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INTRODUCTION One method for assessing the in vitro response to CFTR-modulating compounds is by analysis of epithelial monolayers in an Ussing chamber,where the apical and basolateral surfaces are isolated and the potential difference,short-circuit current,and transepithelial resistance can be monitored. The effect of a chloride ion gradient across airway epithelia on transepithelial chloride transport and the magnitude of CFTR modulator efficacy were examined. METHODS CFTR-mediated changes in the potential difference and transepithelial currents of primary human nasal epithelial cell cultures were quantified in Ussing chambers with either symmetrical solutions or reduced chloride solutions in the apical chamber. CFTR activity in homozygous F508del CFTR epithelia was rescued by treatment with VX-661,C4/C18,4-phenylbutyrate (4-PBA) for 24 hr at 37°C or by incubation at 29°C for 48 hr. RESULTS Imposing a chloride gradient increased CFTR-mediated and CaCC-mediated ion transport. Treatment of F508del CFTR homozygous cells with CFTR modulating compounds increased CFTR activity,which was significantly more evident in the presence of a chloride gradient. This observation was recapitulated with temperature-mediated F508del CFTR correction. CONCLUSIONS Imposing a chloride gradient during Ussing chamber measurements resulted in increased CFTR-mediated ion transport in expanded non-CF and F508del CFTR homozygous epithelia. In F508del CFTR homozygous epithelia,the magnitude of response to CFTR modulating compounds or low temperature was greater when assayed with a chloride gradient compared to symmetrical chloride,resulting in an apparent increase in measured efficacy. Future work may direct which methodologies utilized to quantify CFTR modulator response in vitro are most appropriate for the estimation of in vivo efficacy. View Publication

In order to overcome the challenges associated with a limited number of airway epithelial cells that can be obtained from clinical sampling and their restrained capacity to divide ex vivo,miniaturization of respiratory drug discovery assays is of pivotal importance. Thus,a 96-well microplate system was developed where primary human small airway epithelial (hSAE) cells were cultured at an air-liquid interface (ALI). After four weeks of ALI culture,a pseudostratified epithelium containing basal,club,goblet and ciliated cells was produced. The 96-well ALI cultures displayed a cellular composition,ciliary beating frequency,and intercellular tight junctions similar to 24-well conditions. A novel custom-made device for 96-parallelized transepithelial electric resistance (TEER) measurements,together with dextran permeability measurements,confirmed that the 96-well culture developed a tight barrier function during ALI differentiation. 96-well hSAE cultures were responsive to transforming growth factor $\beta$1 (TGF-$\beta$1) and tumor necrosis factor $\alpha$ (TNF-$\alpha$) in a concentration dependent manner. Thus,the miniaturized cellular model system enables the recapitulation of a physiologically responsive,differentiated small airway epithelium,and a robotic integration provides a medium throughput approach towards pharmaceutical drug discovery,for instance,in respect of fibrotic distal airway/lung diseases. View Publication

NAD is a multifunctional molecule involved in both metabolic processes and signaling pathways. Such signalling pathways consume NAD which is replenished via one of several biosynthesis pathways. We show that influx of NAD across the plasma membrane may be able to contribute to the homeostasis of intracellular NAD levels. Indeed,extracellular application of NAD was able to replete NAD levels that had been lowered pharmacologically using the novel drug FK866 and was also able to rescue cells from FK866-induced cell death. A marked lag between the drop in NAD levels and cell death prompted us to investigate the mechanism of cell death. We were unable to find evidence of apoptosis as assessed by immunoblotting for the Caspase 3 activation fragment and immunostaining for cytochrome C and AIF translocation. We,therefore,investigated whether autophagy was initiated by FK866. Indeed,we were able to observe the formation of LC3-positive vesicles that had fused with lysosomes in FK866-treated but not control cells. Furthermore,this autophagic phenotype could be reverted by the addition of NAD to the extracellular medium. View Publication

Cellular immunotherapeutics targeting the human papillomavirus (HPV)-16 E6 and E7 proteins have achieved limited success in HPV-positive oropharyngeal cancer (OPC). Here we have conducted proteome-wide profiling of HPV-16-specific T cell responses in a cohort of 66 patients with HPV-associated OPC and 22 healthy individuals. Unexpectedly,HPV-specific T cell responses from OPC patients were not constrained to the E6 and E7 antigens; they also recognized E1,E2,E4,E5,and L1 proteins as dominant targets for virus-specific CD8+ and CD4+ T cells. Multivariate analysis incorporating tumor staging,treatment status,and smoking history revealed that treatment status had the most significant impact on HPV-specific CD8+ and CD4+ T cell immunity. Specifically,the breadth and overall strength of HPV-specific T cell responses were significantly higher before the commencement of curative therapy than after therapy. These data provide the first glimpse of the overall human T cell response to HPV in a clinical setting and offer groundbreaking insight into future development of cellular immunotherapies for HPV-associated OPC patients. View Publication

A minor haplotype of the 10q26 locus conveys the strongest genetic risk for age-related macular degeneration (AMD). Here,we examined the mechanisms underlying this susceptibility. We found that monocytes from homozygous carriers of the 10q26 AMD-risk haplotype expressed high amounts of the serine peptidase HTRA1,and HTRA1 located to mononuclear phagocytes (MPs) in eyes of non-carriers with AMD. HTRA1 induced the persistence of monocytes in the subretinal space and exacerbated pathogenic inflammation by hydrolyzing thrombospondin 1 (TSP1),which separated the two CD47-binding sites within TSP1 that are necessary for efficient CD47 activation. This HTRA1-induced inhibition of CD47 signaling induced the expression of pro-inflammatory osteopontin (OPN). OPN expression increased in early monocyte-derived macrophages in 10q26 risk carriers. In models of subretinal inflammation and AMD,OPN deletion or pharmacological inhibition reversed HTRA1-induced pathogenic MP persistence. Our findings argue for the therapeutic potential of CD47 agonists and OPN inhibitors for the treatment of AMD. View Publication

BACKGROUND Mammalian target of rapamycin complex 1 (mTORC1) is a protein kinase that relays nutrient availability signals to control numerous cellular functions including autophagy,a process of cellular self-eating activated by nutrient depletion. Addressing the therapeutic potential of modulating mTORC1 signaling and autophagy in human disease requires active chemicals with pharmacologically desirable properties. METHODOLOGY/PRINCIPAL FINDINGS Using an automated cell-based assay,we screened a collection of {\textgreater}3,500 chemicals and identified three approved drugs (perhexiline,niclosamide,amiodarone) and one pharmacological reagent (rottlerin) capable of rapidly increasing autophagosome content. Biochemical assays showed that the four compounds stimulate autophagy and inhibit mTORC1 signaling in cells maintained in nutrient-rich conditions. The compounds did not inhibit mTORC2,which also contains mTOR as a catalytic subunit,suggesting that they do not inhibit mTOR catalytic activity but rather inhibit signaling to mTORC1. mTORC1 inhibition and autophagosome accumulation induced by perhexiline,niclosamide or rottlerin were rapidly reversed upon drug withdrawal whereas amiodarone inhibited mTORC1 essentially irreversibly. TSC2,a negative regulator of mTORC1,was required for inhibition of mTORC1 signaling by rottlerin but not for mTORC1 inhibition by perhexiline,niclosamide and amiodarone. Transient exposure of immortalized mouse embryo fibroblasts to these drugs was not toxic in nutrient-rich conditions but led to rapid cell death by apoptosis in starvation conditions,by a mechanism determined in large part by the tuberous sclerosis complex protein TSC2,an upstream regulator of mTORC1. By contrast,transient exposure to the mTORC1 inhibitor rapamycin caused essentially irreversible mTORC1 inhibition,sustained inhibition of cell growth and no selective cell killing in starvation. CONCLUSION/SIGNIFICANCE The observation that drugs already approved for human use can reversibly inhibit mTORC1 and stimulate autophagy should greatly facilitate the preclinical and clinical testing of mTORC1 inhibition for indications such as tuberous sclerosis,diabetes,cardiovascular disease and cancer. View Publication

Aberrant activation of the hedgehog (Hh) signaling pathway has been implicated in the epithelial-to-mesenchymal transition (EMT) and cancer stem-like cell (CSC) maintenance; both processes can result in tumor progression and treatment resistance in several types of human cancer. Hh cooperates with the epidermal growth factor receptor (EGFR) signaling pathway in embryogenesis. We found that the Hh signaling pathway was silenced in EGFR-TKI-sensitive non-small-cell lung cancer (NSCLC) cells,while it was inappropriately activated in EGFR-TKI-resistant NSCLC cells,accompanied by EMT induction and ABCG2 overexpression. Upregulation of Hh signaling through extrinsic SHH exposure downregulated E-cadherin expression and elevated Snail and ABCG2 expression,resulting in gefitinib tolerance (P {\textless} 0.001) in EGFR-TKI-sensitive cells. Blockade of the Hh signaling pathway using the SMO antagonist SANT-1 restored E-cadherin expression and downregulate Snail and ABCG2 in EGFR-TKI-resistant cells. A combination of SANT-1 and gefitinib markedly inhibited tumorigenesis and proliferation in EGFR-TKI-resistant cells (P {\textless} 0.001). These findings indicate that hyperactivity of Hh signaling resulted in EGFR-TKI resistance,by EMT introduction and ABCG2 upregulation,and blockade of Hh signaling synergistically increased sensitivity to EGFR-TKIs in primary and secondary resistant NSCLC cells. E-cadherin expression may be a potential biomarker of the suitability of the combined application of an Hh inhibitor and EGFR-TKIs in EGFR-TKI-resistant NSCLCs. View Publication

Although initial treatment of ovarian cancer is successful,tumors typically relapse and become resistant to treatment. Because of poor infiltration of effector T cells,patients are mostly unresponsive to immunotherapy. Plasma gelsolin (pGSN) is transported by exosomes (small extracellular vesicle,sEV) and plays a key role in ovarian cancer chemoresistance,yet little is known about its role in immunosurveillance. Here,we report the immunomodulatory roles of sEV-pGSN in ovarian cancer chemoresistance. In chemosensitive conditions,secretion of sEV-pGSN was low,allowing for optimal CD8+ T-cell function. This resulted in increased T-cell secretion of IFN$\gamma$,which reduced intracellular glutathione (GSH) production and sensitized chemosensitive cells to cis-diaminedichloroplatinum (CDDP)-induced apoptosis. In chemoresistant conditions,increased secretion of sEV-pGSN by ovarian cancer cells induced apoptosis in CD8+ T cells. IFN$\gamma$ secretion was therefore reduced,resulting in high GSH production and resistance to CDDP-induced death in ovarian cancer cells. These findings support our hypothesis that sEV-pGSN attenuates immunosurveillance and regulates GSH biosynthesis,a phenomenon that contributes to chemoresistance in ovarian cancer. SIGNIFICANCE: These findings provide new insight into pGSN-mediated immune cell dysfunction in ovarian cancer chemoresistance and demonstrate how this dysfunction can be exploited to enhance immunotherapy. View Publication

Spontaneous control of human immunodeficiency virus (HIV) is generally associated with an enhanced capacity of CD8+ T cells to eliminate infected CD4+ T cells,but the molecular characteristics of these highly functional CD8+ T cells are largely unknown. In the present study,using single-cell analysis,it was shown that HIV-specific,central memory CD8+ T cells from spontaneous HIV controllers (HICs) and antiretrovirally treated non-controllers have opposing transcriptomic profiles. Genes linked to effector functions and survival are upregulated in cells from HICs. In contrast,genes associated with activation,exhaustion and glycolysis are upregulated in cells from non-controllers. It was shown that HIV-specific CD8+ T cells from non-controllers are largely glucose dependent,whereas those from HICs have more diverse metabolic resources that enhance both their survival potential and their capacity to develop anti-HIV effector functions. The functional efficiency of the HIV-specific CD8+ T cell response in HICs is thus engraved in their memory population and related to their metabolic programme. Metabolic reprogramming in vitro through interleukin-15 treatment abrogated the glucose dependency and enhanced the antiviral potency of HIV-specific CD8+ T cells from non-controllers. View Publication

Several HIV-1 and SIV vaccine candidates have shown partial protection against viral challenges in rhesus macaques. However,the protective efficacy of vaccine-elicited polyclonal antibodies has not previously been demonstrated in adoptive transfer studies in nonhuman primates. In this study,we show that passive transfer of purified antibodies from vaccinated macaques can protect naive animals against SIVmac251 challenges. We vaccinated 30 rhesus macaques with Ad26-SIV Env/Gag/Pol and SIV Env gp140 protein vaccines and assessed the induction of antibody responses and a putative protective signature. This signature included multiple antibody functions and correlated with upregulation of interferon pathways in vaccinated animals. Adoptive transfer of purified immunoglobulin G (IgG) from the vaccinated animals with the most robust protective signatures provided partial protection against SIVmac251 challenges in naive recipient rhesus macaques. These data demonstrate the protective efficacy of purified vaccine-elicited antiviral antibodies in this model,even in the absence of virus neutralization. View Publication