The depletion of FOXM1 or ABCC5 with siRNA significantly blocked drug efflux and increased the intracellular concentrations of paclitaxel, thereby promoting paclitaxel-induced cell death. Siomycin A, a FOXM1 inhibitor, significantly enhanced in vitro cell killing by paclitaxel in drug-resistant NPC cells. This study is the first to identify the roles of FOXM1 in drug efflux and paclitaxel resistance by regulating the gene transcription of abcc5, one of the ABC transporters. Small molecular inhibitors of FOXM1 or ABCC5 have the potential to overcome paclitaxel chemoresistance in NPC patients.Paclitaxel is clinically used as a first-line chemotherapeutic regimen for several cancer types, including head and neck cancers. However, acquired drug resistance results in the failure of therapy, metastasis and relapse. The drug efflux mediated by ATP-binding cassette (ABC) transporters and the survival signals activated by forkhead box (FOX) molecules are critical in the development of paclitaxel drug resistance. Whether FOX molecules promote paclitaxel resistance through drug efflux remains unknown. In this study, we developed several types of paclitaxel-resistant (TR) nasopharyngeal carcinoma (NPC) cells. These TR NPC cells acquired cancer stem cell (CSC) phenotypes and underwent epithelial to mesenchymal transition (EMT), and developed multidrug resistance. TR cells exhibited stronger drug efflux than parental NPC cells, leading to the reduction of intracellular drug concentrations and drug insensitivity. After screening the gene expression of ABC transporters and FOX molecules, we found that FOXM1 and ABCC5 were consistently overexpressed in the TR NPC cells and in patient tumor tissues. Further studies demonstrated that FOXM1 regulated  Its annual incidence has been reported to be over 20/100000 in the Cantonese population in China.4 and it has been commonly used to treat many cancer types, including breast, head and neck, lung and melanoma,5 as a first-line chemotherapeutic drug,. Paclitaxel, alone or in combined with cisplatin and/or 5-FU, has been tested as an adjuvant chemotherapeutic agent for advanced NPC patients in clinical trials.8 NPC patients are sensitive to chemotherapy at the beginning of treatment, but develop acquired resistance shortly after, resulting in therapy failure.10 The molecular mechanisms of drug resistance are elusive. Previous studies have implicated many factors contributing to chemoresistance, including increased drug efflux or decreased drug influx, abnormal cellular metabolism, mutations of drug target genes, hyperactive DNA repair machines, deregulation of Bcl-2 apoptosis molecules, cell senescence, autophagy, tumor microenvironment and more.12 Paclitaxel drug resistance has been closely associated with the modification of microtubulin in cancer cells, which may be induced by the point mutations, expression deregulation or posttranscriptional tubulin alterations.12 Hyperactive drug efflux and multidrug resistance (MDR) because of the overexpression of ATP-binding cassette (ABC) transporters, such as MRP1 and MDR1, are significant factors in the development of paclitaxel resistance.13Paclitaxel was approved by FDA to treat ovarian cancer in 199215 Paclitaxel induces the expression of FOXO3a, and confers paclitaxel resistance by elevating BH3-only Bcl-2, or activating MAPK/JNK signaling.16 Among FOX molecules, FOXM1 has a critical role in the development of paclitaxel resistance.18 The overexpression of FOXM1 results in paclitaxel resistance, and FOXM1 depletion increases paclitaxel sensitivity.17 FOXM1 is a cell growth-specific transcription factor that regulates target genes controlling the G1/S and G2/M cell cycle transition.20 The exact mechanisms of FOXM1 involvement in paclitaxel resistance remain undefined.The molecules of Forkhead box (FOX) family are reportedly involved in taxane-associated drug resistance.21 CNE2TR cells exhibited resistance to paclitaxel, acquired the characteristics of cancer stem cells (CSCs) and underwent epithelial to mesenchymal transition (EMT). The paclitaxel-resistant cells developed MDR by decreasing the intracellular drug concentrations. FOXM1 and the ABC transporter ABCC5 were consistently overexpressed in paclitaxel-resistant NPC cells and tumor tissues. We explored whether FOXM1 contributed to paclitaxel resistance by regulating abcc5 gene transcription and protein expression, thereby increased drug efflux. We also tested whether a FOXM1 inhibitor used as a chemosensitizer may restore paclitaxel sensitivity in cancer cells.In this study, we developed several types of paclitaxel-resistant CNE2TR NPC cells by intermittently treating the cells with low doses of paclitaxel.21 The resistance of CNE2TR cells to paclitaxel was assessed by colony formation assay and apoptosis detection assay. Paclitaxel 30, 50, 70 and 100 ng/ml killed many more CNE2 cells than CNE2TR cells  compared with parental CNE2 or CNE1 cells. E-cadherin decreased, whereas Vimentin, Snail and ZEB1 markedly increased  also were resistant to other chemotherapeutic drugs, that is, developed MDR. To verify this hypothesis, we tested the drug sensitivity of CNE2 and CNE2TR cells to the chemotherapeutic drugs cisplatin and chlorambucil in addition to paclitaxel. The sensitivities of CNE2TR and CNE2 cells to the three drugs were very consistent. Paclitaxel, cisplatin and chlorambucil induced more cell killing in CNE2 than CNE2TR. The IC50 of chemotherapeutic drugs in CNE2TR cells was much higher than CNE2 . The resAs paclitaxel-resistant CNE2TR cells developed resistance to other chemotherapeutic drugs, we hypothesized that common pathways of MDR were probably activated in CNE2TR cells. We first tested the drug efflux of paclitaxel in CNE2TR cells compared with parental CNE2 cells. These cells were treated with paclitaxel, and cell culture media were harvested, for testing extracellular drug concentrations. Cells were prepared by ultrasonic homogenization after thorough washing, and intracellular drug concentrations were measured by UPLC-MS . As show15 We compared their expression levels in CNE2TR and CNE2 cells. Higher expression levels of FOXO3a, FOXM1 and FOXC2 were detected in CNE2TR than CNE2 at both mRNA and protein levels  and all four levels   .19 Recent whole-genome studies suggested that abcc5 may be a target gene regulated by FOXM1, as ChIP-seq data showed a very strong binding peak in the enhancer/promoter areas of the abcc5 gene.23 We tested whether FOXM1 regulates the transcription of abcc5 gene. In CNE2TR cells, the mRNA and protein levels of ABCC5 obviously decreased when FOXM1 was depleted by siRNA AA(C/T). The core sequence of FHK consensus binding motif taaAGGAaac at the promoter of abcc5 gene was mutated to gcgAGGAaac or taaAGGAgct  containing 10% fetal bovine serum with 1% penicillin/streptomycin  at 37 °C in a 5% CO2 incubator. Paclitaxel-resistant cells were maintained in culture medium supplemented with 1 nM paclitaxel .The CNE2 and CNE1 NPC cell lines were obtained from the Cancer Research Institute of Central South University, Changsha, China. The paclitaxel-resistant cell sublines CNE2TR, CNE1/T and SKOV3R were established by intermittently exposing the parental cells to gradually increasing concentrations of paclitaxel.α-MCS-IRES-Bsd lentiviral vector . The FOXM1-expressing or control lentivirus was produced by co-transferring the plasmids with VSV-G, Rev, and Gag-Pol to HEK-293 T cells. FOXM1-overexpressing CNE2 cells were generated by infecting CNE2 cells with lentivirus for 48 h, followed by selection using 8 μg/ml BSD for 2 weeks.Human FOXM1 cDNA was synthesized and then cloned into the pLV-EF1μg/ml puromycin for 2 weeks.The following primers were designed to synthesize FOXM1 shRNA: upper: 5'-GATCCGCTCTTCTCCCTCAGATATATTCAAGAGATATATCTGAGGGAGAAGAGTTTTTTG-3'; lower: 5'-AATTCAAAAAACTCTTCTCCCTCAGATATATCTCTTGAATATATCTGAGGGAGAAGAGCG-3'). The shRNA was synthesized and cloned into Lenti-X shRNA Expression Systems . The FOXM1 knockdown or control lentivirus was produced by co-transferring the plasmids with VSV-G, Rev, and Gag-Pol to HEK-293 T cells. FOXM1 stable knockdown CNE2TR cells were generated by lentivirus infection for 48 h, followed by selection using 2 μl of MTS solution [3--5-(3-carboxymethoxyphenyl) -2-(4-sulfophenyl)-2Htetrazolium]  was added to each well, and cultured at 37 °C in 5% CO2 for 4 h. Absorbance at 490 nm was measured by spectrometer. Each treatment was performed in quintuplicate. The relative viability of the cancer cells was calculated.Cells were cultured overnight (5000 per well) in 96-well flat-bottomed microtiter plates exposed to paclitaxel at 50 or 200 ng/ml. At individual time points of 0, 24, 48 and 72 h, 20 CNE2 or CNE2TR cells were treated with stepwise concentrations of paclitaxel for 48 h. To test the sensitivity of CNE2TR cells to paclitaxel when FOXM1 or ABCC5 was knocked down, we first transfected FOXM1 or ABCC5 siRNA to CNE2TR cells for 24 h, followed by paclitaxel treatment of (100 ng/ml) for an additional 48 h. CNE2TR cells were treated with stepwise concentrations of siomycin A  for 48 h, and DMSO was used as a negative control. The cells were re-seeded in six-well plates (1000 cells per well) after treatments as above, and then cultured for 15 days for colony formation. Each treatment was performed in triplicate. The cell colonies were fixed in 3.7% paraformaldehyde and stained by 0.05% crystal violet solution. The dishes were photographed after staining. The cells were cleaved by 10% SDS, and the cell survival ratio was assessed by measuring absorbance at 570 nm.The total RNA was isolated by the RNAiso Plus method following the manufacturer's protocol. The dissolved RNA sample was measured on a spectrophotometer to determine concentration and quality before its conversion to cDNA. Quantitative real-time PCR was then performed using the CFX96TM Real-Time PCR Detection System  according to the manufacturer's instructions. Values were expressed as fold changes compared with the corresponding values for the control using the 2 –ΔΔCt method.μg) were separated by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes . The blots were blocked for 1 h, and then incubated with the primary antibodies at 4 °C overnight. The blots were incubated for 1 h with horseradish peroxidase-conjugated secondary antibodies . The protein was visualized with a Pierce ECL Western Blotting Substrate detection system . β-Actin was used as the loading control. The primary antibodies used in this study included anti-FOXM1, anti-FOXO3a, anti-p53, anti-ABCA2, anti-SOX2, anti-ALDH1, anti-GAPDH and anti-β-actin purchased from Santa Cruz ; anti-FOXO1, anti-FOXC2 and anti-ABCC5 purchased from Abcam ; and anti-E-cadherin, anti-vimentin, anti-Snail, anti-ZEB-1, anti-p38, anti-p44/42, anti-AKT, anti-AKT473 and anti-AKT308 purchased from Cell Signaling Technology .The cells were harvested with RIPA buffer. The protein samples  or Thermo Scientific Dharmacon  using DharmaFECT Transfection Reagents  according to the manufacturer's instructions.The sequences of siRNAs used in this study were: siRNA FOXM1 (sense: 5′-CUCUUCUCCCUCAGAUAUATT-3′), siRNA FOXO1 (sense: 5′-GCAGUAGAUACAGAUUGUATT-3′), siRNA FOXO3a (sense:5′-GCAUUAGGCAUAUAAAUGUTT-3′), siRNA FOXC2 (sense:5′-AGAAGGACGUGUCCAAGGATT-3′), siRNA ABCC5  non-target negative control siRNA (5′-UUCUCCGAACGUGUCACGUTT-3′), which has been confirmed to have minimal targeting of known genes.Cells were suspended in PBS and incubated with anti-CD44  and anti-CD133 . Positive-staining cells were analyzed by Flow cytometry . To evaluate cell apoptosis, cells were collected and washed twice with PBS while spinning at 1000 r.p.m. for 10 min. Cell pellets were resuspended in a FITC-labeled Annexin V and propidium iodide (PI) staining solution  and incubated for 15 min at room temperature. The apoptotic cells were then analyzed by flow cytometry (Millipore).abcc5 gene promoter was cloned by PCR, and subcloned into pGL3-TATA vector. A FHK consensus binding motif was mutated using a Quickchange site-directed mutagenesis kit (Thermo Fisher Scientific). CNE2TR cells were co-transfected with the gene promoter (WT or MUT), siFOXM1 RNA and pRL-SV40 (Promega) using the TransIT-X2 Dynamic Delivery System . The firefly/Renilla luciferase activities were detected by the Dual-Glo Luciferase reporter assay system (Promega) according to the manufacturer's manual 48 h after transfection. Luminescence was then measured using the Perkin Elmer (EnSpire 2300) Multilabel Reader .The human 5 cells per well) in six-well plates. A confluent monolayer of CNE2TR and CNE2 cells was scratched using a sterile 200 μl plastic pipette tip. Displaced cells were moved with three washes, and the cell gaps were monitored at 24, 48 and 72 h after scratching. The cell gap was quantified by Image Pro Plus software  and data are presented as cell gap distance. CNE2 and CNE2TR cells were cultured in serum-free RPMI 1640 medium for 48 h, the cells were re-plated on transwell plate inserts with serum-free media, and normal culture media with 10% FBS was put in the bottom wells. The invasive cells on the membrane were stained by Violet Crystal 24 h after cell plating. Each treatment was performed in triplicate.CNE2 and CNE2TR cells were plated (2 × 106 each well in six-well plate) were treated with paclitaxel  for 2 h. The cell culture media were harvested to test extracellular drug concentrations. The cells were prepared by ultrasonic homogenization after thorough washing, and 500 μl supernatants were mixed with methanol. The samples were dried in a vacuum-dryer and re-dissolved in 200 μl HPLC-grade methanol. The drugs were measured by liquid chromatography (LC)/electrospray ionization (ESI)-tandem MS using the UltiMate 3000 UPLC system online coupled to an linear trap quadrupole-Orbitrap Velos Pro mass spectrometer (Thermo Fisher Scientifc).CNE2 and CNE2TR cells (2 × 10μl) were separated by a C18 RP LC column  and eluted with a binary system consisting of solvent A (0.1% formic acid in aqueous phase) and solvent B (0.1% formic acid in methanol) at 1:1 with a flow rate of 0.3 μl/min. An ESI source was applied and operated in the positive ion mode. The capillary voltage was 3.5 kV. For MS conditions, nitrogen (N2) was used as the sheath gas at a flow rate of 35 l/min. The aux gas (N2) flow rate was 8 l/min. The molecular weight of paclitaxel is 876. The method was linear in stepwise concentrations of 110, 130, 220, 440, 880 and 1760 ng/ml with the coefficient correlation of 0.999. The procedure was repeated in three biological replicates.The analytes  for 24 h. The rhodamine 123-positive cells were analyzed by flow cytometry. The cells were seeded and fixed on a glass slide, and the nuclei were stained with DAPI. The green fluorescence of cells inside the cytoplasm or nuclei was observed by confocal laser scanning microscopy .Paraffin sections of NPC tumor tissues were obtained from the Department of Pathology, Xiangya Hospital, Central South University. The slides were incubated with primary antibody against FOXM1 (Santa Cruz) and ABCC5 , respectively. For negative control, isotype-matched antibodies were applied. FOXM1 staining was detected in both nuclear and cytoplasmic compartments, whereas ABCC5 staining was detected in both cytoplasm and membrane. The IHC results were judged from 5 to 10 random fields (× 400) by two independent senior pathologists. The degree of staining was classified into four stages . The ratio of positive cells was divided into three stages . The overall standard including staining degree and ratio of positive cells: +percent of positive cells × 1, ++ percent of positive cells × 2 and +++ percent of positive cells × 3. The overall results: +<1, ++1 to 1.5, +++>1.5.in vitro experiments were done either in triplicate or in quintuplicate. The analyses were conducted with Graphpad 6.0 software . The results are described as the mean±S.D. with analysis by one-way ANOVA or two-way ANOVA, and protein and mRNA expression levels were compared by two-sided Student's t-tests. The correlation between FOXM1 and ABCC5 expression in tumor tissues was assessed by Pearson correlation or Gamma correlation, and kappa analysis. A P-value of <0.05 was considered to be statistically significant . The correlation of FOXM1 and ABCC5 was regarded as low when the correlation factor was less or equal to 0.4, intermediate when it was between 0.4 and 0,75, and high when it was over or equal to 0.75.All


Clopidogrel, a P2Y12 antagonist, is widely used for the treatment of thrombosis and stroke, blocking P2Y12 receptor-mediated platelet aggregation. Diquafosol, a long acting P2Y2 receptor agonist, is being used for the treatment of dry eye. P2X3 receptor antagonists have been developed that are orally bioavailable and stable in vivo and are currently in clinical trials for the treatment of chronic cough, bladder incontinence, visceral pain and hypertension. Antagonists to P2X7 receptors are being investigated for the treatment of inflammatory disorders, including neurodegenerative diseases. Other investigations are in progress for the use of purinergic agents for the treatment of osteoporosis, myocardial infarction, irritable bowel syndrome, epilepsy, atherosclerosis, depression, autism, diabetes, and cancer.Purinergic signalling, i.e., the role of nucleotides as extracellular signalling molecules, was proposed in 1972. However, this concept was not well accepted until the early 1990’s when receptor subtypes for purines and pyrimidines were cloned and characterised, which includes four subtypes of the P1 (adenosine) receptor, seven subtypes of P2X ion channel receptors and 8 subtypes of the P2Y G protein-coupled receptor. Early studies were largely concerned with the physiology, pharmacology and biochemistry of purinergic signalling. More recently, the focus has been on the pathophysiology and therapeutic potential. There was early recognition of the use of P1 receptor agonists for the treatment of supraventricular tachycardia and A Purinergic signalling, i.e., nucleotides as extracellular signalling molecules, was proposed in 1972 . HoweverMore recently, the focus has been on the pathophysiology and therapeutic potential of both P1  and P2  receptorInvestigations into purinergic signalling and its roles in disorders of the CNS have been reported, for instance following surgery, stroke, accidents and ischemia, neurodegenerative diseases , multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), epilepsy and neuropsychiatric disorders . Reviews covering this topic are available , includi2AR has also been claimed as a promising therapeutic agent for the prevention and treatment of neurodegenerative diseases  modulation as a therapeutic strategy against AD  may be a good candidate for traumatic spinal cord injury treatment  mice and murine cell lines, it was concluded that P2Y2R play a neuroprotective role in neurological disorders, especially AD  were found to antagonise responses mediated by P2XR. These antipsychotic drugs act therapeutically by inhibiting dopaminergic hyperactivity by suppressing P2X-mediated effects see . Brain p2AR from astrocytes interferes with glutamate homeostasis resulting in cognitive and psychomotor impairment in schizophrenia  and P2X7R in mood disorders are available -1β in the brain contributed to anxiety  receptors modulate the drug-seeking effects of methamphetamine  and in a rat model   gene expression and raised CD73 activity. Adenosine therapy mediated by A failure . A1R agomyocyte) . A2BR ag in rats . CD73 ann injury . Inflammn injury .6R could be a therapeutic target to regulate cardiac hypertrophy  concluded that infusion for 3 h of adenosine resulted in a reduction of infarct size . Protectfarction . Microglfarction . Loss offarction . CD39 refarction . P2Y12R farction . The KATfarction .2AR involves abnormal calcium handling in AF. Prevention of A2AR activation in patients with AF may sustain uniform beat-to-beat responses at higher beating frequencies . Up-regulation of Aquencies . Adenosiquencies , althougquencies . ATP-indquencies . The effed in AF . The roled in AF .1R  is clinically prescribed to slow conduction time via the atrioventricular node, via A1R . Treatme1R . To prov1R .Cardiomyopathy can be an inherited disease, but can occur as a result of vitamin B deficiency, amyloidosis, alcoholism or viral infections. ATP synthase disruption contributes to diabetic cardiomyopathy . P2X7R i11R agonists reduce cardiac fibrosis ATP released as a cotransmitter from sympathetic nerves together with noradrenaline (NA) contributes, via P2X1R, to the vasoconstriction that results from increased sympathetic vasomotor activity in hypertension. Therefore, P2X1R antagonists should be useful for the treatment of hypertension, especially since there is a substantial increase in ATP relative to NA released from sympathetic nerves in spontaneously hypertensive rats .(2)1, P2Y2, and P2X4 agonists would cause increased vasodilation in hypertension.Release of ATP from endothelial cells by shear stress as a result of changes in blood flow acts on P2YR and P2X4R on endothelial cells to release nitric oxide (NO) resulting in vasodilation. Introduction of P2Y(3)Brain stem and hypothalamic neurons mediate sympathetic nerve activity. Recent studies show that P2X3R antagonists are antihypertensive, due to reduced sympathetic nerve activity as a result of increased peripheral P2X3R-mediated carotid body chemoreceptor reflexes .(4)P2X7R in the kidney contribute to the pathophysiology of hypertension and P2X7R antagonists may have promise as clinical antihypertensive agents.(5)In hypertensive patients adenosine activates the vascular renin-angiotensin system. P1R agonists have been suggested for the treatment of hypertension .6R were shown to age-dependently promote vascular remodelling in a mouse model, an effect inhibited by MRS2578, suggesting that P2Y6R are a therapeutic target for the prevention of age-related hypertension  and ATP in atherosclerosis , followed by vasodilation (reactive hyperaemia) associated with pain. A purinergic hypothesis for migraine was proposed in 1981 . It was Adenosine may also be involved in migraine. Adenosine infusion resulted in symptoms that were migraine-like and withdrawal of the P1R antagonists theophylline and caffeine also resulted in migraine-like symptoms. Clinical trials with the adenosine uptake inhibitor dipyridamole, that results in increased extracellular adenosine, were halted due to the increase of migraine attacks in all patients. Overactive P2YR on glial cells may contribute to pain transduction during migraine. Reviews have been published about the role of purinergic signalling in the aetiology of migraine and the potential of purinergic compounds .1R-mediated sympathetic neurotransmission and ATP-mediated endothelial vasodilatation in mesenteric arteries of streptozotocin (STZ) diabetic rats , dyspnea, asthma, cystic fibrosis (CF), allergy, infection and injury. Purine nucleotide release from airway epithelial cells is raised in inflammatory processes and has an important role in the pathophysiology of chronic lung disease. P2X7R are a therapeutic target in lung hypersensitivity reactions seen in chronic inflammatory responses. P2X7R modulate lung inflammatory, fibrotic and functional changes in silicosis, an occupational lung disease . P2X4R mammation . P2Y2R hpression . Nucleotytokines . Anti-in2A, A2B, and A3R were all claimed to mediate inhibition of allergic airway inflammation and mast cells were shown to be involved. Expression of adenosine receptors on monocytes from patients with asthma contributes to the progression of the disease   , althougsis (TB) . In infesis (TB) . Polymorsis (TB) . Data hasis (TB) . Selectisis (TB) . P2X7R c in vivo . The rol in vivo . The autStreptococcus pneumoniae infection of the lungs by pulmonary neutrophil recruitment regulation -2′-deoxy-ATP], VX-770  and nitrate ions (NO3-). The authors concluded that future therapeutic developments might include the complementary use of ATP analogues with VX-770.Cystic fibrosis is hereditary as a result of a loss of function gene mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Some outstanding reviews about purinergic signalling in CF have been published . The regl repair . With thl repair . Gating - secretion across CF airway epithelia is restored by ATP by triggering calcium entry via P2XR. It was suggested that P2X4/6 heteromultimer receptors might be involved. CFTR activity is necessary for ATP release following erythrocyte deformation. CFTR is reduced or absent in CF and mechanical deformation of erythrocytes does not induce ATP release. Increasing nucleotide release via motion could have therapeutic implications for CF patients. Women with CF showed reduced survival compared with males. Oestrogen may lower breathing-induced release of ATP and ATP receptor-mediated [Ca2+]i increase that induces Cl- secretion. Anti-oestrogens may therefore be beneficial in the treatment of CF.Cl1R agonists release [Ca2+]i and activate Cl- and K+ currents in CF epithelial cells of the airways. A1 and A2AR participate in regulation of Cl- secretion in CF airway epithelial cells. Bronchoalveolar lavage from CF patients contained high concentrations of adenosine, correlating with higher 5′-nucleotidase and lower ADA activity. Adenosine regulates CFTR via A2BR. The exhaled breath condensate biomarker, adenosine, tracks changes in lung function in CF (Aon in CF . There ion in CF .2 and/or P2Y4) that mediate increases in [Ca2+]i. In A549 cells there is calcium-dependent UTP and ATP release (with subsequent increase in adenosine levels). Erythromycin selectively inhibits influx of Ca2+ induced via P2X4R stimulation in A549 lung tumour cells. A549 cells express P2Y2, P2Y4, and P2Y6, and P2X4R. ATP induces dose-dependent inhibition of growth of cell lines, including human mesothelioma (MER082), human papillary lung adenocarcinoma (H441), human squamous cell lung carcinoma (H520), human large cell lung carcinoma (H460), human small cell lung carcinoma (GLC4) and the PC14 lung adenocarcinoma cell line. ATP was released from Calu-3 cells derived from human lung adenocarcinoma, probably in response to P2Y2R activation. UTP, ATP and uridine diphosphate stimulate proliferation of lung tumour A549 cells through P2Y2 and P2Y6R.Human lung A549 epithelial-like adenocarcinoma cells express P2UR  reduced rejection of airway allografts by stimulating Asurvival .Purinergic signalling is involved in the physiology of the nasal organs, ear, eye and tongue. Purinergic drugs are being explored for corneal injury, retinal detachment, glaucoma, dry eye, retinitis, uveitis, rhinosinusitis, diabetic retinopathy, macular degeneration, noxious odour damage, Ménière’s disease, sensorineural deafness, tinnitus and taste defects .The early literature up to 2006 was reviewed  and therATP channel opener, lowers intraocular pressure  from S. mansoni was the first non-vertebrate ATP-gated ion channel, which could be an alternative drug target to treat schistosomiasis  and Crohn’s disease are the main types. P2Y in rats  and are  in rats . ATP act in rats . Oestrog in rats . The rol in rats .2A and A3R expression . P2X7R are expressed weakly in healthy glomerulus, but after glomerular injury  are upregulated, mostly in podocytes, but also in mesangial and endothelial cells . P2X7R p2BR-mediated IL-6 induction contributes to renal fibrogenesis and this receptor has therapeutic potential for treatment of chronic kidney disease -1 and PC-2 proteins. Nucleotide release is involved in both fluid flow and pressure responses and its role in altered mechanosensory transduction in PKD is considered in a review , the purinergic component can be as much as 40% and is consequently a therapeutic target. Botulinum neurotoxin type A (BTXA) is often used to treat bladder incontinence, where it inhibits ATP release as well as acetylcholine (ACh) release both from parasympathetic nerves and urothelial cells. Bladder epithelial cell release of ATP from patients with IC was substantially higher compared to healthy controls. Dysfunction in micturition involves P2X3R in rats with chronic spinal cord-injury, suggesting that P2X3R antagonists could be used to treat neurogenic bladder dysfunction.There are valuable reviews that discuss the roles of purinergic signalling in OAB . OAB syn2AR antagonists occurs, probably by acting in the CNS to regulate the micturition reflex  is either neurogenic or idiopathic in origin. Neurogenic detrusor instability can occur as a result of a stroke, or pelvic or spinal cord injury, as well as in PD and MS. Neurogenic bladders are hyper-responsive to ATP, via P2X3R.Expression of P2X2R in both urothelium and detrusor muscle of suprasacral spinal cord injury patients are similar to P2X2R expression in bladder tissue from idiopathic overactive bladder patients. ATP levels increased in the rat spinal cord-injured bladder that activates P2X3 and P2X2/3R on afferent nerve fibres resulting in neurogenic bladder overactivity . P2YR agUrothelial cell ATP release (both resting and evoked) was substantially more from rat bladders following chronic spinal cord injury, contributing to bladder hyperactivity development. In a mouse bladder overactivity model, bradykinin facilitated the release of ATP from nerve terminals via prejunctional receptors .In the absence of P2X3R in KO mice, there is bladder hyperactivity . AF-219,Idiopathic detrusor instability patients show abnormal bladder purinergic transmission, which may contribute to the symptoms of OAB . There iThe possible roles of ATP and adenosine in IC/bladder pain syndrome are discussed in a review . Hypoosm2R expression is lost.Contractions of the rat bladder mediated by parasympathetic nerve stimulation and release of ATP and ACh, were reduced in cyclophosphamide-induced cystitis , while P1R blockade during the initial phase of IC/bladder pain syndrome was suggested as a treatment for this condition . The A1R in rat urinary bladder is decreased in cyclophosphamide-induced cystitis   .1R, in pigs, guinea-pigs, rabbits and hamsters. The responses to non-adrenergic, non-cholinergic nerve stimulation were blocked by α,β-meATP, indicating that the P2X1R subtype was involved.The principal non-adrenergic, non-cholinergic inhibitory transmitter to the urethra is NO, but a small component of purinergic neurotransmission is also involved . ATP cauATP channels in the urethra is available , lowered detrusor tone leading to increased bladder capacity and decreased voiding efficiency. Some sensory functions in the urethra are mediated by afferent fibres that express P2X3R . A revievailable .The ureter motor innervation is sparse, perhaps because peristaltic activity is not neurogenic, but myogenic. The dominant nerves in the ureter are sensory, confined mainly to the suburothelial plexus. These nerves contribute to vesicoureteral reflux that activates reflexes that modulate urine delivery to the bladder.2BR. In the ureter, ATP, α,β-meATP and adenosine evoked transient decreases in the frequency of peristalsis. In the rat ureter expression of P2X1R was shown on smooth muscle, while P2X5 and perhaps P2X7R are expressed on urothelium and P2X6R in the layer beneath the urothelium. P2X3R are localised on subepithelial sensory nerves that mediate nociception produced by release of ATP following distension of urothelial cells  express purinergic receptors: P2Yvailable .1R KO mice α-naphthylisothiocyanate-induced cholestatic liver injury was decreased. CD39-deficient mice treated with adenosine, probably via A2AR, were protected from reperfusion injury  cells reduced hepatic ischaemia/reperfusion injury, indicating that during liver regeneration ATP modulates NK cell function. UTP acting via P2Y2 and/or P2Y4R before induction of ischaemia attenuates post-ischaemic hepatocyte apoptosis resulting in a reduction of liver damage  and hepatocholangiocarcinoma. ATP increases calcium uptake by rat hepatoma cells, probably via P2Yma cells , which mma cells . Carcinoma cells . CD39 KOstigated . P2X3R ostigated . Inhibit3R agonist, CF101, inhibited liver metastasis (following colon carcinoma)  . CF102, patients . A2BR ararcinoma . Mouse rarcinoma . Liver marcinoma . In hepaarcinoma .Reviews are available about purinergic signalling in the reproductive system in both health and disease .4R, whereas ADP, after breakdown of ATP released from endothelial cells, acts via P2Y1R on endothelial cells to produce relaxation via NO  ATP increased apoptotic cell death in PC-3 cells, probably via P2X7R. CD73 KO mice resist prostate carcinogenesis and CD73 promoted de novo prostate tumorigenesis. Anti-CD73 monoclonal antibodies decreased tumour growth and metastasis in the prostate -prostate .There are many polymorphisms of the P2X7R , which, 3R activation by IB-MECA inhibited proliferation of prostate cancer cells and induced cell cycle arrest and apoptosis . SSc is a severe disease of the connective tissue of affected organs, including the skin. Adenosine Aeroderma . Fibrobl1R, and subsequent release of proinflammatory cytokines. Skin inflammation is reduced by P2X7R antagonists.The skin is an immune defence organ. Chemical, immune-specific or physical insults evoke increased expression of proinflammatory mediators, in particular keratinocytes release chemokines. Release of ATP is associated with inflammation of the skin, as well as increase in expression of, in particular, P2X7 and perhaps P2Y6R mediate UV radiation-induced inflammatory responses  KO mice, rapid ATP release from keratinocytes is triggered by irritant chemicals causing exacerbated skin inflammation. Ultraviolet (UV) radiation evokes ATP release from keratinocytes and P2Yesponses . ATP, whP levels . P2X3 an2B and particularly A2AR agonists promote cutaneous wound healing in both healthy and diabetic conditions. A2AR agonists also promote collagen production by dermal fibroblasts. Adenosine inhibits proliferation of vascular smooth muscle cells via A2R activation. A review discusses the involvement of A2A and A2BR signalling in wound healing and fibrosis  and both ATP and adenosine contribute to cell proliferation and migration during angiogenesis. In cultured porcine artery smooth muscle cells, ADP and ATP stimulate DNA synthesis and cell proliferation. ATP and UTP are mitogenic in human vascular smooth muscle cells and P2Y1, and P2Y2R expression was altered in the epidermis. P2Y1R expression was increased in the basal proliferating layer of keratinocytes in the regenerating epidermis, while P2Y2R were significantly decreased. ATP and UTP, probably via P2Y2R, increased proliferation of the MCS-P5 murine keratinocyte cell line, and enhanced wound healing in mice  at the epidermis suprabasal layer. In lesions P2YThe skin protects the water-rich internal organs from environmental dryness, with the stratum corneum being critical to the water-impermeable barrier. Topical application of ATP and α,β-meATP via P2XR delayed barrier recovery, damaged by surfactant, organic solvent or tape stripping. P2R antagonists, however, accelerated barrier repair. In mice skin wounds healed faster following treatment with ATP-encapsulated fusogenic lipid vesicles rather than with lipid vesicles. PPADS, a P2XR antagonist, accelerates skin barrier repair and prevented epidermal hyperplasia.2 administration following burn injury reversed the damage to the intravascular clearing of lipid emulsions within the reticuloendothelial immune system. Glucose metabolism was affected by ATP in thermally injured skin.There is increased concentration of adenosine in burn blister fluid, and depletion of ATP in skin after thermal injury. ATP has a protective effect against skin burns. ATP-MgClBurn injury produces severe pain and the relieving effects of tetramethylpyrazine, a Chinese medicine, are claimed to be due to it acting as a P2X3R antagonist . Skin P2Skin pain is associated with changes in sympathetic nerve activity and ATP released from rat sympathetic nerves as a cotransmitter with NA may modulate cutaneous nociceptors. P2X3R are expressed on sensory neurons in DRG, nodose and trigeminal ganglia that innervate cutaneous tissues. ATP and ADP stimulate skin afferent nerve terminals and skin cell damage stimulates nociceptive sensory nerves via ATP release. In rat skin with carrageenan-induced inflammation, nociceptors can be selectively activated with α,β-meATP, indicating the involvement of P2X3R. ATP contributes to the enhanced sensitivity of inflamed skin nociceptors, leading to heat hyperalgesia. P2X3R antagonists are anti-nociceptive. UV light evokes hypersensitivity of skin to ATP-induced pain. After subcutaneous bee venom injection, there is reduction of prolonged pain by PPADS, and it was suggested that stimulation of P2XR in the spinal cord contributes to cutaneous pain. The terminals of nociceptive neurons in the skin are targets for P2X3R antagonists. UTP sensitises a subpopulation of cutaneous C-fibre nociceptors to mechanical stimuli. There is increased P2X3R expression on calcitonin gene-related peptide-positive sensory fibres during the growth of tumours, mediating nociception. Increased expression of skin glial cell neurotrophic factor increases the mechanical sensitivity of nociceptive afferents expressing P2X3R.1R prevents thermal hyperalgesia (Ecto-5′-nucleotidase (CD73) is located on nociceptive terminals and epidermal keratinocytes in the epidermis, which, by hydrolysing AMP to adenosine, is suggested to be anti-nociceptive. Blockade of peripheral P2Yralgesia . Mechaniralgesia .Reviews with coverage of purinergic signalling and cutaneous pain are available .2A antagonists prevents radiation dermatitis  carcinoma cell line, A431, express P2R that induce increases in [CaO-(4-Benzoylbenzoyl) ATP, a potent P2X7R agonist, reduced skin carcinoma and papilloma and formation (ATP via P2X7R caused apoptosis of A431 cells and UTP and adenosine (after ATP breakdown) also induced cell death . 2′(3′)-ormation . ADA in ormation .in vivo. CD39 is over-expressed in differentiated human melanomas. Expression of P2X7R was increased in superficial spreading melanoma patients, which were later shown to be functional and may be a therapeutic target for melanoma therapy  and P2X7R mediate cell death. Therefore, P2Y2R antagonists and P2X5 and P2X7R agonists have therapeutic potential for the treatment of skin cancer.In conclusion, P2YReviews that include discussion of the pathophysiology of musculoskeletal diseases have been published . There aATP was used early to treat myopathies, although the mechanism of action was not known. Allopurinol has been used for the treatment of Duchenne muscular dystrophy (DMD). It counteracts low purine nucleotide degradation levels that occur in Duchenne muscle, but chronic administration of allopurinol failed to improve DMD symptoms. Lymphoblastoid cells from DMD patients were found to be highly sensitive to ATP stimulation.mdx model of DMD, lacking the dystrophin protein, mimics muscle damage and subsequent regeneration. Sequential expression of P2X5, P2Y1 and P2X2R was described during muscle regeneration in the mdx model  influx causes tetanic failure in MG  is characterised by allodynia, as well as mood disorders. In fibromyalgia patients, there are decreased levels of ATP in platelets perhaps contributing to the pathogenesis of the disease .2R gene were linked to bone mineral density and the risk of osteoporosis in Dutch fracture patients , leads to a high risk of bone fracture. The P2X7R is considered to be important in relation to the treatment of osteoporosis . P2X7R ppatients . It was patients . P2Y13R oporosis .Trans-differentiation of osteoblasts to adipocytes, which involves A2BR, may contribute to the pathogenesis of osteoporosis  receptors were cloned from osteoclastoma. The expression of P2Y2R from human osteoclasts from bone giant cell tumour was later reported. Butyl benzyl phthalate, which interferes with mammalian ion channel receptors, inhibited ATP-induced cell proliferation via P2XR in human osteosarcoma HOS cells  involves osteolytic bone lesions, due largely to enhanced osteoclast activity. Antiation . 8-AminoA major cause of severe combined immunodeficiency are genetic defects in the ADA gene. Lack of ADA causes accumulation of adenosine. Bone defects as a result of reduced osteoclastogenesis together with a defect in osteoblast function leading to low bone formation were seen in about a half of early-onset ADA-deficient patients . Further4A diminished the expression of the achondroplasic FGFR3 receptor and P2Y1, P2Y2, P2Y6, and P2Y11R are expressed by achondroplasic chondrocytes mediating the action of Ap4A . Ap of Ap4A . Ap4A redwarfism .There is an increase in osteoclast numbers in Paget’s disease, leading to an increase in bone resorption and a high turnover of bone. Bisphosphonates have been employed as a treatment for Paget’s disease and P2X7 antagonists have also been considered .1R are highly expressed in OPLL cells. Mechanical stress and ATP increase the levels of osteopontin and alkaline phosphatase mRNA in OPLL cells, effects that can be inhibited by MRS2179, a selective P2Y1R antagonist. Over-expression of P2Y1R in OPLL-induced mineralisation resulted in ectopic bone formation in the spinal ligament cells of patients with OPLL  is increased. P2Yith OPLL .12R antagonists, such as clopidogrel and ticagrelor, are currently in wide use for stroke and thrombosis, as are P2Y2R agonists for dry eye and A1R agonists for supraventricular tachycardia. The use of P2X7R antagonists for the treatment of inflammatory diseases is promising, but the presence of polymorphic variations of this receptor is holding up the development of selective antagonists appropriate for each disease. P2X3R antagonists are in clinical trials for use against visceral pain, chronic cough and hypertension. A2AR agonists are in use for the treatment of PD, and perhaps soon in wider use. P2X1R antagonists are being investigated for treatment of bladder disorders and hypertension, while P2X4, P2X7 and A3R antagonists are being explored for neuropathic pain. Treatments with inhalation of ectonucleotidases to alter the balance of ATP and adenosine and inhibitors of ATP release, are also a therapeutic approach being explored. The development of novel purinergic compounds by medicinal chemists that are orally available and stable in vivo would be a significant advantage in developing therapeutic approaches, including centrally penetrant P2X7R antagonists (Clinical interventions involving purinergic signalling are just beginning. However, the beginning and future of purinergic compounds for the treatment of a wide range of diseases is described in this review. P2Yagonists .The majority of the therapeutic approaches for many heart disorders based on purinergic signalling manipulation are not fully understood yet and strategies to overcome the side-effects of treatment need to be considered. The pathophysiological roles of purinergic signalling in blood vessels are clearer and it plays an important role in controlling vascular tone and remodelling. Immunologic factors related to purinergic signalling are attracting more attention and should be considered . Human ein vivo (see Although still in its infancy, clinical use of purinergic compounds has started. Several relevant pharmacological interventions are currently in clinical use. The lack of more established purinergic therapies may be due to there being relatively few receptor subtype-specific agonists and antagonists that are both effective and stable vivo see . In someThe author confirms being the sole contributor of this work and approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer SS and handling Editor declared their shared affiliation.


Previous studies on influenza A(H1N1)pdm09 candidate vaccine viruses (CVVs) that had adapted to growth in embryonated chicken eggs by the acquisition of amino acid substitutions at HA positions 222 or 223 showed that improved protein yield could be conferred by additional amino acid substitutions in the haemagglutinin (HA) that arose naturally during passaging of the virus in eggs. In this study we investigated, by means of reverse genetics, the ability of a non-egg adapted (cell-like) A(H1N1)pdm09 virus to egg-adapt at HA loci other than 222/223, introducing amino acid substitutions previously identified as egg adaptations in pre-H1N1pdm09 H1N1 viruses and assessing their effect on protein yield and antigenicity. We also investigated the effect on the protein yield of these substitutions in viruses that had A(H1N1)pdm09 internal genes rather than the traditional PR8 internal genes of a CVV. The data show that a cell-like A/Christchurch/16/2010 can be egg-adapted via amino acid substitutions in at least three alternative HA loci , in viruses with either PR8 or A/California/7/2009 internal genes, but that the effects on protein yield vary depending on the amino acid substitution and the internal genes of the virus. Since CVVs need to produce high protein yields to be suitable for vaccine manufacture, the findings of this study will assist in the future characterisation of both wild type viruses and lab-derived CVVs for vaccine use. Influenza is an annual global threat to public health, mitigated by seasonal vaccination. Due to antigenic drift of influenza viruses, there is a need to continually assess features of the viruses causing disease and, when necessary, update the virus strain(s) being used to manufacture vaccine. Each new candidate vaccine virus (CVV) needs to be adapted to growth in embryonated hens’ eggs, whilst maintaining the appropriate antigenicity. Such egg-adapted viruses generally have amino acid substitutions around the receptor binding site of the haemagglutinin (HA) that can impact on viral attributes such as antigenicity and growth , 2.AH1N1)pdm09 viruses continue to circulate and are included in seasonal influenza vaccine. It is expected that H1N1 virus will undergo antigenic drift and new egg isolates will be required for inclusion in future seasonal vaccines. This study was conducted to investigate various substitutions that could lead to egg-adaptation of the H1N1 virus and the impact of these on antigenicity, growth of virus and yield of antigen. A comparison of HA structures showed that known egg adaptation loci of pre-H1N1pdm09 viruses map to the same positions around the receptor binding site in H1N1pdm09 viruses. In absence of other information on egg-adaptive mutations of H1N1pdm viruses, it was logical to assess the pre-2009 egg-adaptive sites. Therefore known substitutions that occurred during egg-adaptation of A(H1N1)pdm09 viruses N1pdm09 v,4 at thePaired viruses were generated with both PR8 or A/California/7/2009 internal genes and the effect of the HA substitutions on viruses resembling either laboratory derived CVVs or naturally occurring viruses assessed.All viruses were engineered from A/Christchurch/16/2010 (Chr/16) A(H1N1)pdm09, which is antigenically equivalent to A/California/7/2009 , the A(H1N1)pdm09 reference strain, despite differing from it at passage E2 by 9 amino acid residues in HA  and 6 residues in neuraminidase (NA). .−5 in PBS’A’ at 35°C for 72 hours. Allantoic fluid was harvested from chilled eggs and virus growth was assessed using the haemagglutination (HA) assay following standard protocols with 0.7% turkey RBC. Embryonated hens’ eggs used in this study were exempt from ethical approval as they were not within the final third of their incubation period  Act 1986 Amendment Regulations 2013).Viruses were grown in 10 or 11 day old embryonated hens’ eggs at a dilution of 102 for 72 hours. Cell medium was clarified at 1500rpm/5mins/4°C in a JS5.3 (Avanti) rotor and virus growth assessed by HA assay.Viruses were grown in MDCK cells, in MEM/1% L-glutamine/1.5% HEPES/2% bovine albumin and 2.5μg/ml TPCK trypsin (Worthington), according to standard protocols. Cells were incubated at 35°C/5%COMutations were introduced into the HA gene of A/Christchurch/16/2010 as shown in Viruses were derived using these HA genes and the NA gene from A/Christchurch/16/2010, using reverse genetics as previously described . A totalViruses were assayed in a haemagglutination inhibition (HI) assay according to standard protocols  using 0.Virus concentrates were prepared from batches of 30 eggs as described in . Final vTotal protein content of virus concentrates was assayed using a BCA assay kit (Pierce) according to manufacturer’s instructions.SDS-PAGE analysis including deglycosylation was carried out as previously described . QuantitSingle radial diffusion (SRD) assays were performed as previously described . Virus cInfectivity assays were performed as previously described . The infStatistical analysis was performed using Minitab 16 software. Analysis of variance (ANOVA) was performed using the general linear model with the Tukey method for pairwise comparisons and 95% confidence limits .This study uses H1N1)pdm09 numbering. Substitutions at HA residues 222 or 223 have been shown previously to be associated with egg adaptation and in non-egg adapted virus these positions are expected to be 222D, 223Q. The HA gene of wt egg adapted A/Christchurch/16/2010 (with egg adaptation 222N) was engineered to contain the ‘cell-like’ residue 222D N1pdm09 n and resiA panel of viruses with PR8 internal genes was assessed for the genetic stability of potential egg adaptive substitutions introduced into a DQ HA gene . VirusesWe concluded from these data that HA amino acid substitutions I216R, D187S and D187V were single-site egg adaptations compatible with the cell–like 222D/223Q motif. We also concluded that S190R may also be compatible with the cell like 222D/223Q motif and therefore should be included in the viruses created for yield studies.HA genes were constructed containing 222D/223Q plus amino acid substitutions identified from the genetic stability data. They were rescued along with A/Christchurch/16/2010 NA gene and the six internal genes of either PR8 or the A(H1N1)pdm09 reference virus A/California/7/2009 . In addiTotal viral protein yield, %HA content, mgs HA protein and mgs functional HA protein are shown in All viruses with a PR8 backbone had higher yield  than viruses with an A(H1N1)pdm09 backbone, irrespective of the HA sequence  between viruses with 222N/223Q but different sources of PR8 internal genes  by any measure of yield .There was no significant difference in HA content across the panel of viruses. Thus the HA yield  shows a similar trend to that of total protein yield although not all differences were statistically significant. NIBRG-259 had the highest yield (5.5mg HA/ml) and was statistically significantly different to NIBRG-257 and NIBRG-236 (~3.5mg HA/ml each). The yield of functional HA of PR8 backbone viruses, as measured by SRD , ranges from ~3.5mg HA/ml (NIB-74xp) to ~5.5mg HA/ml (NIBRG-259) with no significant differences between them. Their SRD HA yield however was significantly different to that of wt Chr/16 (0.5mg HA/ml). The data show that when PR8 internal genes are present, the yield of 222D/223Q viruses is equivalent to, or even higher than the 222N/223Q viruses .Viruses with A(H1N1)pdm09 derived internal genes gave much lower protein yields in all assays compared to viruses with PR8 internal genes, irrespective of the 222/223 motif or other HA substitutions  or Cal/7 backbone (NIBRG-260), are significantly different to each other (p<0.05) in total protein and HA yield , implying that the presence of different A(H1N1)pdm09 backbones affects yield.For HA protein yield, DQ viruses range from ~0.5mg/100 eggs (NIBRG-261) to 1.5mg/100 eggs (NIBRG-264); however, these four viruses are not statistically significantly different from Chr/16 (p>0.05). NIBRG-260 is significantly different from both wt Chr/16 and NIB-74xp (3.5mg). These data reflect those for total protein yield.The pattern of yield of functional HA protein for these viruses (9 to 109.5) between different HAs, whilst the titre for wt Chr/16 is 3 logs lower . Titres of all PR8 backbone viruses are not significantly different from each other but are significantly different to wt Chr/16 (p<0.05).Infectivity titres are shown in 6 for wt Chr/16  to 108.5 for NIBRG-260 and NIBRG-263 . Titres for Chr/16 and NIB-74xp are significantly different, reflecting their 3 log difference in infectivity, with each of them significantly different to all other viruses in the data set. Of the remaining viruses, NIBRG-260  is significantly different to NIBRG-262  and NIBRG-264 , but not to NIBRG-263  and NIBRG-261 , whilst NIBRG-262 is also significantly different to NIBRG-263. The data show that replacing the wt Chr/16 backbone with a Cal/7 backbone increases the infectivity of a ‘wt-like’ NQ virus, and that further replacement of the NQ motif with a DQ motif can result in equivocal infectivity depending on the additional egg adaptive amino acid substitution.Viruses with an A(H1N1)pdm09 backbone  show a wider variation in infectivity, from 10A Haemagglutination inhibition assay was used to assess potential antigenic changes conferred by the amino acid substitutions; all viruses had HI titres within twofold of the titres obtained with wt Chr/16 and so it was concluded that there were no antigenic differences .In the development of seasonal candidate vaccine viruses (CVVs) for egg vaccine production, viruses undergo natural selection in the egg although some of the amino acid substitutions selected for in eggs can affect the viruses’ antigenicity. However, the impact of egg-adaptive substitutions on yield, another important phenotype of CVVs, is not well understood. This study was designed to assess the impact of a variety of egg-adapting mutations, introduced rationally by reverse genetics, on the protein and HA yield, and the infectivity, of seasonal H1N1pdm09 CVV.Previous studies on A(H1N1)pdm09 viruses indicate HA substitutions D222N and Q223R to be egg-adapting changes with the DQ sequence being present in non-egg-adapted viruses. A/Christchurch/16/2010 is antigenically similar to A/California/07/2009, the current WHO recommended virus for vaccine, and the CVV NIB-74xp was prepared from it. For this study, the Chr/16 HA was engineered to contain the non-egg-adapted DQ motif, and substitutions at positions 222, 186, 187, 190 and 216 were introduced into the HA . We showA mutation previously shown to be responsible for improved virus yield in an H1N1pdm09 CVV, K119N, could not be rescued with a PR8 backbone without the virus gaining additional mutations in the HA, but could be obtained with a Cal/7 backbone; however, a virus with a K119N/S190R double substitution conferred egg adaptation with either set of internal genes . This deViruses with N129D or A186D required an initial cell passage prior to successful growth in eggs and probably egg adapted via a subsequent Q223R substitution. These findings concur with other studies where 129D and 186D were found in H1N1pdm09 viruses already egg adapted at 222/223 ,12 or 19The consistently higher protein and HA yields of all PR8 backbone viruses compared to wt Chr/16 was undoubtedly due to the inclusion of highly egg adapted PR8 internal genes, which is well established as the best way (to date) to improve the yield of wild type viruses for vaccine production in eggs ,15,16. TThe data presented here suggest that a D222N substitution in A(H1N1)pdm09 viruses impacts both egg adaptation and yield, since viruses with the same Cal/7 backbone and an alternative egg adaptation substitution generally had poorer yields. However, amongst the 222D/223Q viruses with Cal/07 backbones, the yield was variable and one virus  had similar yield to NIBRG-260 (NQ). This is possibly due to altered receptor binding involving residue 187; it has been reported that a DQ (non-egg adapted) motif preferentially binds α2,6 linked glycans mediated through residues 187D and 222D ,18,12,19The nature of the 187 residue is also important for yield, since only 187V (NIBRG-264) and not 187S (NIBRG-261) showed improved yield with a Cal/07 backbone. It has been reported that negatively charged residues in A(H1N1)pdm09 HA possibly decrease HA-sialic acid interaction and facilitate release of progeny for multiple cycle replication, which may increase the yield of virus ; howeverThis study has identified various HA residues in addition to 222 and 223 associated with the egg-adaptation of an H1N1pdm09 virus. These alternative residues cluster around the receptor binding site and include residues  identified in the egg adaptation of pre-2009 H1N1 viruses. Importantly, these egg-adapting substitutions do not affect the antigenicity of the virus. Egg-adapting substitutions reduce the HA content in viruses with A(H1N1)pdm09 internal genes although in many cases there is an overall increase in yield of total viral and HA protein. In viruses with PR8 internal genes, the effect of egg-adapting substitutions on yield is less pronounced. Nonetheless, data from this study will enhance the assessment of A(H1N1)pdm09 wild-type strains for CVV preparation by widening the choice of isolates that potentially have improved growth and yield in eggs. The virus with one particular substitution, I126R (NIBRG-259), outperformed all other viruses studied, even although the yield enhancement was not statistically significantly different from two other viruses. However, the absolute yield of total viral protein (12.5 mg/100 eggs) would have highlighted this virus as the best CVV of the viruses studied since during CVV development for seasonal vaccine, minimal  studies on protein and/or antigen yield are performed due to time constraints. Thus when a future drifted antigenic variant A(H1N1)pdm09 is identified for inclusion in vaccine, egg-adapted viruses with an I126R substitution should be considered as prime candidates for CVV development.


Further, the W452W149PB2 virus also induced the highest virus titers in lungs at all time points and the highest levels of inflammatory cytokine responses among all viruses tested. This high virulence phenotype was also confirmed by high viral titers in the respiratory tracts of infected ferrets. Further, a mini-genome assay revealed that W452W149PB2 has significantly increased polymerase activity (p < 0.001). Taken together, our study demonstrates that a single gene substitution from other avian influenza viruses can alter the pathogenicity of recent H5N8 viruses, and therefore emphasizes the need for intensive monitoring of reassortment events among co-circulating avian and mammalian viruses.Recently identified highly pathogenic avian influenza (HPAI) H5N8 viruses (clade 2.3.4.4) are relatively low to moderately pathogenic in mammalian hosts compared with HPAI H5N1 viruses. In this study, we generated reassortant viruses comprised of A/MD/Korea/W452/2014(H5N8) with substitution of individual genes from A/EM/Korea/W149/2006(H5N1) to understand the contribution of each viral gene to virulence in mammals. Substituting the PB2 gene segment or the NA gene segment of the H5N8 virus by that from the H5N1 virus resulted in significantly enhanced pathogenicity compared with the parental H5N8 virus in mice. Of note, substitution of the PB2 gene segment of the H5N8 virus by that from the H5N1 virus resulted in a 1000-fold increase in virulence for mice compared with the parental virus (MLD Since the mid-1990s highly pathogenic avian influenza (HPAI) A(H5N1) viruses have ravaged domestic poultry in Asia. The unprecedented spread of these viruses toward Africa and Europe since 2003 has given rise to at least 10 distinct phylogenetic clades based on the antigenicity of their hemagglutinin (HA) genes.A newly emerged, HPAI A(H5N8) virus caused poultry outbreaks in the Republic of Korea in mid-January 2014.Although HPAI H5N8 is highly pathogenic in chickens, it has thus far been extremely poor at inducing remarkable clinical illness in domestic ducks and ferrets, which may allow establishment of the virus in these hosts.50 of 105.8 EID50).Although most HPAI H5N1 viruses cause severe clinical symptoms and high mortality in experiment animal models including mice, the recent H5N8 viruses showed only moderate virulence in mice, similar to the 2009 pH1N1 viruses  virus were altered in mammals following reassortment events with the HPAI H5N1 (W149. Clade 2.2) virus, variants were plaque purified in Madin-Darby canine kidney (MDCK) cells following co-infection and were subsequently genotyped by RT-PCR using custom-designed gene-specific primers. Reassortants were then confirmed by full-length sequencing. The resulting plaque-purified reassortant viruses and selected single gene-reassortants used in this study are listed in 50) end point. Each reassortant virus containing the PB1, HA, NP, M, or NS gene of W452 showed similar growth properties to the parental W452 virus in embryonated chicken eggs  substitution virus (W452W149NA) exhibited reduced viral titers compared with the parental W452 virus (107 vs. 108.8 EID50/ml).To make the virus stock, each virus was propagated into 10-day-old embryonated chicken eggs and titered via the 50% egg infectious dose (EIDken eggs . Insertiin vitro growth properties of these viruses correspond with the pathogenic properties of H5N8 (W452) in mammalian hosts, their 50% mouse lethal doses were determined  , while all mice infected with parental H5N1 (W149) virus succumbed to death or were killed due to more than 25% weight loss and/or severe clinical signs within 8 dpi  (W149PB2 and W452W149NA) were included in the highly pathogenic group. Notably, mice infected with the W452W149PB2 virus experienced rapid weight loss of more than 25%, 100% mortality, and an MLD50 of 102.5 EID50, which is even lower than that of the wild-type H5N1(W149) virus (103.3 EID50 of MLD50)  in infected mice, in spite of the fact that it showed a reduced yield in embryonated eggs (107.0 EID50/ml). Reassortant viruses including W452W149PA and W452W149HA induced body weight loss and mortality in a manner similar to the wild-type H5N8 virus with moderate virulence (ranging from 105.7 to 105.8 EID50 of MLD50)  (To verify whether the different termined . In addi3 EID50) . For the3 EID50) , (ii) mo3 EID50)  and (iii3 EID50) . To compf MLD50) . The W45f MLD50) , Table 15 EID50) , Table 1W149PB2 virus peaked at 3 dpi (106.7 EID50/g) and was at least as high as that of the H5N1 (W149) virus (W149NA virus showed a low (104.8 EID50) MLD50  virus . Althoug0) MLD50 , the lun0) MLD50  and B. Til 9 dpi . In addiil 9 dpi . In contil 9 dpi .Figure W149PB2 virus was detected in all organs tested. These results clearly demonstrate that the PB2 gene from the HPAI H5N1 (W149) virus substantially alters virus replication and expand the tissue tropism in the infected host.Multiple organ failure is a typical feature of HPAI H5N1-infected mice and is the cause of the high mortality rate.W149PB2, and W452W149NA) compared with the other 2 groups (W149PA and W452W149HA) induced low to intermediate expression of cytokines with relatively delayed kinetics (5 to 7 dpi) with the expression levels gradually increasing up to 7 dpi  showed low levels of cytokine expression compared with W452 at 5 dpi  from 3 dpi until death . Moreover, the expression of IL-15 and MIP1β were highest at 3 dpi in the highly pathogenic viruses while the moderately and low-pathogenic virus-infected mice showed mildly elevated to normal expression levels . Further, expression of IL-2, IL-5, and IL-13 showed increases during the virus infections in each group . Together these data suggest that elevated expression of pro-inflammatory cytokines and chemokines is closely associated with the high virulence of the W452W149PB2 and W452W149NA reassortants.To elucidate the relationship between cytokine production and viral pathogenicity, we measured induction of cytokines in the bronchoalveolar lavage BAL) fluid of infected mice at 1, 3, 5, 7, and 9 dpi  showed histopathological damage, including serious exudate-filled alveolar spaces (W149NA demonstrated the most extensive infiltration of inflammatory cells (W149PA and W452W149HA) demonstrated inflammation in limited areas of the lungs , and normal human bronchial epithelial (NHBE) cells (W149PB2 viruses showed at least 100-fold higher mean peak titers (p < 0.05) than W452 in all infected cells. In MDCK cells, W452W149PB2, and to a lesser extent the viruses possessing the PA or HA of W149 replicated more efficiently than parental W452 virus  (To examine the E) cells . Viral tk titer) A-C.FiguW149PB2, and W452W149PAviruses reached higher mean peak titers (106.5 to 109.25 EID50/ml) than the W452 virus (105.25 EID50/ml) at 72 hours post infection (hpi) (W149HA and W452W149NA demonstrated similar peak titers to W452 (P>0.05), and the other viruses showed reduced mean peak titers (102.75 to 4.25 EID50/ml) (W149NA demonstrated higher titers (103.5 EID50/ml) at 24 h p.i. Overall, these results demonstrate that in the W452 backbone a single gene substitution from W149 can alter the in vitro growth characteristics of H5N8 (W452) virus in the 3 mammalian cell lines examined.In 2 systems of human cells , these viruses demonstrated delayed and reduced titers compared with those of MDCK cells D to I. Ion (hpi) D-E. MoreID50/ml) F. Viral ID50/ml)  G-I. IntW149PB2, W452W149HA, and W452W149NA viruses demonstrated increased virulence with low MLD50 compared with the other reassortant viruses. Since the ferret is recognized as the most suitable host for influenza virus studies, the W452W149PB2, W452W149HA and W452W149NA viruses were intranasally inoculated into groups (n = 15) of ferrets at 106 EID50 to investigate whether these viruses could efficiently replicate and be pathogenic in this model. One day after virus infection, 3 naïve ferrets were added into cages adjacent to infected animals to evaluate any possible indirect droplet transmission. For comparison, we also tested W149 and W452 viruses. Nasal washes from each group of ferrets were collected at 1, 3, 5, and 7 dpi for virus titration. In addition, 3 ferrets from each group were killed on 1, 3, 5, and 7 dpi (3 ferrets/dpi) to assess the viral titers and tissue distribution of the virus.In mice the W4525.8 EID50/Swab at 5 dpi, while the W452 virus showed the highest virus mean peak titer at 1 dpi (103.1 EID50/Swab), which gradually decreased until 7 dpi (only one ferret showed a titer of 10l.5 EID50/Swab(1/3))  with levels at 7 dpi of 102.8 EID50/Swab )  and B. Fab(1/3))  and G. ID50/Swab . FurtherD50/Swab  and G. H52 virus  and E. Fecovered  and GFiNone of the nasal washes from contact ferrets in any group were positive for virus during the duration of experiment (18 days) . MoreoveW149PB2, W452W149HA, and W452W149NA viruses could also influence virus replication properties outside of the respiratory organs. In agreement with the previous study, the H5N1(W149) virus was recovered from organs outside the respiratory tract, such as spleen, kidney and intestine  with comparable or decreased titers compared with W452 virus  viruses without any genetic incompatibility since all 8 combinations of single-gene reassortants were selected from virus co-infections of MDCK cells by plaque purificationAn outbreak of novel reassortant HPAI H5N8 virus, which is a member of clade 2.3.4.4, was reported in 2014 in South Korea and subsequently spread into nations in Asia (China and Japan), Europe , and North America (Canada and the United States).in vitro, increased virulence, an expanded range of tissue tropism, and in vivo growth properties at levels similar to those of H5N1(W149). This was especially true of the H5N8 (W452) virus containing the PB2 gene from W149 virus. In fact, the W452W149PB2 virus showed 1,000-fold higher virulence than W452 virus  and 10–100 fold higher viral lung titers at all-time points in mice with systemic infection. Reciprocally, the W149W452PB2 virus showed more than 10-fold attenuation in virulence compared with W149 virus   . Many stW149NA, showed relatively low viral titers in eggs (10–100 fold lower than the other reassortant) and viral growth, but was categorized into the highly pathogenic group due to the low MLD50 (4.8 log10EID50). Further study will be necessary to verify this, but it appears likely that HPAI H5 viruses belonging to clade 2.3.4.4 may begin to switch their neuraminidase gene with that of other viruses to obtain one more suitable for their HA and NA balance, such as H5N8.In China, an H5N1 inactivated vaccine has been compulsorily used to control widespread HPAI H5N1 throughout the country.cytokine storm and is followed by massive innate immune responses associated with acute respiratory distress syndrome, a typical medical condition of highly pathogenic influenza infection that results in high morbidity and mortality.The unbalanced production of inflammatory cytokines and chemokines observed during influenza virus infections has been suggested to be a mechanism of pathogenesis for these viruses.W149PB2 virus in mammalian hosts, we performed ferret infection and indirect transmission studies comparing these to wild-type W149 and W452 viruses. Similar to the results of the mouse study, the W452W149PB2 virus exhibited increased virus titers in nasal washes compared with the W452 virus; however, there was no evidence of aerosol transmission to naive contact ferrets (W149PB2 virus induced significantly high and prolonged virus titers in infected ferrets as well as wide organ distribution compared with the W452 virus (W149PB2 and W452 viruses (HA clade 2.3.4.4) were (101.2 to 102.8 EID50/g)  suggesting that such a reassortant is a potential threat to public health (To verify the increased virulence and growth properties of the W452 ferrets . Further52 virus . InteresEID50/g) . These rc health .Trojan horse and mixing vessel for the creation of novel reassortants with pandemic potential. Furthermore, the rapid and wide spread of H5N8 virus to Asia and Europe and the first outbreak of these viruses in North America has the potential for the creation of novel viruses with newly-introduced genes.The current study focused on the investigation of virulence properties of the reassortant H5N8 viruses in mice and ferrets, which are appropriate models for the evaluation of viral pathogenesis.50 end point. The handling of viruses was performed in an enhanced biosafety level 3 (BSL3) containment laboratory as approved by the Korean Centers for Disease Control and Prevention (KCDC-14–3–07).To obtain reassortant viruses containing single gene changes from A/EM/Korea/W149/06[W149(H5N1)] into the A/MD/Korea/W452/2014[W452(H5N8)] backbone, the 2 viruses were mixed (1:3 ratio) and was used to infect MDCK  cells, and then plaque purification was conducted. Briefly, a monolayer of MDCK cells in a 6-well plate were infected with serially diluted viruses for 1 hour, washed twice with phosphate buffered saline  and overlaid with a 0.7% agarose-EMEM media with or without L-1-tosylamide-2-phenylmethyl chloromethyl ketone (TPCK)-treated trypsin . After 3 d of incubation, plaque colonies were picked and resuspended in medium and injected into 10-day-old embryonated chicken eggs . After 30 hours of incubation, viruses were harvested and full-length gene segments sequenced to confirm the origin of each segment. Confirmed viruses were stored at -80°C and titrated via the EID50) of each virus, 8-week-old female BALB/c mice  for each virus were anaesthetized and inoculated intranasally with 10-fold serially diluted virus in a 30 ul volume. The mice were monitored daily for 14 d and checked for changes in body weight. The infected mice were killed if they lost more than 25% of their initial body weight.To determine the 50% mouse lethal dose . Tissues were homogenized with an equal volume (ml/g) of PBS containing antibiotics . Homogenates were cleared by centrifugation, and then viral titers in the 10-fold serial dilutions of supernatants were measured by determining the EID50. The remaining mice were used for H&E staining at 6 dpi.Additionally, groups of mice (n = 38 per group) were inoculated intranasally with 10BAL fluid from mouse lungs was collected at 1, 3, 5, 7, and 9 d after each virus infection or from the uninfected control group. The BAL solution was then centrifuged at 12,000 rpm for 5 min at 4°C and stored at −80°C until the analysis.A multiplex biometric immunoassay was adopted for cytokine measurement according to the manufacturer's instructions , The concentrations of TNF-α, lL-1β, IL-6, IL-12p70, IL-15/IL15R, INF-γ, IL-18, IL-2, IL-5, IL-13, GM-CSF, and MIP-1β were measured. Briefly, BAL fluid samples were incubated with antibody-coupled beads corresponding to the various cytokines. Complexes were washed, then incubated with biotin-conjugated secondary antibody, followed by another wash and incubation with phycoerythrin reporter conjugated-streptavidin. Assays were measured by detection of the fluorescent reporter signal and identification of beads on a Bio-Plex 200 system and data were analyzed using the associated Bio-Plex Manager software version 4.1.1 .Luciferase mini-genome reporter assays were performed using a luciferase reporter plasmid (pHW72-Luci) containing the influenza A virus M – driven noncoding region.50.MDCK, A549  and NHBE cells were cultured in 6-well plates and then infected with reassortant viruses at a multiplicity of infection (MOI) of 0.01 or 0.1 for 1 hour, washed and incubated at 35°C in a 5% CO2 atmosphere in the presence of TPCK-treated trypsin (2 ug/ml). Cell culture supernatants were collected at 12, 24, 48, and 72 hpi and virus titers were determined by EID6.0 EID50 of W452, W149 and W452W149PB2 viruses under anesthesia. Three ferrets per group were killed at 1, 3, 5, and 7 dpi for virological and pathogenesis experiments. After 24 h, naïve ferrets (n = 3) were co-housed in the other half of a cage  with a ferret in each inoculated group, with the ferrets only separated by 2 stainless steel grids allowing for airborne virus transmission. Ferret body weight and temperature were monitored every other day until 7 dpi. Nasal washes were collected every other day from the inoculated ferrets and daily from 1 day post-exposure from the contact ferrets until 18 d post contact (dpc) and blood was collected from contact groups at 18 dpc. Virus titrations of nasal washes and various tissue organs were determined by EID50 assays.Female ferrets, 18- to 20- weeks old and sero-negative for influenza A viruses (ID Bio Corporation) (n = 15/group), were intranasally inoculated with 10W149PB2 W452W149HA, and W452W149NA viruses were harvested at 6 dpi or 5 dpi, respectively. The samples were fixed in 10% neutral-buffered formalin and embedded in paraffin. Histological assessment was conducted using standard hematoxylin and eosin staining and light microscopy (magnification x400) or stained with NP polyclonal antibodies. The slides were viewed using an Olympus η 71  microscope and DP controller software to capture images.Mouse lungs infected with each virus and ferret lungs infected with W452, W149, W452p < 0.05, **or †† indicates ** p < 0.01, and *** or ††† indicates p < 0.001). All statistical analyses were performed with using GraphPad Prism version 5.00 for Windows .To assess significant differences in viral titers and cytokine expression levels between groups or across time points W452-infected samples were compared. Asterisks indicate the statistical significance between W452 and other viruses determined by One-way ANOVA and subsequent Dunnett's test. Further, daggers indicate the statistical significance across time points. * or † indicates All animal experiments, including use of embryonated chicken eggs, were approved by the Medical Research Institute, a member of Laboratory Animal Research Center of Chungbuk National University (LARC)  and were conducted in strict accordance and adherence to relevant policies regarding animal handling as mandated under the Guidelines for Animal Use and Care of the Korea Center for Disease Control (K-CDC).


NKG2DL-expressing T cells were strikingly enriched within HBV-infected livers compared with the periphery or to healthy livers (p < 0.001). NKG2D+NK cells were also increased and preferentially activated in the HBV-infected liver (p < 0.001), in direct proportion to the percentage of MICA/B-expressing CD4 T cells colocated within freshly isolated liver tissue (p < 0.001). This suggests that NKG2DL induced on T cells within a diseased organ can calibrate NKG2D-dependent activation of local NK cells; furthermore, NKG2D blockade could rescue HBV-specific and MICA/B-expressing T cells from HBV-infected livers. To our knowledge, this is the first ex vivo demonstration that non-virally infected human T cells can express NKG2DL, with implications for stress surveillance by the large number of NKG2D-expressing NK cells sequestered in the liver.NK cells, which are highly enriched in the liver, are potent regulators of antiviral T cells and immunopathology in persistent viral infection. We investigated the role of the NKG2D axis in T cell/NK cell interactions in hepatitis B. Activated and hepatitis B virus (HBV)–specific T cells, particularly the CD4 fraction, expressed NKG2D ligands (NKG2DL), which were not found on T cells from healthy controls ( Natural killer cells are well known for their capacity to kill virally infected and transformed cells, but also have potent regulatory capacity –3. In pa+TbetloEomeshi NK cells residing in human liver that are not present in the circulation  patients recruited from the Mortimer Market Clinic , the Royal Free and University College London hospitals and the Royal London Hospital . A total of 46 healthy control blood samples were obtained from staff or students at University College London. Surplus liver tissue was obtained from 36 CHB patients undergoing diagnostic liver biopsies. Healthy liver remote from the tumor site was obtained from 11 non-HBV infected patients undergoing tumor resection for colorectal metastases. Nine transplant perfusates from cadaveric donor livers were collected during liver transplant surgery under the standard graft preparation protocols. The study was approved by the relevant ethical review boards and informed consent was obtained in writing. All CHB patients had detectable hepatitis B surface Ag and DNA for >6 mo, were anti-hepatitis C virus, anti-δ virus and anti-HIV–Ab negative and treatment naive. Liver inflammation was determined by serum alanine transaminase (ALT). Details of the clinical characteristics of all HBV patients are included in PBMC isolated from whole blood by standard gradient centrifugation on Ficoll-Paque Plus . Intrahepatic lymphocytes (IHL) were obtained following gentle mechanical disruption of liver tissue prior to passing through a 70 μm cell strainer (BD Biosciences) and multiple washes with RPMI 1640 (Invitrogen) as previously described , 20. IHLFor phenotypic analysis, PBMC and IHL were washed with PBS and stained with a fixable Live/Dead blue dye (Invitrogen) at 4°C for 10 min, prior to Fc receptor blocking with FcR blocking reagent . Surface staining was performed at 4°C for 30 min in the presence of saturating concentrations of mAbs, or isotype matched controls. The following Abs were used for the sequential gating strategy : CD3-PE-A 2B4 cell line carrying GFP–conjugated NF-AT transfected with the human NKG2D/DAP10-CD3ζ complex as previously described  was usedNK cells and CD4 T cells were isolated from PBMC (>95% purity and viability)  according to manufacturer’s instructions.2O2 solution  at varying concentrations (0.25–1 mM) for 0.5–2 h, followed by surface staining for NKG2DL as described above.The presence of NKG2DL on PBMC or isolated CD4 T cells from CHB patients was investigated in cocultures with GFP reporter cells. Cells were cocultured with 2B4 transfectants overnight in RPMI 1640 containing 5% FCS, and GFP expression analyzed by flow cytometry. Purified CD4 T cells or PBMC from healthy controls were rested for 48 h prior to the addition of HTo measure NK cell capacity to degranulate, purified NK cells were cocultured at 1:1 ratio with target cells (C1R/C1R-MICA) at 37°C for 6 h, in the presence of anti-CD107a-APC (BD Biosciences). Brefeldin A  and Monensin  were added after 1 h. Where indicated, an anti-NKG2D blocking Ab or control Ab mAb  was added at the onset of culture. NKG2D blockade did not affect NK cell viability.PBMC were stimulated with HBV overlapping peptides (OLP)  in the presence of 40 IU IL-2 (Miltenyi Biotec) in RPMI 1640 complete medium for 10 d at 37°C. IL-2 and medium were refreshed on day 4 of culture. On day 9, PBMC were restimulated with 1 μg/ml OLP overnight, in the presence of Brefeldin A (1 μg/ml). Virus-specific T cells were identified via ICS for corresponding IFN-γ production. To examine the effect of blocking NKG2D on virus specific CD4 T cells, NKG2D blocking  or an isotype-matched control mAb were added with peptide at onset of culture and cells were treated as described above.For overnight stimulation of PBMC or IHL, 10 μg/ml HBV OLPs were added and the cells were incubated at 37°C in the presence of Brefeldin A (1 μg/ml) (added after 1 h). HBV-specific T cells were identified by ICS for IFN-γ.U test (for two groups), the Wilcoxon signed rank test (for two paired groups), Kruskal–Wallis (for >2 non-paired groups) or Friedman (for >2 paired groups) one-way tests were used as appropriate. Correlations between variables were analyzed using Spearman’s rank correlation coefficient (r). A p value < 0.05 was considered significant for all tests. All figures are marked as follows: *p < 0.05, **p < 0.005, ***p < 0.001.The non-parametric Mann–Whitney +NK cells in the HBV-infected liver. To investigate NKG2DL expression, CD4 and CD8 T cells from healthy controls and a well-characterized cohort of patients with CHB  fraction compared with HLA-DR− T cells, particularly in the CD4 compartment  T cells within the NKG2DL-expressing fraction  failed to reproducibly induce NKG2DL on T cells (data not shown).Previous reports have shown upregulation of NKG2DL following in vitro induction of T cell proliferation . Howeverfraction . Reactivfraction , 42 and fraction , and arefraction , 45. A pcontrols  and to acontrols . The H2Od ULBP-1 , mimicki+ versus HLA-DR− CD4 and CD8, +) fraction, MICA/B was expressed on more CD4 than CD8 T cells and on more intrahepatic than peripheral T cells . We observed a striking increase in the proportion of T cells expressing MICA/B directly ex vivo from the liver compared with the periphery of patients with CHB. By contrast, intrahepatic T cells from control livers had low expression of MICA/B , 2B. The T cells , consist− T cells  than peripheral NK cells in CHB, and was at a similarly high level on NK cells in healthy livers  in CHB than healthy donors . The NKG2Dy donors . NKG2D+Nl livers , 5C.+NK cells in the HBV-infected liver could reflect activation by NKG2DL-expressing cells, we initially employed an in vitro system. A lymphoblastoid cell line transfected with MICA*008  NKG2D+NK cells was seen in the periphery, and even more robustly within the liver (r = 0.72, p = 0.0003, To further probe the link between NKG2DL-expressing T cells and NK cell activation, we quantitated these two parameters in parallel using direct ex vivo analysis of blood and liver biopsy tissue from 20 patients with CHB. A highly significant correlation between the percentage of CD4 T cells expressing MICA/B and the proportion of activated (HLA-DRTaken together, our analysis of tissue samples from the site of HBV pathogenesis revealed enhanced NKG2D expression and selective activation of NKG2D-expressing NK cells, in association with induction of NKG2DL on liver-infiltrating CD4 T cells. Their close ex vivo correlations suggest NKG2DL-expressing CD4 T cells may be able to calibrate NK cell activation.To our knowledge, in this study we provide the first direct ex vivo evidence that uninfected human T cells are capable of expressing selective NKG2DL, and suggest that this allows them to engage in cross-talk with NKG2D-expressing NK cells. We demonstrate that the NKG2DL MICA/B is preferentially expressed on activated and HBV-specific CD4 T cells, particularly those within the HBV-infected liver. NKG2D expression is increased on NK cells from the HBV-infected liver and their enhanced activation correlates with the proportion of intrahepatic CD4 T cells expressing MICA/B. Our data reveal the novel paradigm of NKG2DL-expressing T cells sequestered in diseased livers; this raises the possibility that they will be enriched among tissue-resident populations, and contribute to organ-specific pathogenesis in other settings.There are eight different NKG2DL in humans, which also exhibit high allelic polymorphism, providing an intricate system for signaling cellular stress and fine-tuning the response of NKG2D-bearing cells . We obseIn terms of types of triggers of cellular stress, mitogen- or Ag-driven activation and proliferation have been shown to induce NKG2DL on human T cells in vitro , which iNKG2D-expressing NK cells can be sensitive to relatively small changes in target cell MICA/B expression . This waThe NKG2D receptor on NK cells is primarily known for its role in the recognition of transformed and infected cells; in this study we have demonstrated its capacity to act as a sensitive detector of T cell stress in the liver. Although previous studies have shown that engagement by either cell-bound , 58 or s+ NK cells is reminiscent of our previous finding of increased TRAIL+ NK cells and TRAIL-R2+ T cells colocated within the intrahepatic compartment (Our finding that NKG2D blockade of liver-infiltrating lymphocytes rescued HBV-specific and MICA/B-expressing CD4 but not CD8 T cells is in line with the fact that MICA/B was expressed at significantly higher levels on global, activated and HBV-specific CD4 than CD8 cells within the liver; however, it may also be attributable to CD8 T cells possessing additional mechanisms to protect them from NKG2D-mediated NK cell killing . Our finpartment . This copartment ] or the partment . NK cellpartment , 61, theData Supplement


The United States 2017–18 influenza season  was a high severity season with high levels of outpatient clinic and emergency department visits for influenza-like illness (ILI), high influenza-related hospitalization rates, and elevated and geographically widespread influenza activity across the country for an extended period. Nationally, ILI activity began increasing in November, reaching an extended period of high activity during January–February, and remaining elevated through March. Influenza A(H3N2) viruses predominated through February and were predominant overall for the season; influenza B viruses predominated from March onward. This report summarizes U.S. influenza activityhttps://stacks.cdc.gov/view/cdc/54973), including 151,413 (67.6%) for influenza A and 72,700 (32.4%) for influenza B. Nationally, the percentage of clinical laboratory–tested specimens positive for influenza virus peaked for 5 consecutive weeks during January 13–February 10 (surveillance weeks 2–6) (range = 26.1%–26.9%). Regionally,CDC receives influenza test results from public health and clinical laboratories located in all 50 states, Puerto Rico, and the District of Columbia through U.S. World Health Organization (WHO) Collaborating Laboratories and the National Respiratory and Enteric Virus Surveillance System (NREVSS). During October 1, 2017–May 19, 2018, clinical laboratories tested 1,210,053 specimens for influenza virus; 224,113 (18.5%) tested positive . Among the 37,681 seasonal influenza A viruses subtyped, 31,977 (84.9%) were influenza A(H3N2), and 5,704 (15.1%) were influenza A(H1N1)pdm09. Influenza B lineage information was available for 11,950 (77.2%) influenza B viruses; 10,612 (88.8%) were B/Yamagata lineage, and 1,338 (11.2%) were B/Victoria lineage. Whereas influenza A(H3N2) viruses accounted for the majority of circulating viruses, the proportion of influenza A viruses subtyped as A(H1N1)pdm09 ranged regionally from 9.0% in the central United States to approximately 24% in the northwestern and southeastern United States. Influenza B viruses were more commonly reported than were influenza A viruses from early March to late May (weeks 9–20). The proportion of influenza B viruses reported regionally ranged from 23.0% in the Midwest to 40.6% in the northwestern United States.Public health laboratories tested 98,446 specimens during October 1, 2017–May 19, 2018; 53,790 (54.6%) were positive for influenza viruses, including 38,303 (71.2%) positive for influenza A and 15,487 (28.8%) for influenza B  patients who tested positive for seasonal influenza virus by public health laboratories and for whom age data were available, 3,802 (8.1%) were aged 0–4 years; 11,550 (24.5%), 5–24 years; 15,597 (33.1%), 25–64 years; and 16,172 (34.3%), ≥65 years. Influenza A(H3N2) viruses predominated among all age groups, ranging from 51.2% of viruses among persons aged 5–24 years to 70.0% among persons aged ≥65 years. The largest proportion of reported influenza B viruses (36.5%) occurred in persons aged 5–24 years.Public health laboratories participating as U.S. WHO collaborating laboratories submit a subset of influenza-positive respiratory specimens to CDC for virus characterization through three National Influenza Reference Centers in the California, New York, and Wisconsin state public health laboratories. CDC characterizes influenza viruses through genomic sequencing and antigenic characterization . This process evaluates whether genetic changes in circulating viruses have led to antigenic drift away from the vaccine reference virus.Influenza-positive specimens are sequenced using next-generation sequencing (NGS)CDC evaluates the antigenic similarityCDC has genetically characterized 3,329 influenza viruses collected since October 1, 2017, including 832 influenza A(H1N1)pdm09 viruses, 1,313 influenza A(H3N2) viruses, and 1,184 influenza B viruses. A subset of these viruses was also antigenically characterized.https://stacks.cdc.gov/view/cdc/54975). This has been the predominant HA clade in the United States since the 2015–16 season  gene segments from 832 A(H1N1)pdm09 viruses collected since October 1, 2017, showed that all viruses belonged to subclade 6B.1 , with 3C.2a predominating. Viruses with the 3C.2a HA emerged at the end of the 2013–14 season and have remained the predominant clade since the 2014–15 season  viruses were sequenced, and phylogenetic analysis of the HA gene segments indicated that multiple clades/subclades were cocirculating , which also predominated in the 2016–17 season  viruses had a six-nucleotide deletion in the HA gene segment (encoding amino acids 162 and 163). Viruses like these, previously abbreviated as V1A-2Del and now designated as V1A.1, were first reported during the 2016–17 season . Among 1,147 influenza A(H1N1)pdm09 viruses tested for oseltamivir and peramivir susceptibility, 11 (1.0%) were resistant to both drugs and contain a known marker of resistance in the neuraminidase gene segment (H275Y). Among 786 influenza A(H1N1)pdm09 viruses also tested for zanamivir susceptibility, no resistant viruses were detected. All 2,354 influenza A(H3N2) viruses tested for oseltamivir and zanamivir susceptibility were susceptible to both medications. No peramivir-resistant viruses were detected among 1,248 A(H3N2) viruses tested. All 1,118 influenza B viruses tested were susceptible to all three medications. High levels of resistance to the adamantanes (amantadine and rimantadine) persist among influenza A viruses. Adamantane drugs are not recommended for use against influenza at this time.The Food and Drug Administration’s Vaccines and Related Biologic Products Advisory Committee recommended that the 2018–19 trivalent vaccine to be used in the United States contain an A/Michigan/45/2015 A(H1N1)pdm09-like virus, an A/Singapore/INFIMH-16–0019/2016 A(H3N2)-like virus, and a B/Colorado/06/2017-like (B/Victoria lineage) virus ,Complete medical chart abstraction data in FluSurv-NET will not be finalized until later in 2018; however, as of June 1, 2018, data were available for 7,584 (24.9%) hospitalized adults and children with laboratory-confirmed influenza. Among 6,910 hospitalized adults with information on underlying medical conditions, 6,385 (92.4%) had at least one reported underlying medical condition that placed them at high riskCDC tracks pneumonia and influenza (P&I)–attributed deaths through CDC’s National Center for Health Statistics (NCHS) Mortality Reporting System. The percentages of deaths attributed to P&I are released 2 weeks after the week of death to allow for collection of sufficient data to produce a stable P&I mortality percentage. During the 2017–18 season, based on data from NCHS, the proportion of deaths attributed to P&I was at or above the epidemic thresholdhttps://www.cdc.gov/flu/professionals/classifies-flu-severity.htm). These ITs help assess the historic chance that surveillance system data will exceed a certain threshold. CDC then classifies the severity of the current influenza season by determining which IT was crossed by at least two of the peak values from the above indicators. Based on this method, the severity of the 2017–18 season was classified as high severity overall and high severity for each age group . This is the first time that each age group was classified as high in the same season, in a retrospective analysis going back to the 2003–04 season  the percentage of visits to outpatient clinics for ILI from ILINet, 2) the rates of influenza-associated hospitalizations from FluSurv-Net, and 3) the percentage of deaths resulting from pneumonia or influenza from NCHS  were associated with infection with an influenza A(H3N2) virus, 31 (18%) with an influenza A(H1N1)pdm09 virus, 36 (21%) with an influenza A virus for which no subtyping was performed, 64 (37%) with an influenza B virus, two (1%) with an influenza A and B coinfection, and two (1%) with an influenza virus for which the type was not determined. The mean age of the pediatric deaths reported this season was 7.1 years (range = 8 weeks–17 years); 97 (57%) children died after admission to the hospital. Among the 154 children with a known medical history, 79 (51%) had at least one underlying medical condition recognized by the Advisory Committee on Immunization Practices (ACIP) as placing them at high risk for influenza-related complications. Among the 138 children who were eligible for influenza vaccination (age ≥6 months at date of onset) and for whom vaccination status was known, 30 (22%) had received at least 1 dose of influenza vaccine before illness onset . Since influenza-associated pediatric mortality became a nationally notifiable condition in 2004, the total number of influenza-associated pediatric deaths per season has ranged from 37 during the 2011–12 season to 171 during the 2012–13 season, excluding the 2009 pandemic, during which 358 pediatric deaths were reported to CDC during April 15, 2009–October 2, 2010.The 2017–18 influenza season was a high severity, A(H3N2)-predominant season. In 2017, CDC began using a new methodology to classify seasonal severity and applied the methodology to the 2003–04 through 2016–17 seasons. The 2017–18 season is the third overall high severity season since 2003–04 and the first classified as high severity for all age groups  was the predominant subtype circulating, there was substantial diversity in type and subtype of influenza infections leading to death in children. Less than one fourth (22%) of vaccine-eligible children who died from influenza this season had received influenza vaccine before illness onset.Analysis of the influenza A(H3N2), A(H1N1)pdm09, and B/Yamagata lineage viruses showed that circulating viruses were antigenically similar to the cell-grown reference viruses representing the 2017–18 Northern Hemisphere influenza vaccine viruses. The majority of U.S.-produced influenza vaccines use egg-based manufacturing and viruses adapted for growth in eggs. Amino acid changes in these egg-adapted viruses might contribute to differences in antigenicity from circulating viruses. Although this can occur in all types/subtypes, it was most evident in circulating A(H3N2) viruses, where half showed reduced inhibition by antisera to the egg-adapted vaccine reference virus. Whereas the overall number of circulating B/Victoria viruses was low, a substantial amount of antigenic drift from the vaccine reference virus B/Brisbane/60/2008 was observed.Interim estimates of the effectiveness of the 2017–18 inactivated influenza vaccines against medically attended respiratory illness published in February 2018 were 36%  overall, 25% (CI = 13%–36%) against illness caused by influenza A(H3N2) viruses, 67% (CI = 54%–76%) against illness caused by influenza A(H1N1)pdm09, and 42% (CI = 25%–56%) against illness caused by influenza B viruses -predominant seasons have also been associated with increased hospitalizations and deaths; however, the 2017–18 season followed an A(H3N2)-predominant season, and all severity indicators were higher than during the 2016–17 season. The majority of A(H3N2) viruses were genetically characterized as 3C.2a clade, similar, but genetically distinct from the 3C.2a1 subclade that predominated during the 2016–17 season, and from the viruses that circulated during Australia’s 2017 influenza season . Additional information regarding influenza viruses, influenza surveillance, influenza vaccine, influenza antiviral medications, and novel influenza A infections in humans is available online (https://www.cdc.gov/flu).Influenza surveillance reports for the United States are posted online weekly -predominant season. Influenza activity indicators were notable for the volume and intensity of influenza cases that occurred in most of the country at the same time. Record hospitalization rates and high numbers of influenza-associated pediatric deaths also were reported.Receiving a seasonal flu vaccine each year remains the best way to protect against seasonal influenza and its potentially severe consequences. Testing for seasonal influenza viruses and monitoring for novel influenza A virus infections should continue year-round.