, human, porcine, and bovine chondrocytes, and rat neurons.