Two metabolites (nFFA and an N‐oxide or a hydroxylamine) were detected in human liver microsomes versus six in dog and seven in rat liver microsomes; no metabolite was unique to humans. Selective CYP inhibitor studies showed FFA metabolism partially inhibited by quinidine , phencyclidine , and furafylline  and, to a lesser extent (<15%), by tienilic acid (CYP2C9), esomeprazole (CYP2C19), and troleandomycin (CYP3A4/5). Incubation of nFFA with rCYP1A2, rCYP2B6, rCYP2C19, and rCYP2D6 resulted in 10%−20% metabolism and no clear inhibition of nFFA metabolism by any CYP‐selective inhibitor. Reaction phenotyping showed metabolism of FFA by recombinant human cytochrome P450 (rCYP) enzymes rCYP2B6 , rCYP1A2 (22%−23%), rCYP2C19 (49%−50%), and rCYP2D6 (59%−97%). Neither FFA nor nFFA was a drug transporter substrate. Results show FFA metabolism to nFFA occurs through multiple pathways of elimination. FFA dose adjustments may be needed when administered with strong inhibitors or inducers of multiple enzymes involved in FFA metabolism .Fenfluramine (FFA) has potent antiseizure activity in severe, pharmacoresistant childhood‐onset developmental and epileptic encephalopathies . To assess risk of drug interaction affecting pharmacokinetics of FFA and its major metabolite, norfenfluramine (nFFA), we conducted in vitro metabolite characterization, reaction phenotyping, and drug transporter−mediated cellular uptake studies. FFA showed low in vitro clearance in human liver S9 fractions and in intestinal S9 fractions in all three species tested (t Reaction phenotyping showed metabolism of the antiseizure medication fenfluramine by recombinant human cytochrome P450 (rCYP) enzymes rCYP2B6, rCYP1A2, rCYP2C19, and rCYP2D6. Fenfluramine dose may need to be adjusted when co‐administered with strong inhibitors or inducers of these enzymes . ASMantiseizure medicationAUCarea under the concentration curveBCAbicinchoninic acidBCRPbreast cancer resistance proteinCDDCDKL5 deficiency disorderCIDcollision‐induced dissociationintCLintrinsic clearancemaxCmaximum drug concentrationCYPcytochrome P450DDIdrug‐drug interactionDSDravet syndromeEDTAethylenediaminetetraacetic acidFDAUnited States Food and Drug AdministrationFFAfenfluraminemffraction metabolizedHEKhuman embryonic kidney cellselk−1)rate constant of elimination (minLC‐MS/MSliquid chromatography/tandem mass spectrometryLGSLennox‐Gastaut syndromem/zmass‐to‐charge ratioMDCKIIMadin‐Darby canine kidney cellsMS/MStandem mass spectrometryNADPnicotinamide adenine dinucleotide phosphateNADPHnicotinamide adenine dinucleotide phosphate (hydrogen)nFFAnorfenfluramineOATorganic anion transporterOCTorganic cation transporterP‐gpP‐glycoprotein multidrug transporterrCYPrecombinant cytochrome P4501/2thalf‐life (in vitro)TEERtransepithelial electrical resistanceRtretention timeUDPGAuridine diphosphate glucuronic acidUGTuridine diphosphate glucuronosyltransferaseFenfluramine reduced convulsive seizure frequency in patients with Dravet syndrome and other developmental and epileptic encephalopathies. These patients take multiple concurrent antiseizure medications, emphasizing the importance of evaluating fenfluramine’s drug‐drug interaction potential. We characterized the victim potential of fenfluramine and its active metabolite, norfenfluramine, by reaction phenotyping. Fenfluramine dose adjustments may be needed when administered with strong inhibitors or inducers of CYP2D6, CYP2B6, CYP1A2 and, to a lesser extent, CYP2C9, CYP2C19, and CYP3A4/5. A companion paper evaluates fenfluramine’s perpetrator potential.1Fenfluramine (FFA), as designated by International Union of Pharmacology (IUPHAR),norfenfluramine (nFFA), modulate serotonergic neurotransmission. In clinical trials, FFA has shown efficacy for treatment of seizures associated with Dravet syndrome (DS), Lennox‐Gastaut syndrome (LGS), and CDKL5 deficiency disorder (CDD). All are pediatric developmental and epileptic encephalopathies characterized by pharmacoresistant seizures of multiple types.https://www.fintepla.com/). The severity and pharmacoresistance of these conditions often require multi−ASM regimens. Resultant drug‐drug interactions (DDIs) may lead to loss of efficacy or to toxicity by reducing or elevating plasma drug levels to subtherapeutic or toxic concentrations, respectively.Characterizing the metabolic stability, metabolites, and substrate potential for drug transporters of antiseizure medications (ASM) and the enzymes that catalyze their biotransformation and elimination can help predict pharmacokinetic DDIs. Early literature dating to the 1960s and 1970s—when FFA was marketed as an anorectic agent—describes FFA and nFFA metabolism in multiple species,cytochrome P450 (CYP450) enzymes mediate phase 1 biotransformation and elimination reactions for most orally administered xenobiotics in the liver and, to a lesser extent, in the intestine.Seven m) >25% by a single enzyme or clearance pathway have high victim potential.In the context of ASM polypharmacy, ASMs with fraction metabolized . CYP enzymes mediating FFA and nFFA metabolism were identified via recombinant proteins and direct or metabolism‐dependent inhibitors. A companion manuscript characterizes the perpetrator potential of FFA.22.16, gemfibrozil glucuronide, and esomeprazole were received from Toronto Research Chemicals, Inc. . Tienilic acid was purchased from Cypex, Ltd., in Dundee, Scotland, and troleandomycin triacetate from Enzo Life Sciences, Inc., in Farmingdale, New York. The deuterium‐labeled metabolite used as the internal standard for FFA was 1’‐hydroxymidazolam‐d4 . All other reagents and solvents were of analytical grade. FFA and nFFA, or 1‐(3‐(trifluoromethyl)phenyl)propan‐2‐amine hydrochloride, were obtained from IPCA Onyx Scientific Ltd. .Diethyldithiocarbamate trihydrate, ethylenediaminetetraacetic acid (EDTA), furafylline, glucose‐6‐phosphate, glucose‐6‐phosphate dehydrogenase, ketoconazole, letrozole, magnesium chloride, nicotinamide adenine dinucleotide phosphate (NADP), paroxetine maleate salt, phencyclidine hydrochloride, quinidine, sucrose, and tris(hydroxymethyl)aminomethane hydrochloride, or tromethane hydrochloride base, were obtained from Sigma‐Aldrich , in St. Louis, Missouri. The deuterium‐labeled metabolites used as an internal standard for nFFA, hydroxybupropion‐d2.2Escherichia coli transfected with empty expression plasmid or plasmid expressing nicotinamide adenine dinucleotide phosphate (hydrogen) (NADPH)‐CYP450 oxidoreductase without CYP450 enzymes were obtained from Cypex, Ltd. Metabolite identification and stability studies used human liver S9 fractions  from pooled human liver (n = 50) and intestine (n = 10), pooled canine liver (n = 8) and intestine (n = 7), and pooled rat liver (n = 454) and intestine (n = 199). All rCYP450 enzymes except rCYP1A2 and rCYP2D6 were expressed with cytochrome b5.Microsomes from a mixed‐gender pool of 200 non‐transplantable human livers  were characterized as described.2.34 internal standard.LC‐MS/MS analyses to assess metabolic stability and identify the transporter substrate were performed using an API 4000 QTRAP mass spectrometer  with electrospray ionization in positive mode. Chromatography was performed using an Atlantis dC18 analytical column . Mobile phases were 0.2% v/v formic acid in water and 0.2% v/v formic acid in acetonitrile. For metabolic stability experiments, FFA and metabolites were eluted at a flow rate of 0.6 mL/min, with a linear gradient of 20%−80% acetonitrile from 0.30−2.0 min. For transporter substrate experiments, the gradient was slightly modified to achieve suitable chromatography. The mass transitions monitored were 232.0 > 159.0 for FFA and 348.0 > 330.0 for 1ʹ‐OH‐midazolam‐dFor metabolic characterization experiments, LC‐MS/MS analyses were performed using an Acquity Ultra Performance Liquid Chromatography system with an in‐line photodiode array detector and ethylene bridged hybrid C18 columns  coupled to a Synapt G2 HDMS QTOF (Waters Corporation). Mobile phases were 0.1% v/v formic acid in water and 0.1% v/v formic acid in methanol. FFA and metabolites were eluted at a flow rate of 0.5 mL/min with a linear gradient of 2.0%−35% methanol from 1.0−10.0 min. The mass spectrometer was operated in positive mode with electrospray ionization. Data were acquired over the range 50−1200 m/z (mass‐to‐charge ratio) using capillary voltage, 3.5 kV; extraction cone voltage, 4.0 V; source temperature, 120°C; and desolvation temperature, 350°C. A continuous lock mass reference compound, fexofenadine, was sampled at 10‐s intervals for centroid data mass correction. Data were processed with MetaboLynx XS, a component of MassLynx (v. 4.1) software (Waters Corporation).For FFA and nFFA reaction phenotyping experiments, LC‐MS/MS experiments were performed using an API 4000 QTRAP or QTRAP 5500 mass spectrometer (SCIEX) with positive electrospray ionization. Chromatography was performed using an Atlantis dC18 analytical column . Mobile phases were 0.2% v/v formic acid in water and 0.2% v/v formic acid in methanol. FFA and nFFA were eluted at a flow rate of 0.5 mL/min with a linear gradient of 40%−95% methanol from 0.20−2.1 min. The mass transitions monitored were 232.0>109.0 for FFA, 204.0>159.0 for nFFA, 348.0>330.0 for 1’‐hydroxymidazolam‐d4 internal standard (FFA reaction phenotyping), and 262.0>244.0 for hydroxybupropion‐d6 internal standard (nFFA reaction phenotyping). Analyst Instrument Control and Data Processing software (v. 1.6.1) was used for collecting and integrating data (SCIEX). Calibration curves ranging from 1.25 µM−0.01 µM were used to quantify nFFA. Relative quantitation was used to approximate the amount of FFA test material used.2.4The line of best‐fit for calibration standards was calculated by weighted (1/x) linear regression based on peak‐area ratios of analyte to the internal standard using Analyst Instrument Control and Data Processing software . Mean analyte peak‐area ratio at each time point was normalized to controls at t = 0 min (100%).2.52) (3 mM), and EDTA , in the presence or absence of a three‐component cofactor mixture  [8 mM], and 3’‐phosphoadenosine 5’‐phosphosulfate [0.5 mM]). The NADPH‐generating system consisted of NADP , glucose‐6‐phosphate , and glucose‐6‐phosphate dehydrogenase (1 U/mL). Reactions were initiated by adding the cofactor mix and were stopped at 0, 30, 60, or 120 min by adding an equal volume of acetonitrile. Cofactor controls were incubations performed in parallel in the absence of cofactor for 120 min. Additional incubations were performed with midazolam (10 μM) as a positive control to establish the metabolic competence of test systems. Samples were centrifuged at 920 × g for 10 min at 10°C. Supernatant fractions were analyzed by LC‐MS/MS.FFA (1 or 10 µM) was incubated with liver and intestinal S9 fractions (2 mg protein/mL) at 37 ± 1°C in 0.2‐mL incubation mixtures containing potassium phosphate buffer , magnesium chloride  was determined from the time course of substrate loss based on single exponential decay (A(t)=A0(‐kelt)). Half‐life in vitro was determined by the following equation: t1/2 = 0.693/kel. The in vitro intrinsic clearance (CLint) was calculated from the rate constant of elimination as follows:To characterize substrate loss and metabolic stability, duplicate samples were analyzed by LC‐MS/MS to determine the change in substrate concentration over the incubation period. The rate constant of elimination .2.6CYP2B6, rCYP2C8, rCYP2C9, rCYP2C19, rCYP2D6, or rCYP3A4, and incubation at 37°C. The 200‐μL incubation mixtures contained potassium phosphate buffer , MgCl2 (3 mM), and EDTA , and an NADPH‐generating system. Incubations of FFA with control bactosomes and membranes containing human NADPH‐CYP450 oxidoreductase, but not human CYP450 (reductase control), served as negative controls. FFA or nFFA was added to the incubation mixtures in water (1% v/v). Reactions were initiated by adding the NADPH‐generating system and were terminated at 0 and 60 min by adding 175 μL acetonitrile stop reagent containing an internal standard . At termination, each sample volume was normalized to the volume of standards (400 μL) with 25 μL of standard blank (methanol). Samples were centrifuged (920 × g for 10 min at 10°C). Supernatant fractions were analyzed by LC‐MS/MS.Methods used to evaluate the role of CYP450s in the metabolism of FFA have been described.2.7To determine the metabolic role of individual CYP450 enzymes more quantitatively, FFA (1 μM) or nFFA (0.1 μM) was manually incubated in duplicate with human liver microsomes (1 mg protein/mL) for 60 min (FFA), or for 0 and 120 min (nFFA), in the presence of direct‐acting or metabolism‐dependent chemical inhibitors. Direct‐acting chemical inhibitors were letrozole , quinidine , and ketoconazole . Metabolism‐dependent inhibitors were furafylline , phencyclidine , gemfibrozil glucuronide , tienilic acid , esomeprazole , paroxetine , diethyldithiocarbamate , and troleandomycin . Solvent controls were processed in parallel with each inhibitor. Under the conditions described above, incubations were conducted and reactions terminated at 0 or 120 min. Metabolite formation for chemical inhibitors was normalized over time to metabolite formation in the respective solvent controls for each inhibitor. Assays were conducted in duplicate due to the low biological and technical variability expected in this assay; statistical analysis was not possible with duplicate samples.2.8breast cancer resistance protein (BCRP), organic anion transporter (OAT)1, OAT3, organic cation transporter (OCT)2, MATE1, MATE2‐K, and P‐glycoprotein multidrug transporter , according to published methods.3H]‐p‐aminohippurate probe (1 μM) and probenecid inhibitor (100 μM); for HEK293‐OAT3, [3H]‐estrone‐3‐sulfate probe (50 nM) and probenecid inhibitor (100 μM); for HEK293‐OCT2, [14C]‐metformin probe (10 μM) and quinidine inhibitor (300 µM); and for HEK293‐MATE1 or HEK293‐MATE2‐K, [14C]‐metformin probe (10 μM) and cimetidine inhibitor  or pyrimethamine inhibitor (0.3 μM). BCRP substrate determination was made by bi‐directional permeability across MDCKII‐BCRP and control cells cultured on 24‐well transwell plates. Incubation medium containing probe substrate was added. Ten minutes later, transepithelial electrical resistance (TEER) was recorded and cells were preincubated at 37 ± 2°C for 30−60 min before FFA or nFFA or positive control substrate with the solvent control or positive control inhibitor was added to the donor  chamber. Incubation medium with solvent control or positive control inhibitor was added to the receiver  chamber, and aliquots were collected from the donor (20 µL) at 0 and 120 min, or from receiver compartments (100 µL) at 0, 15, 30, and 120 min, and were replaced with comparable volumes of incubation medium. Samples containing FFA, nFFA, or probe substrate were mixed with internal standard and analyzed by LC‐MS/MS. For OAT‐, OCT‐, and MATE‐HEK293 cells, substrate was identified by determining the accumulation of FFA and nFFA in transporter‐expressing cells. After incubation with FFA, nFFA, or radiolabeled positive control substrate in the presence or absence of positive control inhibitor, incubation medium was removed and cells were rinsed with 1 mL of ice‐cold phosphate buffered saline , washed, and for radiolabeled compounds, resuspended in sodium hydroxide (0.1 M). An aliquot was added to 96‐well plate containing scintillation fluid that was analyzed by a MicroBeta2 scintillation counter . Protein concentration was determined by a bicinchoninic acid (BCA) protein assay according to the manufacturer’s protocol . Non‐radiolabeled compounds were resuspended in a 50:50 v/v methanol:water solution containing internal standard and were analyzed by LC‐MS/MS. Assays were run in triplicate.FFA and nFFA were evaluated as substrates of the transporters 2.9http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY,Key protein targets and ligands in this article are hyperlinked to corresponding entries in 33.11/2 = 35.4 min; CLint = 9.8 µL/min/mg) ≈ dog  >> human . Substrate loss in rat and dog but not in human S9 liver fractions was NADPH‐dependent , rat intestinal S9 fractions showed slightly more substrate loss than other species  were detected  of FFA was observed in all liver and intestinal fractions Table . FFA and3.3FFA substrate loss and nFFA formation were characterized over time in human liver microsomes (1 mg protein/mL) at substrate concentrations of 1−100 µM Figure . FFA subDisappearance of FFA (%) and formation of the metabolite product nFFA (pmol) at 1 μM were observed with rCYP2D6 , rCYP2C19 , and rCYP1A2 ; other rCYP450s resulted in <10% FFA loss and <10 pmol nFFA formation Figure . At 10 μ3.4nFFA (0.1 μM and 1 μM) was incubated with a panel of rCYP450s . rCYP1A2, rCYP2B6, rCYP2C19, and rCYP2D6 each metabolized nFFA by 10%−20%, with smaller contributions from the other enzymes Figure . The low3.5FFA and nFFA were not substrates of the BCRP, OAT1, OAT3, OCT2, MATE1, MATE2‐K, or P‐gp transporters . The absolute oral bioavailability of FFA is reported to be 68%−83%,In a companion study to the current analysis, neither FFA nor nFFA significantly inhibited or induced CYP450 enzymes, suggesting minimal perpetrator potential at intended clinical doses  .In a recent clinical DDI study, FFA was co‐administered with a DS ASM regimen of stiripentol, clobazam, and valproate in healthy subjects.0‐24 (Prescribing information: https://www.fintepla.com/). The 166% increase is approximately 2.7‐fold which means stiripentol/clobazam took away 69% (100% [1/2.7]) of clearance. In addition, renal clearance accounts for 3% to 10% of the dose excreted as FFA (Prescribing information: https://www.fintepla.com/). As stiripentol/clobazam do not inhibit MAO or renal clearance, approximately 80% of the clearance is accounted for in vivo; the maximum possible amount remaining for metabolism by MAO would be 20% and unlikely to be of clinical significance. Taken together, these results suggest that nFFA and C1−C6 require CYP450s for formation, with the glucuronidated metabolites requiring sequential phase 1 and phase 2 reactions. The appearance of NADPH‐independent substrate loss in rat S9 fractions at the 10‐µM but not the 1‐µM dose suggests that doses used are resulting in CYP450‐independent metabolism in rat and other species evaluated, as was observed in another study.In metabolic stability experiments, FFA showed some NADPH‐independent metabolism in human liver and intestinal S9 fractions, but formation of nFFA and all metabolites formed in liver microsomes from all species required NADPH and/or UDPGA (both components of the cofactor mix). There was no evidence of chemical hydrolysis or non‐enzymatic reactions. Liver microsomes contain CYP450s and uridine diphosphate glucuronosyltransferases (UGTs) that mediate phase 1 and 2 biotransformation reactions requiring NADPH or UDPGA, respectively, whereas S9 fractions contain both microsomal and cytosolic components, including NADPH‐independent mediators of phase 1 and 2 biotransformation reactions.A proposed metabolic pathway for FFA is shown in Figure FFA was not a likely substrate of any of the drug transporters investigated . Overexpression of transporters in the brain and excretory organs has led to some subtherapeutic ASM plasma levels, especially in refractory epilepsies.This study is limited by its in vitro design. In addition, pharmacogenetic variants in CYP450s and drug transporters could affect metabolism, although data indicate that polymorphisms of individual CYP450s are unlikely to significantly influence FFA pharmacokinetics . The high concentration of phencyclidine used as an inhibitor of CYP2B6 may also have the potential to inhibit CYP2D6. Finally, the reason for the non‐NADPH‐dependent loss of substrate (observed most notably in the human liver preparations compared with other species) remains to be established, especially since the MS data indicate NADPH dependency. The most likely explanations include residual NADPH and UDPGA in the fresh S9 preparation and the high signal‐to‐noise ratio afforded by MS compared to the metabolic stability experiments.It should be noted that some drug metabolism studies incorporate relative activity factor analyses to account for system variations and relative enzyme abundance when using a recombinant CYP approach. For the FFA reaction phenotyping we followed current FDA recommendations that two methods are to be used: enzyme inhibition in HLM/hepatocytes and the recombinant CYPs. We agree that application of the relative activity factor improves extrapolation of in vitro results to in vivo. The results of the two methods we report converge on three major CYPs with indication of possible minor contributions from additional enzymes. The three major enzymes comprise inducible and non‐inducible CYPs, as well as polymorphically and non‐polymorphically expressed CYPs. We consider this information sufficient for addressing a concern of potential susceptibility of FFA to DDI due to a single or a limited number of clearance pathways. Were the results different, application of the relative activity factor would have been considered.The apparent half‐lives of the parent drug loss were significantly different between 1 and 10 µM and were variable in the species tested, suggesting potentially different apparent Km values among species. To derive the intrinsic clearance from apparent half‐life obtained at a single concentration, it is important to ensure that the concentration is well‐below the apparent Km; alternatively, both 1 and 10 µM should be considered. In human liver S9 incubations, at 10 μM FFA with cofactors, the percent of the drug remaining was 24.4%, 8.8%, and 9.9% at 120, 60, and 30 min, respectively. At 1 μM FFA, the percent of the drug remaining was 20.9%, 11.1%, and 1.9% at 120, 60, and 30 min, respectively. In the absence of the cofactors the percent of the drug remaining was 22.9% and 21.6% in incubations of 10 and 1 μM FFA, respectively. These data are fairly close for the two drug concentrations examined. Aside from that observation, we proceeded on the side of caution and used the data for the lower concentration of the drug, 1 μM, for the evaluation of the half‐life of FFA. Our data support differences in FFA half‐life values obtained in the rat and dog were larger than in the human at two concentrations of the drug.In the metabolic stability and metabolite identification experiments, UDPGA was included as the cofactor for UGT, but pore forming agents  were not included in the incubations. The effect of alamethicin on the activity of UGT enzymes is attributed to alamethicin‐mediated formation of pores in the microsomal membrane and an increased access of the UGT substrate to the microsomal lumen where the enzyme binding site is exposed. The addition of alamethicin could increase enzymatic activity of the UGT enzymes. In our studies, FFA was found not to be a substrate for the uptake transporters recommended in the current FDA guidance; therefore, providing an additional route of access of the drug to the enzyme could overestimate its UGT‐mediated clearance.In conclusion, the in vitro studies described in the current report suggest that FFA may have victim potential, which can manifest when the drug is co‐administered with other ASM regimens containing drugs with substantial effects on combinations of CYP450 family enzymes. The CYP450s with victim potential are CYP2D6, CYP2B6, CYP1A2, and (to a lesser degree) CYP2C9, CYP2C19, and CYPA4/5.P.M. and B.B. are employees of, and own stock in, Zogenix, Inc., S.S. is a consultant for Zogenix, Inc., B.W.O., P.B.L., S.M., and M.C. are consultants for Zogenix, Inc., and are employees of Sekisui XenoTech.Participated in research design: BWO, Conducted experiments: SM, Contributed new reagents or analytical tools: N/A, Performed data analysis: SM, Wrote or contributed to the writing of the manuscript: PM, SS, BB, MC, PBL, SM, BWO.


In comparison, KOH etching for 5 h resulted sparser but nonetheless disordered surface morphology with only 8 spikes per µm2. The NaOH surface was more effective at eliminating Gram-negative pathogens, while the KOH surface was more effective against the Gram-positive strains. These findings may guide further research and development of bactericidal titanium surfaces which are optimised for the predominant pathogens associated with the intended application.Inspired by observations that the natural topography observed on cicada and dragonfly wings may be lethal to bacteria, researchers have sought to reproduce these nanostructures on biomaterials with the goal of reducing implant-associated infections. Titanium and its alloys are widely employed biomaterials with excellent properties but are susceptible to bacterial colonisation. Hydrothermal etching is a simple, cost-effective procedure which fabricates nanoscale protrusions of various dimensions upon titanium, depending on the etching parameters used. We investigated the role of etching time and the choice of cation (sodium and potassium) in the alkaline heat treatment on the topographical, physical, and bactericidal properties of the resulting modified titanium surfaces. Optimal etching times were 4 h for sodium hydroxide (NaOH) and 5 h for potassium hydroxide (KOH). NaOH etching for 4 h produced dense, but somewhat ordered, surface nanofeatures with 75 nanospikes per µm Worldwide, there is a growing demand for implantable medical devices, in part due to an aging population made possible by advancements in medicine and technology . TitaniuTo tackle the burden of IAI, much research has focused on the modification of implanted biomaterials to grant them with anti-infective properties. For example, surfaces can be coated with antimicrobial peptides or drug-eluting compounds ,26,27,28Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), and Pseudomonas aeruginosa (P. aeruginosa). The Gram-positive coccus S. aureus was chosen due to its ubiquitous association with IAIs [E. coli and P. aeruginosa were chosen as representative Gram-negatives which frequently appear in implant infections [P. aeruginosa rods are typically longer than E. coli rods  [The purpose of this study is to examine the role of etchant type and processing time on the morphology of the resultant surfaces and their antibacterial capacity. Potassium hydroxide (KOH) and sodium hydroxide (NaOH) were used as etchants. Etching time was 1, 3, 4, and 5 h. The modified surface was challenged with three clinically relevant pathogens: ith IAIs , and welith IAIs  combinedith IAIs . E. colifections ,39. P. actively) ,41 which2) were received polished at a Ra of 0.5 µm  SDN BHD, Kedah, Malaysia). To create the nanostructured surface, the discs were hydrothermally etched at 150 °C, using either 1 M KOH or 1 M NaOH in a stainless-steel reactor . Next, the reactors were cooled, and the samples were cleaned in ultrapure water. Ti-6Al-4V discs were then dried, followed by heat treatment inside an oven and allowed to cool down overnight. The as-received titanium alloy discs were used as controls (AR-Ti) and hydrothermally etched discs, using 1 M NaOH and 1 M KOH aqueous etching solutions, were then cleaned and sterilised at 121 °C for 20 min prior to use. Samples were fabricated using both alkaline solutions, (1 M KOH and 1 M NaOH) with etching times of 1, 3, 4, and 5 h.Ti-6Al-4V discs , whereas the diameter was measured at mid-height in parallel orientation with the basal plane, and a correction factor of x/cos (45°) to amend dimensional distortion during linear measurements, where x equals the length of nanostructures. Spike height and diameter at mid height were expressed as mean ± standard deviation, and n = 5. The spacing between nanostructures was determined from four zero-degree tilted SEM images, considering the nanostructure tips in a 25 µm2 area and presented as mean ± standard deviation. Density was calculated by counting spike tip and expressing as nanostructures per μm2. The nanostructures dimensions were determined using ImageJ software version 1.53f51 .Scanning electron microscope (SEM) characterisation was performed on samples fabricated using both alkaline etchant solutions (1 M KOH and 1 M NaOH) with etching times of 1, 3, 4, and 5 h. The morphology and distribution of nanostructures upon the surface of the titanium substrate were analysed on a field emission gun scanning electron microscope Zeiss Merlin FEG-SEM , equipped with a secondary electron (SE) detector, at 2 KV with magnification from 5–50 K. The stage was tilted at 45 degrees for imaging of the nanostructures, and orthogonal when analysing density of individual nanostructures and imaging bacteria on the surfaces. The height of the nanostructures was determined by the linear distance between a basal plane and the highest point of each spike .Atomic force microscopy (AFM) was used to acquire 25 µmChemical composition of the upper 10 nm layer of AR-Ti, NaOH-4h, and KOH-5h were analysed using X-ray photoelectron spectroscopy (XPS). XPS survey spectra were collected using a Kratos AXIS Ultra DLD spectrometer  equipped with a magnetically confined charge compensation system, with monochromatic AlKα radiation (hν = 1486.7 eV). The sample area analysed was 300 μm × 700 μm at a pass energy of 160 eV. Data analysis was performed with CasaXPS software . All binding energies were referenced to the low energy, aliphatic C 1 s peak at 285.0 eV.Surface wettability was evaluated for AR-Ti, NaOH-4h, and KOH-5h using contact angle evaluated by the sessile drop method using a contact angle goniometer model RD-SDM02 . The contact angle from probe liquid, ultrapure water (4 μL) was measured by a tangent fitting method using plugin Contact_Angle.jar for Image J software version 1.53f51 .Escherichia coli ATCC 11303 (E. coli), Pseudomonas aeruginosa ATCC 15692 (P. aeruginosa), and Staphylococcus aureus ATCC 25923 (S. aureus) were cultured in tryptic soy broth  and incubated overnight at 37 °C. Prior to starting bacterial experiments, it was determined that an optical density of 1 measured at 600 nm (OD600) was approximately 1 × 109 CFU/mL6 CFU/mL, and 1 mL added to wells containing titanium discs  in triplicate, and incubated for 20 h at 37 °C in a humid chamber. Next, the samples were stained with LIVE/DEAD® BacLight™ Bacterial Viability Kit  as per the manufacturer’s instructions and analysed by an Olympus FV3000 laser confocal microscope . Samples were inverted onto a glass coverslip and four randomly selected regions were imaged. SYTO 9 and propidium iodide fluorescence excitation/emissions were monitored at 480/500 nm and 490/635 nm, respectively. The viability was determined using ImageJ software v1.53a .To compare the different etching times  and etchants (NaOH and KOH), overnight cultures of bacteria were adjusted to a concentration of 1 × 10E. coli, P. aeruginosa, and S. aureus incubated on AR-Ti, NaOH-, and KOH-etched samples were fixed for 1 h with 4% paraformaldehyde, 1.25% glutaraldehyde, and 4% sucrose in PBS. Following fixation, they were washed in PBS, followed by dehydration in an ascending ethanol order from 50% (v/v) to absolute ethanol and further dried using hexamethyldisilazane . After the dehydration process, the discs were mounted on aluminium stubs using double-sided carbon tape, sputter-coated with 2 nm platinum, and examined using a Zeiss Merlin FEG-SEM .Overnight cultures of v/v fetal bovine serum  and 1% (v/v) penicillin/streptomycin . Tissue culture plate (TCP) and AR-Ti discs were used as positive controls. HDF cells were seeded at a density of 2.5 × 104 cells/well. Short-term cytocompatibility was assessed using the resazurin assay . A stock solution of 100 µg/mL resazurin was prepared in phosphate buffered saline (PBS) and diluted to a final working concentration of 10 µg/mL. Briefly, the cells were incubated on TCP, AR-Ti, NaOH-4h, and KOH-5h for 48 h at 37 °C and 5% CO2, after which the culture medium was replaced with 250 μL media containing 10% resazurin of the stock solution and incubated for 1 h. Next, 100 µL of the supernatant was transferred into a 96 well plate and the fluorescent intensity was recorded using a plate reader. Resazurin is a fluorescent assay that detects cellular metabolic activity. The blue nonfluorescent resazurin reagent is reduced to highly fluorescent resorufin by dehydrogenase enzymes in metabolically active cells. The resorufin formed in the assay was quantified by measuring the fluorescent intensity using a plate reader . Percent of cell viability is calculated using the following formula, Primary human derived dermal fibroblast cells (HDF) were seeded on AR-Ti, NaOH-4h, and KOH-5h samples in a 48 well plate. Cultures were maintained in Dulbecco’s modified Eagle medium  supplemented with 10% hed work . Samplest-test followed by post hoc analysis using a Tukey’s multiple comparisons test. To compare nanostructure topographical dimensions, a Student’s t-test was performed. Statistical analysis was performed using GraphPad Prism version 8.3.0 . All experiments were undertaken in triplicate and reported as mean and standard deviation. In all experiments, significance was set at p < 0.05.Statistical analysis for surface parameters and viability was carried out using one-way ANOVA or Student’s A range of surface nanostructures were created on titanium alloy discs using hydrothermal etching with either NaOH or KOH aqueous solutions. Surfaces were etched for 1, 3, 4, or 5 h to investigate the changes in geometrical and morphological characteristics to investigate the nanostructure evolution with time. Surfaces were then annealed to improve corrosion resistance, alloy plasticity, abrasion resistance, and potential for bone ongrowth, and reduce surface stress developed during fabrication ,44. The 2. The results are presented in p < 0.0001). The nanostructure diameter at mid-height, followed an upward trend for NaOH, with a significant increase between 1 h and 3 h etching times , and 1 h and 5 h etching times . However, the nanostructures’ diameter at mid-height remained almost constant from 1 to 5 h in the case of KOH-etching . The spacing between nanostructures remained unchanged for etching times on the NaOH-etched surfaces , and increased on KOH-etched surfaces . This indicates that there was no new nanostructure formation over time, only evolution of those that had already formed at the beginning of the hydrothermal process, the main outcome being the increase in nanostructure height. The density of the nanostructures was lower on the KOH-etched surface (8 ± 2 spikes/ µm2 at 5 h etching time) compared to NaOH-etched surface (32 ± 9 spikes/µm2 at 5 h etching time). The SEM micrographs were used to measure spike height, diameter at mid-height, spacing between, and density per µmE. coli, P. aeruginosa, and S. aureus on the surfaces generated by NaOH and KOH hydrothermal etching for 1, 3, 4, and 5 h are shown in S. aureus. This is somewhat expected as Gram-positive bacteria possess a thicker peptidoglycan layer which confers greater structural support compared to the relatively fragile Gram-negative species [S. aureus, a high proportion of damaged cells were observed on the KOH-5h surface . However, although the spike heights were not significantly different, the KOH-5h shows a much greater range in measured nanostructure height (175 nm) compared to the NaOH-4h (80 nm). This irregularity of spike heights can be clearly observed in both the SEM and AFM micrographs (p = 0.02) and greater spike density (p = 0.0001) when compared to the KOH-5h surface.To find a potential explanation for the observed bactericidal selectivity between NaOH-4h and KOH-5h, we first compared the spike dimensions of the fabricated nanostructures . We foun2) occurring during the hydrothermal etching process. Furthermore, the thicker oxide layer also resulted in a reduction in the surface concentration of vanadium and aluminium as XPS sampling depth is limited to the outermost 10 nm of the surface. This may prove to be advantageous for implantable biomaterials as wear particles and leaching of vanadium and aluminium from Ti-6Al-4V have raised concerns due to their potential toxicity and DNA damaging effects [Elemental composition detected on the AR-Ti, NaOH-4h, and KOH-5h surface using XPS is presented in  effects ,50,51. Ap < 0.0001, indicative of significantly increased hydrophilicity (p > 0.05). Such superhydrophilic behaviour is the result of the combination of surface roughness at the nanoscale and a hydrophilic TiO2 surface layer, consistent with the Wenzel and Cassie Baxter theory [To compare the hydrophilicity of samples before and after hydrothermal etching, water contact angles (WCA) were measured. The WCA for NaOH-4h surface (7.6° ± 1.2°) and KOH-5h (9.7° ± 1.1°) was significantly lower than that for the AR-Ti surface (76.0° ± 5.0°), hilicity B. There r theory ,53,54. Ar theory ,56.2 respectively, whereas for the NaOH-4h surface, these parameters were 88.4 nm, 71.4 nm, and 79.9 µm2, respectively. Compared to the NaOH-4h surface, the KOH-5h surface had a similar RMS value of 88.5 nm, and a decrease in both Ra and SA  incubated for 48 h are shown in p > 0.05 . These r2, compared to the sparser KOH-5h with a density of approximately 8 spikes per μm2 suggesting that a greater density of spikes supports bactericidal activity against Gram-negative rods, while a sparser nanostructures array supports killing of Gram-positive cocci. Short-term in vitro cytocompatibility study using HDF cells incubated on NaOH-4h and KOH-5h surfaces showed no unfavourable effects on morphology, viability, and proliferation. Further research is required to fully understand the mechanisms supporting the antibacterial properties of the nanostructured surface. However, this study highlights that with appropriate optimisation, substrates with selective antibacterial activity could be potentially achieved. We investigated the role of alkaline etchants and hydrothermal processing duration on the resultant surface nanotopography outcomes for titanium alloy discs. Nanostructured surfaces were challenged with three clinically relevant pathogens to determine their bactericidal potential. Our bacteriological analysis allowed us to narrow down the etching parameters to two favourable processing conditions involving either NaOH-etching for 4 h or KOH-etching for 5 h. NaOH-4h treatment exhibited spikes that are densely packed with a mean of 75 spikes per μm


Physical rehabilitation interventions address functional deficits caused by impairments that affect someone’s performance. Whilst rehabilitation is important, it is assumed that these services are either minimal or nonexistent in low-resource settings. Our data expand on the data from the Situation Assessment of Rehabilitation in the Republic of Rwanda report to describe rehabilitation services and who access them at public and semiprivate facilities (primarily funded by the private sector).This article describes the use of the outpatient physical rehabilitation services across nine health facilities, the characteristics of adults attending these health facilities and some of the facilitators and barriers they encounter when attending rehabilitation.Data were collected between September and December 2018 from the heads of departments and adult patients attending outpatient rehabilitation services funded by the government, international nongovernmental organisations or faith-based organisations.Two hundred and thirteen adults were recruited from nine facilities. There is a sixfold difference in the number of rehabilitation personnel between public and semiprivate hospitals in these facilities’ catchment areas. However, most participants were recruited at public facilities (186 [87%]), primarily with physical disorders. Patients reported that family support (94%) was the most crucial facilitator for attending rehabilitation, whilst transportation cost (96%) was a significant barrier.Rehabilitation service availability for Rwandan adults with disabilities is limited. Whilst family support helps patients attend rehabilitation, transportation costs remain a significant barrier to people attending rehabilitation. Strategies to address these issues include developing triage protocols, training community health workers and families.Data on rehabilitation service provision in Rwanda and most African countries are either non-existent or very limited. These data contain important information regarding the services provided and the people who used them across different health facilities (public versus private) and urban versus rural settings). To improve rehabilitation service provision, we first need to understand the current situation. These data are an important step to better understanding rehabilitation in Rwanda. Adult physical functioning is affected by a variety of health conditions, such as noncommunicable diseases (NCDs) or injuries Manini . NoncommIn response to the growing need for rehabilitation, in 2017, the World Health Organization (WHO) launched the Rehabilitation 2030 initiative. This initiative brought together an international group of key stakeholders in rehabilitation service provision to develop strategies and action plans to provide quality and timely rehabilitation worldwide, particularly in low- and middle-income countries with limited rehabilitation services and the greatest need   and health posts . PhysicaAbout half of Rwanda’s population is over the age of 18 years (7 208 063 out of 13 411 153 people).Physiotherapy (PT) services are available at 53 hospitals and the 4 semiprivate specialised rehabilitation centres. According to the Rwanda Allied Health Professional Council (RAHPC), 360 PTs are registered to practice in Rwanda , 88 of wn = 24) employed in settings supported by INGOs, such as schools and refugee camps. There are 0.02 OTs per 10 000 Rwandans, which is lower than the minimum of 750 per one million (7.5 per 10 000 people) suggested by the World Federation of Occupational Therapists  at one public hospital and two specialised rehabilitation centres. Twenty-six OTs are practicing in Rwanda, with most OTs , in collaboration with the WHO, the United States Agency International Development and the ICRC published a report on the Situation Assessment of Rehabilitation in the Republic of Rwanda. This report describes the insufficient and nonexistent rehabilitation services in Rwanda  and geographical locations . Finally, we use patient-level data to describe the people that use these services and some patients’ facilitators and barriers to attending rehabilitation.Humanity & Inclusion developed, funded and implemented a cross-sectional survey to understand the cost of rehabilitation services in Rwanda. From September to December 2018, in collaboration with the MoH and the National Commission of Persons with Disabilities, HI conducted the survey across 12 rehabilitation facilities supported by public and INGOs or FBOs in both urban and rural settings. However, this article presents data from nine facilities amongst adults with disabilities.The 12 health facilities that provide rehabilitation services were purposively selected. These facilities were selected based on the representation of levels of the health system pyramid , two pubFrom September to December 2018, children and adults with disabilities were randomly selected by site champions across the 12 selected health facilities. All persons with disabilities or caregivers or parents or caregivers of children under 18 years old who agreed to participate and were present when data collectors were at the health facilities were recruited. Of the 385 participants recruited across 12 facilities, this article focuses on the data obtained from 213 adult respondents (18 years or older) with disabilities across 9 facilities. Data on service provision were obtained from the heads of departments.To calculate the overall sample size required across the facilities to answer questions on cost-effectiveness, the Cochran’s formula was used as follows:z = z-score for the standard normal distribution at the significance level of 95%, p = anticipated proportion of people having access to rehabilitation service (50%) and δ = desired precision [5%]):(where n = 213 participants) across 9 facilities.Data were collected on 385 participants; however, data for this article included only the adults with disabilities who completed the survey , a tool developed for use in low- and middle-income countries  and two Rwandan PTs hired by HI coordinated data collection. Participants were recruited by HI research staff between September and December 2018, who were on site three days a week during that period. Data were collected by eight trained rehabilitation professionals using paper-based questionnaires and tablets (where feasible). All data were collected using lists with responses to select from or yes or no responses.The data collectors participated in a three day training programme that included training on administration of the questionnaire, data collection procedures  and ethical norms in research, particularly data collection. Each selected site had a site champion who was briefed on the type of participants needed for the study. Site champions randomly recruited all participants, and those who agreed to participate were referred to persons administering the survey. Eligible participants who were available during the three days of data collection, and agreed to participate, were recruited for the study. The data from the facilities and patients are presented by the source of funding (public or semiprivate) and geographical location . We were interested in assessing the differences between patients with disabilities who attended public and semiprivate facilities in urban and rural areas. Therefore, the data are presented in two sample proportions.p-values for demographic characteristics. All analyses were performed using MS-Excel version 16 and STATA/IC 16 programmes.Descriptive analyses are reported as counts, proportions for all variables and Data were collected from nine facilities; five (56%) were publicly funded, and four (44%) were semiprivately funded. The five publicly funded facilities serve an estimated catchment of 2 050 000 million people and included two university teaching hospitals out of four in the country and three district hospitals out of 42. No provincial referral or specialised hospitals were included. The four semiprivate specialised rehabilitation centres funded by the NGOs or FBOs, in the country were all included. These institutions serve an estimated catchment of 430 000 people . Four ofRehabilitation staff at the nine health facilities include 63 PTs, 16 P&Os and 8 OTs, serving both children and adults. The availability of rehabilitation personnel ranged from 2 PTs at district hospitals to 0 OTs at public hospitals. Whilst we had 13 PTs, 6 P&Os and 5 OTs at semiprivate specialised rehabilitation centres primarily funded by NGOs or FBOs, based on catchment areas, publicly funded facilities have an average of 0.2 rehabilitation staff per 10 000 people, and semiprivately funded facilities have an average of 1.2 rehabilitation personnel per 10 000 people . The avan = 186) and 13% (n = 27) from semiprivately funded facilities. Most participants received PT (n = 190 [89.0%]), with much fewer receiving P&O (n = 24 [11.0%]) or OT (n = 3 [1.4%]), and 8 (4.0%) received more than one service (n = 175 [95.0%]), with few receiving P&O (n = 10 [5.0%]) and OT  and 2 (1.1%) receiving more than one service. At semiprivate facilities, half of the participants received PT and half received P&O , with just two (7.0%) receiving both. Only one (4.0%) participant received OT. In urban settings, the majority of participants received PT (n = 154 [93.0%]), few received P&O (n = 12 [7.0%]) and two (1.2%) received OT (n = 36 [77.0%]), one-quarter of participants received P&O (n = 12 [25.0%]) and one (2.0%) participant received OT.A total of 213 participants were recruited, 87.0% from publicly funded hospitals ( service . A simileived OT . In ruraThere were more men than women (60% vs. 40%) in our sample and more of our participants were unable to work than able to work (51% vs. 27%). The only difference in demographic characteristics for those in public facilities compared to semiprivate facilities was that there were more women (44% vs. 5%), and fewer people were employed (28% vs. 33%) . When coOver 85% of participants considered the following as facilitators to rehabilitation service use: confidence and trust in staff, health coverage, family support and easily accessible services, which did not differ by facility except that patients at semiprivately funded facilities reported more family support than those from public facilities (96% vs. 78%) . The mosWith help from other international agencies, the Rwandan MoH published a report on a Situation Assessment of Rehabilitation in the Republic of Rwanda because of the recognition of the need to better understand rehabilitation service in Rwanda. This report highlights the lack of rehabilitation personnel, such as speech therapists, rehabilitation physicians and nurses  had five times more human resources than those in urban areas. People accessed rehabilitation services primarily for physical disability concerns. Interestingly, the facilitators and barriers to rehabilitation services were similar across facilities, regardless of the funding source or geographic location. Our analysis highlights the most crucial facilitators for accessing rehabilitation, including patients’ confidence and trust in staff, family support and health coverage. At the same time, participants indicated transportation costs, waiting time and inaccessible buildings as some of the significant barriers.It is well known that rehabilitation services are limited in low- and middle-income countries, which makes it imperative to use the existing resources where most needed. We demonstrated that semiprimarily funded facilities had six times more human resources than publicly funded facilities and that rural areas (predominantly semiprivately funded) had five times more human resources than those facilities in urban areas. However, it is unclear whether this distribution of resources reflects the proportion of Rwandans living with disabilities in each of these areas. Whilst rural (primarily semiprivate) facilities are better staffed, they are more difficult to access because patients must pay for services. Thus, the catchment population for the semiprivate facilities is likely much smaller, meaning that staff proportions are likely much higher for semiprivate facilities when considering people’s ability to pay. Whilst this increases the staffing ratios for those who can pay, it negatively affects the services available to those who cannot pay. Whilst numerically it seems that rural areas are better served, there are many additional considerations that suggest this may not be true. In addition to the issue of access because of payment, there is also the issue of distance to travel and costs associated with long-distance travel that may further affect access amongst the population in rural areas.We demonstrated significant differences in some of the demographic characteristics of those who attended public versus semiprivate facilities in urban and rural settings; however, we do not know where the difference lies within these characteristics. We know that participants access rehabilitation services for physical disability concerns. However, it is unclear whether the needs of Rwandans with disability are primarily physical or whether the use of services is driven by the available services. Physiotherapists were the primary rehabilitation personnel available at all facilities, whilst P&Os were available at five facilities, of which one was publicly funded. Occupational therapists were available at one public facility and two semiprivate facilities, and there were no SLT services or physicians and nurses providing rehabilitation services. Whilst there is a clear need for rehabilitation services that address physical disabilities, it is unclear if other rehabilitation needs are not being addressed. Understanding where most Rwandans with disabilities reside and their needs is critical to optimally distribute services; however, with the available funding structure, it is unlikely that redistribution will solve the issues of access to services. In addition to better distribution, implementing the MoH’s new employment health structure proposed in 2020 will increase the overall number of rehabilitation professionals, particularly in areas with no rehabilitation services  financial support for rehabilitation services at public facilities and semiprivate facilities, patients pay 100%; issues such as long waiting times and transportation costs remain significant barriers to accessing services. This is similar to issues noted in other low- and middle-income countries including South Africa and Nigeria . Most data were obtained from urban and public facilities with an under-representation of rural and semiprivate facilities and no representation from referral or provincial hospitals. Therefore, we cannot state that similar patient profiles, barriers or facilitators are experienced in these settings. We were also limited in the number of variables we could include in a regression model if we had to look at predictors of being in a semiprivate or public hospital because we only had 27 people in the semiprivate hospitals. We also calculated the catchment for public and semiprivate facilities using estimates of the population in the area but did not have an idea of the proportion of the population who could afford versus those who cannot afford for semiprivate facilities.Our study describes people who attended a facility, which is influenced by the services provided. We cannot generalise the findings regarding the type of disability because adults with disabilities use the available services (physiotherapy), not necessarily the services they need. Understanding the needs of persons with disabilities who require but cannot attend rehabilitation should be a future research priority. The survey only focused on patients with physical disabilities, which meant that our conclusion was only limited to this population. The survey’s primary purpose was to collect data on cost, which enabled us to only look at the quantity and not the quality of services. As a result, we had no data on the specific conditions , the number of sessions attended, needs and therapy outcomes of people attending facilities that provide rehabilitation services. This information would be beneficial to support the provision of appropriate rehabilitation care.The availability of rehabilitation services for Rwandan adults with physical disabilities is limited, with greater access to services in semiprivate compared to public facilities. Whilst family support help patients attend rehabilitation, transportation costs remain a significant barrier to people attending rehabilitation. Strategies to address these issues are urgently required and should complement the MoH solutions that will be implemented in the next years. These strategies can include developing triage protocols that could be implemented at the referral stage to prevent a constant flow of patients, long waiting lists and time. In addition, community-based interventions provided by families, CHWs, and lay personnel could extend the outreach of basic rehabilitation services to those with long-term health conditions and disabilities living in the community in Rwanda. This would require a major system shift that will require buy-in from multiple stakeholders in multiple systems but could be an efficient and sustainable solution. Finally, service provision could be augmented with the use of low-cost technologies using mobile phones and telerehabilitation (where the internet is available) and could offer additional support for families and CHWs in Rwanda to provide rehabilitation interventions at home or in the community.


Allergic rhinitis (AR) is frequent in children and adolescents and can severely affect their lives. This article describes the development and validation of a questionnaire to assess treatment needs and benefits in children and adolescents, the PBI-AR-K, in a sample of patients receiving grass pollen sublingual immunotherapy.The PBI-AR-K was developed based on an open survey including children and adolescents and expert consensus between methodologists, patients, and physicians. The PBI-AR-K assesses patient needs before the treatment and perceived benefit during or at the end of a treatment. A weighted benefit score can be calculated ranging from 0 to 4 (4 = highest possible benefit). The validation was conducted in children (5–12 years) and adolescents (13–17 years) receiving sublingual immunotherapy. Subscales were developed based on factor analysis. Psychometric properties of items and scales were assessed with descriptive statistics, internal consistency, and convergent validity.The final PBI-AR-K consists of 19 items. For validation, data from 345 patients  was analysed. Factor analyses resulted in four subscales for children and three subscales for adolescents. The items with the highest importance ratings were about choice of leisure activities  and about being free of AR symptoms (adolescents: 3.3). The weighted PBI-AR-K scores reflected considerable patient-reported benefit (2.08–2.82) in both children and adolescents. Internal consistency of all scales was good or acceptable. In the children’s sample, the global scale and three of four subscales were quite consistently correlated with convergent variables, while the subscale ‘treatment burden’ was significantly correlated only with change in average impairments due to rhinitis symptoms. The adolescents’ sample showed more inconsistent results with only change in rhinitis severity being significantly associate with all subscales.The newly developed PBI-AR-K is a reliable and valid questionnaire for use in children; for the use in adolescents, it should be further elaborated. The prevalence of allergic rhinitis (AR) and allergic rhinoconjunctivitis (ARC) increases throughout childhood, peaking in teenage years , 2. AR iAR can be classified according to its severity (mild vs. moderate to severe) and its patterns of occurrence (persistent vs. intermittent) . ModeratTreatment options for AR are the avoidance of allergens and pharmacotherapeutic interventions, both of which aim to reduce symptoms. The only treatment option being able to alter the course of AR is allergen immunotherapy (AIT), which provides short-term as well as long-term benefits in the treatment of AR .Besides clinical efficacy, patient-reported benefits are decisive outcomes for evaluating a treatment. Achieving patient-relevant benefits is a therapy goal in itself; beyond that, it can enhance treatment satisfaction and, hence, patient adherence , which iPBI questionnaires have been developed, standardised, and validated for a wide range of indications, including AR (PBI-AR)  and skinThis article describes the development and evaluation of psychometric properties of a PBI questionnaire in German language specifically designed to assess treatment needs and benefits in children and adolescents with AR, the PBI-AR-K (K for kids).The PBI-AR has been previously developed and validated for the use in adults and contains 25 items . Each PBIn the first step of developing the PBI-AR-K, an open patient survey was conducted. Children and adolescents with physician-confirmed AR and being aged 5 to 17 years were recruited in two federal states in Germany (Hamburg and Schleswig–Holstein). Participants completed a questionnaire with open questions about their most important needs and therapy goals themselves or, if they felt not fluent enough in reading and writing, their parents read out the questionnaire to them and noted their answers.All patient statements from the open survey were discussed in an expert panel including each one methodologists, patients, and physicians. This group categorised all patient statements qualitatively and phrased items thereupon, using easy language to facilitate readability and comprehensibility for children and adolescents. If language and content were appropriate, the same wording as in the adult PBI-AR was used. The wording of the instruction was adapted to the target population in discussions involving teachers, psychologists, and physicians./1889).Psychometric properties of the PBI-AR-K were evaluated as part of a non-interventional study on benefits of sublingual immunotherapy (SLIT) . In thisRecruitment took place over two periods. Each patient was observed during his/her first treatment year , with two visits during the observation period. Visit 1 (study initiation visit) took place at the time of treatment initiation . Visit 2 (study end visit) took place after the end of the respective grass pollen season (2013 or 2014). At both visits, patients completed paper-based questionnaires and physicians completed an electronic case report form. Adolescents aged 13 to 17 years completed the questionnaires on their own, whereas for children aged 5 to 12 years, parents read out the questionnaires and filled in the children’s answers. Table 2). Effect sizes were assumed to indicate small, medium, and strong effects when d = 0.2, d = 0.5, and d = 0.8, respectively, when η2 = 0.01, η2 = 0.06, and η2 = 0.14, respectively, and when r = 0.1, r = 0.3, and r = 0.5, respectively [Means, standard deviations, medians, ranges, and frequencies were calculated to describe sample characteristics and distribution of PBI items and scales. In order to develop subscales, factor analyses with varimax rotation were conducted using the PNQ items. Internal consistency was determined using Cronbach’s alpha based on PNQ items. For convergent validity analysis, we determined the association of PBI-AR-K global and subscale scores with physicians’ and patients’ overall judgement of patients’ well-being (during the first grass pollen season on SLIT) compared to the previous pollen season (before SLIT), change in rhinitis and conjunctivitis severity, change in frequency and duration of rhinitis symptoms, changes in impaired sleep, impaired activities, and impaired performance, change in average impairment due to rhinitis symptoms, and change in peak impairment due to rhinitis symptoms using t-tests, analysis of variance (ANOVA), and Pearson correlation (r) according to the scale level of the data. Results of the t-tests were reported for either equal or inequal variance according to Levene’s tests, with significance levels lower than 0.1 indicating inequal variance . For t-tectively . AnalyseThe open survey was completed by 11 children and adolescents with AR aged between 5 and 17 years. A subsequent expert panel  categorised all statements and developed items. This resulted in the PBI-AR-K encompassing 19 items. Of these, five items had the same wording and ten had the same content as the PBI-AR for adults. Four items were added . All items of the final PBI-AR-K can be seen in Tables The sample consisted of 345 patients with a mean age of 11.1 , of which 60.9% (n = 210) were male. The 223 children had a mean age of 9.2  with a share of 64.6% (n = 144) male participants; the 122 adolescents had a mean age of 14.6  and a share of 54.1% (n = 66) of males. Allergic rhinitis was present in 98.7% (n = 220) of children and in 100% (n = 122) of adolescents, and allergic conjunctivitis in 82.5% (n = 184) and 77.0% (n = 94), respectively. Allergy to grass pollen was diagnosed in 97.8% (n = 218) of children and 99.2% (n = 121) of adolescents; 59.6% (n = 133) and 63.1% (n = 77) had an allergy to rye or cereal pollen, respectively. Concomitant allergies (Table The items with the highest mean importance ratings were ‘to be able to do anything you want in your free time even while having hay fever’ (mean = 3.5) in children and ‘no longer have hay fever symptoms’ (mean = 3.3) in adolescents Table  indicatiThe items with the highest patient-reported treatment benefit as measured with the PBQ Table  were ‘neIn the children’s sample, factor analysis including data from 204 patients revealed four factors with an eigenvalue > 1, explaining 59.8% of all items’ variance. Based on factor loadings, four meaningful subscales could be defined  in children and 2.55  in adolescents with 94.6% and 90.7%, respectively, attaining an at least minimally relevant benefit of 1.0 or higher. In both samples, the subscale ‘treatment burden’ showed the highest mean score with 2.82  in children and 2.68  in adolescents. The remaining subscales also showed mean scores above 2.0 in both children  and adolescents  and adolescents . In both age groups, one subscale showed lower but still acceptable internal consistency: ‘being outdoors’ in children (0.771) and ‘psychosocial burden’ in adolescents (0.786).Analysis of convergent validity Table  revealed2 = 0.76 and r = − 0.30, respectively) and the subscale ‘physical symptoms’ . Besides, the subscale ‘physical symptoms’ showed significant associations with the patients’ global judgement on improvement (η2 = 0.71) and change in rhinitis severity (d = − 0.60). Additionally, the subscale ‘psychosocial burden’ showed significant association with change in the duration of rhinitis symptoms (d = − 0.78) and change in impaired sleep (d = − 0.86).In adolescents, change in average impairment due to rhinitis symptoms (r = − 0.31 to r = − 0.49) and change in conjunctivitis severity (d = − 0.68 to d = 0.81) were significantly associated with all PBI-AR-K scores. Furthermore, change in the frequency of rhinitis symptoms was significantly associated with all scales except the subscale ‘treatment burden’ (d = − 0.91 to d = -1.23). Physicians’ global judgement of patient well-being compared to the previous pollen season as well as change in peak impairment due to rhinitis symptoms were significantly correlated with the global score  and the global scale, all of which were significantly associated with almost all convergent variables. In contrast, the subscale ‘treatment burden’ was significantly correlated only with change in average impairments due to rhinitis symptoms. The low number of significant convergent variables for the treatment-related subscale seems to be plausible, as treatment burden is rather associated with the type of treatment than with the clinical outcomes. The adolescents’ sample showed more inconsistent associations with only change in rhinitis severity showing significant associations with all subscales. Among the subscales, ‘physical symptoms’ was associated with the highest number of convergent variables, which can be explained by the strong focus on symptom-specific convergent variables.The development of paediatric questionnaires is pivotal to assess meaningful data (e.g., Bullinger et al. , Eiser aThe validation of the PBI-AR-K was conducted in a study evaluating the benefits of one AIT, which must be considered a limitation as this may reduce the variance of both patient characteristics and possible patient benefits. Due to the real-world design of this study, no control group was implemented, which is why no comparison with another or no treatment is possible. However, as the purpose of this article is to validate the PBI-AR-K questionnaire, this does not impact the informative value of the results.Since AR is a widespread condition in children and adolescents, the development of age-appropriate tools to assess patient-relevant outcomes is crucial. The results of this study suggest that the newly developed PBI-AR-K is a reliable and valid questionnaire to evaluate treatment needs and benefits of children. For adolescents, results were mixed, suggesting a further need to elaborate in how far the items and subscales of the PBI-AR-K validly reflect AR-specific HRQoL experienced in this age group.


The elevation of body temperature induced by the β-adrenergic agonist CL316243 was higher in ARKO and castrated mice than in the control mice. Similarly, CL316243 induced a greater increase in Uncoupling protein 1 (Ucp1) expression and CREB phosphorylation in the brown adipose tissue of ARKO mice than in that of controls. We determined that activation of AR by dihydrotestosterone suppressed β3-agonist- or forskolin-induced CRE-mediated transcription, which was prevented by AR antagonist. AR activation also suppressed CREB phosphorylation induced by forskolin. Moreover, we found AR nuclear localization, but not transcriptional activity, was necessary for the suppression of CRE-mediated transcription. Finally, modified mammalian two-hybrid and immunoprecipitation analyses suggest nuclear AR and CREB form a protein complex both in the presence and absence of dihydrotestosterone and forskolin. These results suggest androgen–AR signaling suppresses β-adrenoceptor-induced UCP1-mediated brown adipose tissue thermogenesis by suppressing CREB phosphorylation, presumably owing to a protein complex with AR and CREB. This mechanism explains sexual differences in body temperature, at least partially.Thermoregulation is a process by which core body temperature is maintained in mammals. Males typically have a lower body temperature than females. However, the effects of androgens, which show higher levels in males, on adrenergic receptor-mediated thermogenesis remain unclear. Here, we demonstrate that androgen–androgen receptor (AR) signaling suppresses the β-adrenergic agonist-induced rise of core body temperature using castrated and AR knockout (ARKO) male mice. Furthermore,  Vasoconstriction regulates core body temperature by controlling heat loss through the skin, whereas shivering and nonshivering thermogenesis contribute heat production. Thus, while thermoregulation is extremely complex , brown aUcp1 expression in primary brown adipocytes from male mice (in vivo.Androgen receptor (AR) is a member of the nuclear receptor superfamily of ligand-activated transcription factors. Androgens such as testosterone and dihydrotestosterone (DHT) activate AR as a ligand. An increase of testosterone at puberty initiates sexual maturation in males, which contributes to the formation of sex differences in vertebrates , 13, 14.ale mice . Collectale mice , 16, 17.in vivo and dissected the molecular mechanisms underlying the effect of androgens on thermogenesis in vitro.Crosstalk between PKA signaling and AR has been reported, such as PKA signaling enhancing the transcriptional activity of AR . HoweverA). To evaluate the effect of androgen signaling on heat production, changes in body temperature after intraperitoneal injection of CL316243, a highly specific β3-AdR agonist (B). Both saline and CL316243 were administered to individual mice for two consecutive days. Subsequently, the change in body temperature in each mouse was calculated by subtracting the body temperature changes during the saline treatment period from those during the CL316243 treatment period. As shown in Figure 1C, treatment with CL316243 raised the body temperature of castrated mice more than that of their corresponding controls.Male mice castrated at 8 weeks of age showed an increase in core body temperature from the second week after the surgery A. To eva agonist , were de agonist B. Both sA). This difference in body temperature became apparent at 8 weeks of age. Intraperitoneal injection of CL316243 elicited a greater increase in the body temperature of ARKO mice than in that of control mice (B and C). These results indicate that androgen–AR signaling suppresses adrenoceptor-mediated heat production in male mice.To determine the involvement of AR in androgen-suppressed body temperature, ARKO mice were used. Analyses from the weaning period show that the core body temperature of ARKO mice was higher than that of the control mice of the same litter in an age-dependent manner A. This drol mice , B and CA–C). To delineate the mechanism underlying the effect of AR-deficiency on CL316243-mediated increases in body temperature, gene expression profiling in BAT was conducted. Ar expression was eliminated in ARKO mice (D). Ucp1 expression was induced by CL316243 to a greater extent in ARKO mice than in control mice (E). PGC1α was induced by β3-AdR stimuli and contributed to the induction of UCP1 (Pgc1α expression induced by the β3-AdR agonist between ARKO and control mice (F). The expression of β3-AdR and BAT marker genes (Prdm16 and Cidea) was not significantly affected by CL316243 treatment (G–I). In addition, the basal expression of these BAT genes also did not differ between ARKO and control mice, suggesting that AR does not affect the differentiation of BAT. UCP1 protein levels were higher in ARKO mice than in control mice when CL316243 was administered, whereas they did not differ between the control and ARKO mice when CL316243 was not administered (J), likely reflecting mRNA levels.ARKO caused a decrease, no effect, and an increase in body weight, BAT weight, and subcutaneous inguinal white adipose tissue (iWAT) weight, respectively , A–C. ToRKO mice D. Ucp1 erol mice E. PGC1α  of UCP1 , whereasrol mice F. The exreatment , G–I. Innistered J, likelyAr expression was ablated in ARKO mice (K). Ucp1 expression was extremely low in iWAT (data not shown). The expression of UCP1 in iWAT is induced by intermittent β-AdR stimulation  accompanying the browning of the iWAT. In our study, it is assumed that a single administration of the β-agonist CL316243 cannot induce UCP1 expression in iWAT. Unlike in BAT, the β3-AdR agonist-induced Pgc1α expression was higher in ARKO mice than in control mice (L). This inconsistency could be caused by differences in the regulation of Pgc1α by PKA via ATF2 (via CREB in WAT (β3-AdR was not significantly affected by ARKO and CL316243 treatment (M). After β3-AdR stimuli, free fatty acids are produced by the lipolysis of triglycerides and released from WAT. They are utilized in BAT as a fuel for thermogenesis (N).We also examined the effects of ARKO on CL316243 response in iWAT. RKO mice K. Ucp1 erol mice L. This ivia ATF2  in BAT aB in WAT . The expreatment M. After ogenesis . Thus, pogenesis N.A and B). There was a significant difference  in the direct comparison between the treated and untreated control mice. These in vivo studies suggest that AR signaling suppresses β3-agonist-induced Ucp1 expression by suppressing CREB phosphorylation in BAT.We determined the effects of ARKO on β-adrenoceptor-activated PKA signaling by measuring the levels of CREB phosphorylation. In multiple comparisons, CL316243 treatment had no significant effect on CREB phosphorylation in the control mice but led to a significant increase in the ARKO mice; there was a significant difference between the two groups , A and Bin vitro with overexpression experiments. In T37i brown adipocytes, CL316243 activated CRE-mediated transcriptional activity, which was attenuated in the presence of DHT when cells exogenously expressed AR (A). Since similar results were observed with HEK293FT cells (B), subsequent experiments were performed with HEK293FT cells. AR activation also suppressed forskolin-induced CRE activity, which was abrogated by bicalutamide, an AR antagonist (C). Similarly, DHT inhibited the phosphorylation of CREB at Ser133, which was induced by forskolin (D). AR levels were increased by DHT but not affected by forskolin (E). Ucp1 promoter activity was also induced by forskolin, which was attenuated by DHT in AR-overexpressed HEK293FT cells . As CREB regulates Ucp1 expression via the CRE4 site, but not the CRE2 site, of the Ucp1 promoter . AR suppressed the promoter activity of the CRE2 mutant but did not affect the activity of the CRE4 (CREB binding site) mutant. Collectively, these results suggest that agonist-activated AR inhibits CRE4-mediated Ucp1 gene expression by suppressing the activation of CREB.We next examined the mechanism by which AR suppresses CRE activation essed AR A. Since FT cells B, subseqtagonist C. Similaorskolin D. AR levorskolin E. Ucp1 ppromoter , we consA, a K630A/K632A/K633A mutant of AR, which does not enter the nucleus owing to disruption of the nuclear localization signal (B). Other AR mutants, such as L26A/F27A or E897Q, which both lack the N-terminal and C-terminal interaction capacity of AR (A). Collectively, these results indicate that AR nuclear localization, but not transcriptional activity, is required for the AR-mediated repression of CRE.We used AR mutants to further investigate the mechanism by which AR suppressed CRE activation. As shown in Figure 6n signal , was unan signal B. Other ty of AR ; ΔpolyQ+ty of AR ; and C80ty of AR  had no ety of AR A. CollecA). In contrast, Flag-CREB was not coimmunoprecipitated with a mutant AR (K630A/K632A/K633A) lacking nuclear localization ability (B). This suggests that the nuclear AR interacts with CREB. We further assessed the ability of AR to interact with CREB using a modified mammalian two-hybrid assay in which a nuclear receptor ligand dependently transactivates a luciferase reporter gene when the receptor binds to a bait protein. This assay allows the evaluation of binding between a nuclear receptor  and a bait protein  only in the presence of an agonist for a nuclear receptor . When GAL4DBD-fused CREB was expressed, the luciferase reporter was induced, presumably by the transcriptional activity of CREB. This is supported by the finding that reporter gene activity was significantly activated in the presence of forskolin. Moreover, the ability of GAL4DBD-fused CREB to stimulate reporter gene activity was further enhanced by AR in a DHT-dependent manner (C). The binding site of CREB that interacts with AR was determined using a modified mammalian two-hybrid assay. Luciferase reporter activity was significantly enhanced by AR activation when CREB mutants containing the N-terminal region (amino acids 1–101) were used as the bait protein (D). Although AR activation also enhanced luciferase activity when CREB mutants not containing the 1 to 101 region were used as the bait protein , they did not reach statistical significance in multiple comparisons. These results suggest that the N-terminal region of CREB is effective but that multiple regions of CREB may be involved in binding to the AR. It was previously shown that the N-terminal domain (NTD) of AR can form a protein complex with CREB bridged by p300 (E). In contrast, p300 had no effect on the complex formation when the NTD of AR was replaced with full-length AR (F). An immunoprecipitation assay supported the notion that the binding of AR and CREB is not affected by p300 (G).We examined the protein interactions between overexpressed AR and CREB using immunoprecipitation in HEK293FT cells. Flag-CREB was coimmunoprecipitated with both ligand-bound and ligand-unbound AR, irrespective of forskolin stimulation A. In con ability B. This st manner C. The bi protein D. Althou by p300 E. In conength AR F. An imm by p300 G.Figure Ucp1 expression based on studies with primary and immortalized brown adipocytes . The latter was supported by the evidence that BAT β3-AdR protein levels do not differ between young adult male and female young adult rats . These results suggest that full-length AR can form protein complex with CREB even in the absence of p300.AR inhibited CRE-mediated transcription by suppressing CREB phosphorylation, presumably by forming a protein complex with CREB. This action of AR was independent of its transcriptional activity but required AR nuclear localization. This result would lead to an outcome opposite to that of a pharmacological dose of testosterone, which can inhibit PDE activity and increase cAMP levels . In addi., TRPM8 , OXER1 . Collec., TRPM8 . The NTD., TRPM8 , which wvia hyperthermia.Meta-analyses of epidemiological studies indicate that a low testosterone level increases the risks of cardiovascular and all-cause death , 60, altIn this study, we show that androgen reduces adrenaline-induced thermogenesis by suppressing adrenaline-activated CREB phosphorylation in BAT. A limitation of this study is that it was performed in mice, and further studies are needed to determine if the current results observed in mice are consistent in humans, as some differences are seen between mouse and human BAT. Since CREB has various physiological roles , crosstacre; ArΔ/y) were generated using the Cre-loxP system as described previously  and a 12:12-h light: dark cycle (lighting period starting at 08:00) with ad libitum access to standard chow food  and water. Rectal temperature was measured with a thermometer  between 14:00 and 15:00 to evaluate weekly changes. At 12 weeks of age, sham-operated and castrated mice were intraperitoneally injected with vehicle  between 13:30 and 14:30, and body temperature was analyzed for 3 h. The next day, the vehicle was changed to 0.1 mg/kg CL316243 and body temperature was again measured for 3 h. Mice were euthanized at 13 weeks of age under anesthesia 2 h after injection with 0.1 mg/kg CL316243 (between 13:30 and 14:30), and the plasma and organs were obtained for further analysis. Free fatty acid level in plasma was determined using the NEFA C-Test . Mice from the two groups were alternately treated and dissected. All animal experiments were approved by the Animal Care and Use Committee of Osaka Prefecture University  and were performed in compliance with its guidelines.Male global ARKO mice (CAG-eviously . Brieflylox mice  (C57BL/6cre mice  (C57BL/62 and 95% air atmosphere.Mouse brown adipocyte T37i cells (Sigma-Aldrich) or HEK293FT cells (Thermo Fisher Scientific) were cultured in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 Ham (1:1 Mixture) or Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 mg/ml streptomycin. Steroid-free medium was prepared using dextran-coated charcoal-treated 10% fetal bovine serum and phenol red-free medium and used for all experiments for the determination of ligand dependency. Cells were maintained at 37 °C in an atmosphere containing 5% COUcp1 promoter and TK promoter from pUCP1-pro-Luc (Mutant AR expression vectors pcDNA3.1-AR(K630A/K632A/K633A) and pcDNA3.1-ARC806A), in which Ala was substituted for Lys630/Lys632/Lys633 or Cys806, respectively, were constructed by site-directed mutagenesis using specific primers (sense: 5′-CTCTGGGAGCCCGGGCACTGGCAGCACTTGGTAATCTGAAAC-3′ and antisense: 5′-GTTTCAGATTACCAAGTGCTGCCAGTGCCCGGGCTCCCAGAG-3′ or sense: 5′-CCCAGGAATTCCTGGGCATGAAAGCACTGC-3′ and antisense: 5′-GCAGTGCTTTCATGCCCAGGAATTCCTGGG-3′) and pcDNA3.1-AR  as a tem-pro-Luc  were ins-pro-Luc  and CRE406A, in w4 cells/well using steroid-free medium and cultured overnight. For determination of CRE- or androren response element (ARE)-mediated transcriptional activity, cells were transfected with β3-AdR expression vector (pcDNA3.2-β3-AdR-V5 (Renilla luciferase reporter vector (pGL4.74[hRluc/TK] (Promega)) with PEI MAX (Polysciences Inc) and Opti-MEM (Thermo Fisher Scientific) for 24 h. For the mammalian two-hybrid assay, cells were transfected with pGAL-CREB, pcDNA3.1-AR or pACT-AR (NTD)  30 min prior to DHT treatment. Luciferase reporter activity was determined as described previously (HEK293FT cells were seeded in 48-well plates at 6.0 × 103-AdR-V5 ), AR exp3-AdR-V5 , pcDNA3.3-AdR-V5 , 75, pcD3-AdR-V5 , pcDNA3.3-AdR-V5 , pcDNA3.3-AdR-V5 , pcDNA3.3-AdR-V5 , pGL4-AR3-AdR-V5 , or pUCPAR (NTD) , pG5luc eviously . Data arAB_10699020), monoclonal anti-pCREB , polyclonal anti-AR , 1/3000, GL Biochem), and monoclonal anti-β-actin  antibodies. After immunoreaction with horseradish peroxidase-conjugated secondary antibodies , immunoreactive bands were developed using Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore) and detected using an LAS4000 imager . Densitometry measurements were performed by using ImageJ software .BAT was homogenized in RIPA buffer containing phosphatase and protease inhibitors . HEK293FT cells were plated in steroid-free medium and transiently transfected with pcDNA3.1-AR for 24 h. Cells were cultured in the presence of 10 nM DHT for 16 h, followed by stimulation with 10 μM forskolin for 5 min. Cells were washed twice with TBS and harvested and lysed in buffer containing phosphatase and protease inhibitors . After sonication and centrifugation of the tissue and cell culture lysates, the supernatant was subjected to SDS-PAGE, followed by Western blotting with monoclonal anti-CREB  was incubated with 1.5 μg of normal rabbit IgG (FUJIFILM Wako) or affinity purified anti-AR RB3838 IgG overnight at 4 °C, followed by reaction with Dynabeads Protein G (Veritas) for 2 h. The resin was washed four times with the same buffer, and proteins bound to the resin were analyzed by Western blotting.HEK293FT cells were plated in steroid-free medium and transiently transfected with pcDNA3.1-AR or pcDNA3.1-AR (K630A/K632A/K633A), pCF CREB, and pSG5 or pSG5-HA-p300 with PEI MAX for 24 h. Cells were cultured in the presence of 10 nM DHT for 16 h, followed by stimulation with 10 μM forskolin for 5 min. Cells were washed twice with PBS and harvested and lysed in buffer  on ice for 30 min. After centrifugation at 20,000Ex Taq II DNA polymerase (Takara Bio) and Thermal Cycler Dice TP870 (Takara Bio). The sequences of the specific primers for target genes and their annealing temperatures are described in Total RNA was isolated from mouse tissues using Sepasol RNA I super G  by disrupting cells with zirconia beads using a Multi-Beads Shocker (Yasui Kikai Corp), followed by treatment with DNase I. Complementary DNA was synthesized from total RNA using ReverTra Ace (TOYOBO), oligo(dT)20 primer, and random hexamer primers. Real-time PCR was performed using TB Green Premix t test or one-way ANOVA with Tukey-Kramer’s or Dunnett’s post hoc tests using JMP statistical software . Data are shown as means ± SD, and a p-value (p) < 0.05 was considered statistically significant. Significance is indicated in the figures by asterisks or different letters. Bars not sharing the same letter show significant differences between the groups.Data were analyzed by Student’s Data are available within the article or its supplementary materials.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Adrenergic receptors (AR) belong to the G protein-coupled receptor superfamily and regulate migration and proliferation in various cell types. The objective of this study was to evaluate whether β-AR stimulation affects the antiproliferative action of α2-AR agonists on B16F10 cells and, if so, to determine the relative contribution of β-AR subtypes. Using pharmacological approaches, evaluation of Ki-67 expression by flow cytometry and luciferase-based cAMP assay, we found that treatment with isoproterenol, a β-AR agonist, increased cAMP levels in B16F10 melanoma cells without affecting cell proliferation. Propranolol inhibited the cAMP response to isoproterenol. In addition, stimulation of α2-ARs with agonists such as clonidine, a well-known antihypertensive drug, decreased cancer cell proliferation. This effect on cell proliferation was suppressed by treatment with isoproterenol. In turn, the suppressive effects of isoproterenol were abolished by the treatment with either ICI 118,551, a β2-AR antagonist, or propranolol, suggesting that isoproterenol effects are mainly mediated by the β2-AR stimulation. We conclude that the crosstalk between the β2-AR and α2-AR signaling pathways regulates the proliferative activity of B16F10 cells and may therefore represent a therapeutic target for melanoma therapy. Melanoma develops in melanocytes, which are the cells that produce melanin, and is the most aggressive and life-threatening skin cancer. The limited success of classic chemotherapeutics has led to the introduction of targeted therapies aimed at tackling detected oncogenic mutations and/or other DNA aberrations underlying melanomagenesis. Several novel drugs that target the MAPK pathway, the kinase MEK, a member of the MAPK family, or immune checkpoints, key regulators of the immune system, have been developed . HoweverIn recent years, preclinical and clinical studies have provided evidence that catecholamines, which are released in response to physical or emotional stress, can affect the growth and metastasis of many types of cancer, including melanoma. Once released, catecholamines, such as norepinephrine and epinephrine, can activate both β- and α-adrenergic receptors, promoting the physiological responses that follow stimulation of the sympathetic nervous system. β-adrenergic receptors (β-AR), which belong to the guanine nucleotide-binding G protein-coupled receptor superfamily, are expressed in human tissues, such as benign melanocytic naevi, atypical naevi and malignant melanoma, as well as in B16F10 melanoma, the most frequently used syngeneic murine melanoma, as evidenced by qPCR, Western blot and cytofluorimetric analyses ,3,4,5, aIn a recent study , we repoThe objective of this study was threefold: first, determining whether tumor β-ARs are functioning, that is, whether their stimulation causes an increase in cAMP levels; second, determining whether clonidine, the prototypical α2-AR agonist acting on all three α2-AR subtypes, affects B16F10 cell proliferation; third, assessing whether β-AR stimulation by the catecholamine isoproterenol modulates the antiproliferative action of clonidine on B16F10 cells and, if so, evaluating to what extent each β-AR subtype contributes to regulating the proliferative activity of B16F10 cells. Using Ki-67 protein staining, luciferase-based cAMP assay, flow cytometry and B16F10 melanoma cells, we found that activation of β2-ARs with isoproterenol increases cAMP levels in cancer cells, indicating that these receptors are functioning. We also found that β-AR subtype stimulation, on its own, has no effect on cancer cell proliferation, suggesting that β-AR activation has no direct tumor-promoting effects. Finally, we observed that the stimulation of the β2-AR suppresses the antiproliferative effects of α2-AR stimulation on B16F10 cells, whereas both the β1-AR and β3-AR appear to play only a marginal role. Thus, the present study establishes a crosstalk between the β2-AR and α2-AR signaling pathways that is involved in regulating B16F10 melanoma cell proliferation.B16F10 cells express β1-, β2- and β3-ARs, as evidenced by qPCR, Western blot and cytofluorimetric analyses ,5. Here,The cAMP levels increased after isoproterenol treatment A,B. Agonp < 0.05 vs. CTR) and clonidine  compared to control (p > 0.05), it prevented both ST-91  and clonidine  antiproliferative effects . This would seem to indicate that the effect of these drugs was essentially mediated through the α2-AR.Since we could not rule out an effect of ST-91 and CLO also on α1-AR, at least at the concentration of 1 μM, we also used YM-254890 (YM), a selective inhibitor of the Gq protein subfamily. In fact, the α1-AR is associated with the Gq protein, unlike the α2-AR, associated with the Gi protein, and this allowed us to discriminate the effects related to the activation of the two receptors. We did not observe any effect on proliferation, either in cells treated with YM or when YM was administered in dual treatment with ST-91 or CLO .To assess the relative role of each β-AR subtype in modulating the antiproliferative action of the α2-AR agonists, B16F10 cells were treated for 48 h with the following AR ligands: isoproterenol (ISO), ST-91, clonidine (CLO), propranolol (PRO), ICI 118,551 (ICI), isoproterenol plus propranolol (PRO), clonidine plus isoproterenol, or ST-91 plus isoproterenol . We founp > 0.05 vs. CTR) compared to control, suggesting that β3-AR stimulation does not regulate cell proliferation on its own under these experimental conditions  and clonidine  on B16F10 cells  supplemented with 5% FBS  in the presence of penicillin and streptomycin .A B16F10 cell line was cultured at 37 °C in humidified atmosphere containing 5% COFor cAMP experiments, B16F10 cells were infected with Puro-resistant retrovirus encoding the luciferase-based probe GloSensor 22 F, followed by puromycin selection (5 μg/mL). The luciferase-based intracellular cAMP probe GloSensor 22 F was purchased from Promega  and subcloned into puromycin resistance retroviral expression vector pQCXIP .To select cell lines with optimal expression of the biosensor, virally transduced cells were plated at very low density in 15 cm dishes, and 50–100 individual clones from each cell line were isolated with silicon rings for further screening. The cell clones with the best response to forskolin were identified by luminescence recording in 96-well plates as described .B16F10 cells stably expressing GloSensor-22F probe were seeded into 96-well white plastic plates at a density of 2 × 104 cells/well and grown for an additional 24 h. For cAMP assay, the wells were washed once with PBS and further incubated for 60 min in 50 μL PBS containing 25 mM glucose and 2 mM luciferin. Next, 50 μL PBS containing 10 μM rolipram without or with β-adrenergic ligands  diluted in 0.1% BSA in PBS or 100 μM forskolin, used as a positive control, were added to the wells, and the plates were transferred in a luminometer . Luminescence from each well was counted every 30 s with 0.5 s integration time for about 80 min. cAMP response was determined as area under the luminescence–time curves A.Mouse melanoma B16F10 cells were cultured as described above. For proliferation experiments, the cells were treated for 48 h with 1 μM of the β-AR agonist isoproterenol (ISO), the β1- and β2-AR antagonist propranolol, the β2-AR antagonist ICI 118,551 (ICI), the α2-AR agonists ST-91  and clonidine, or the α2-AR antagonist yohimbine (YOH), alone or in different combinations. In another set of experiments, the cells were treated with the non-selective phosphodiesterase inhibitor IBMX (1 μM) or the adenylyl cyclase activator forskolin  for 48 h. To exclude the involvement of the α1-AR in the antiproliferative effects induced by ST-91 and clonidine, we added YM-254890 (1 μM), a selective inhibitor of the Gq protein subfamily able, therefore, to selectively inhibit α1-AR (associated with Gq), to ST-91 or CLO, respectively. Cells left untreated or treated with a drug’s vehicle were considered as the control (CTR). Adrenergic ligands were purchased from Sigma-Aldrich , except for ST-91 and YM-254890 .Proliferative activity was analyzed by Ki-67 nuclear antigen expression using the fluorescein isothiocyanate (FITC) mouse anti-human Ki-67 after cell fixation and permeabilization according to the manufacturer’s protocol . All samples were acquired on a FACSCalibur flow cytometer  equipped with a 488 argon laser and a 635 red diode laser, and at least 10,000 events per sample were run. Data were analyzed using the Cell Quest Pro software . Results were reported as median fluorescence intensity (MFI).v/v) for 10 min at room temperature and air-dried. After 1 h of preincubation with PBS containing 10% of AB human serum, the cells were incubated for 1 h at room temperature with FITC-conjugated mouse anti-human Ki-67 (BD Biosciences). The nuclei were stained with Hoechst 33258 (Sigma-Aldrich) at 37 °C for 15 min. The samples were mounted on glass cover slips with glycerol/PBS (2:1) and observed by intensified video microscopy (IVM) with an Olympus Microphot fluorescence microscope  equipped with a Zeiss CCD camera.For nuclear detection of Ki-67, control and treated cells were fixed in acetone/methanol 1/1 . Unpaired t test was performed to accept or reject the null hypothesis. Differences with