bluuebunny/crossref_metadata_2025_split huggingface dataset:
BLES‐Cath‐2 Intratracheal Administration in an in vivo P. aeruginosa Pneumonia Model
Authors: Nicholson John Michael , Banachewski Brandon , McCaig Lynda , Veldhuizen Edwin , Yamashita Cory , Veldhuizen Ruud 
Published: November 2023

BackgroundIncreasing patterns of antibiotic resistance, especially in individuals with chronic lung disease, have mandated the development of novel therapies. Host‐defense peptides HDP such as cathelicidin‐2 (CATH‐2), possess excellent antimicrobial activities in vitro however in vivo efficacy has not been well established. Furthermore, in order to enhance in vivo distribution, a novel approach using CATH‐2 combined with bovine lipid extract surfactant (BLES) as a delivery vehicle was utilized. It was hypothesized that CATH‐2/ BLES would be superior to CATH‐2 alone in decreasing bacterial burden and improving physiologic parameters in an in vivo model of Pseudomonas aeruginosa (PA) infection with mechanical ventilation.MethodsAdult male Sprague‐Dawley rats were intubated, mechanically ventilated for 30 minutes, followed by an intratracheal inoculum of 8 x 10^8 CFU/mL of PA. One hour following PA administration, rats were randomized to receive an intratracheal administration of either: i) Saline, ii) Air, iii) BLES, iv) Cath‐2, or v) BLES/Cath‐2. Mechanical ventilation continued for three hours during which peak inspiratory pressure (PIP) and arterial blood gases were recorded. At the completion of the ventilation protocol, animals were sacrificed and whole lungs were homogenized and the bacterial burden was assessed by growth analysis of lung homogenates on Cetrimide Agar plates incubated overnight at 37 C. Statistical analysis was performed using a one‐way ANOVA.ResultsPhysiological measurements revealed a significant decline of arterial oxygenation (PaO2) in air and saline control groups. At the three‐hour time point, the BLES/Cath‐2 group had a significantly (p < 0.01) higher PaO2 and compared to the Cath‐2 treated animals. Interestingly, the Cath‐2 group had PIP values at 30 minutes following PA instillation that were significantly higher compared to all groups. Colony growth data demonstrated a significant difference between the Cath‐2 treated animals and the control groups; however there was no difference compared to the BLES/Cath‐2 group.ConclusionOur findings demonstrate that Cath‐2 has excellent anti‐microbial activity against PA in an in vivo model of pneumonia and mechanical ventilation, although significant reduction in lung function was observed. Cath‐2 alone worsens lung function by possibly causing an overwhelming inflammatory response. Furthermore, CATH‐2/ BLES significantly improved physiological parameters compared to Cath‐2 alone. This suggests that the combination of BLES/Cath‐2 allows for both improved lung function and while maintaining antimicrobial activity. Future analysis includes extension of the time points and lavage inflammatory markers.Support or Funding InformationPhysician Services Inc. and the Canadian Cystic Fibrosis foundation
A Novel Assay to Screen Porcine MHC Class II Antibodies in Potential Xenotransplant Recipients
Authors: Ladowski Joseph M , Butler James , Martens Greg , Reyes Luz , Wang Zheng‐Yu , Tune John , Tector Matt , Tector A. Joseph 
Published: November 2023

BackgroundWell‐elucidated in clinical allotransplantation is the role of recipient's donor specific antibodies and graft rejection, specifically the antibodies targeted at Human Leukocyte Antigen (HLA) class II. To determine whether cross‐reactive or preformed antibodies exist to Swine Leukocyte Antigen (SLA) class II, an assay was developed to screen the patients on the Indiana University (IU) transplant waiting list.Methods69 patients on the IU transplant waiting list were selected for study based on their levels of anti‐HLA class II antibodies. Wild‐type pig red blood cells were used to absorb the sera, removing anti‐carbohydrate antibodies and leaving anti‐MHC antibodies. This sera was screened on an immortalized fibroblast cell line transfected with a version of the human class II transactivator (CIITA), producing a constitutively activate SLA class II cell line. A cell that incorporated the antibiotic resistance gene but not the CIITA was chosen as a negative control. Following incubation, the sera was evaluated for presence of IgM and IgG antibody binding by flow cytometry.ResultsCells lacking expression of SLA class II demonstrated decreased IgM and IgG binding compared to cells with 100% SLA class II DR and DQ expression (Figure 1). Of note are several individuals whose sera preferentially binds the class II positive cell (the dots beneath the diagonal line). The difference in binding between a positive and negative cell is statistically different for IgG (p = 0.0048) but not IgM (p = 0.5102). Increased binding is found when evaluating those individuals with anti‐HLA class II antibodies (Figure 2). The statistical significance holds when comparing the change in binding on the SLA class II positive and negative cells for individuals with and without anti‐HLA class II antibodies for IgG (p = 0.0341) and IgM (p = 0.2602).ConclusionsExpression of SLA class II results in increased IgM and IgG human antibody binding, demonstrating the presence of human anti‐pig class II antibodies. The existence of this humoral barrier suggests that antibody‐mediated binding to SLA class II may be an issue faced in clinical xenotransplantation.Support or Funding InformationThis publication was made possible in part by Grant Number UL1 TR001108 (A. Shekhar, PI) from the National Institutes of Health, National Center for Advancing Translational Sciences, Clinical and Translational Sciences Award.
Modeling Chronic Alcohol‐Stimulant Co‐use Shows Dampening of Alcohol Withdrawal Severity
Authors: Nanchanatt Alex J. , Popkin Shayna , Mauterer Madelyn , Rhoads Dennis E. 
Published: November 2023

Co‐use of alcohol and stimulants, including caffeine and amphetamine, may be occurring at an all‐time high level of frequency among students, college age and younger. In previous studies, application of the liquid diet approach allowed us to test alcohol‐caffeine co‐administration to adolescent rats and showed that caffeine reduced alcohol withdrawal severity, but only when adolescents were administered caffeine before and then along with alcohol. The first objective of the present study was to test the hypothesis that amphetamine would have a similar dampening effect on alcohol withdrawal severity. Adolescent rats were assigned randomly to one of 4 groups: pair‐fed control, ethanol, ethanol plus amphetamine, and amphetamine alone. At the doses used, amphetamine had no significant effect on alcohol consumption. After 7 days of alcohol consumption and 6 hours of withdrawal, rats showed significant hypoactivity as measured in computer controlled activity chambers. In sharp contrast, inclusion of amphetamine with alcohol resulted in activity measures indistinguishable from pair‐fed controls. Amphetamine alone had no effect on the activity measures during the same period of administration and withdrawal. After an additional 11–14 days of alcohol consumption, a more severe withdrawal syndrome develops with high frequency of induced running and convulsions (withdrawal severity score = 3.9). Inclusion of amphetamine with ethanol under these conditions resulted in a drastic reduction in the alcohol withdrawal severity score to 1.4. This occurred without the pretreatment period required with caffeine but otherwise amphetamine resulted in a corresponding decrease in alcohol withdrawal severity. The dopamine system plays an important role in the actions of both stimulants. Thus, our second objective in the present study was to determine whether changes in dopamine receptor protein expression correlated with the observed behaviors. Membrane fractions were obtained from cortical and subcortical forebrain preparations and levels of dopamine receptor expression were evaluated by Western blotting. The most interesting results were obtained with expression of D1 receptors in subcortical preparations. D1 receptors were down‐regulated by alcohol, up‐regulated by caffeine and not significantly different from pair‐fed controls under conditions where caffeine decreased alcohol withdrawal. These findings parallel behavioral results and, while these may not be the only receptor changes occurring during administration of these drugs, they lead us to suggest that antagonistic effects of alcohol and stimulants at the level of dopamine D1 receptor expression may serve to reduce the severity of alcohol withdrawal.Support or Funding InformationSupported by the Biology Department of Monmouth University.
mTOR Pathway Inhibition Prevents Neuroinflammation and Neuronal Death in a Mouse Model of Spinal Cord Injury
Authors: Esposito Emanuela , Cordaro Marika , Paterniti Irene , Impellizzeri Daniela , Siracusa Rosalba , Cuzzocrea Salvatore 
Published: November 2023

Mammalian target of rapamycin (mTOR) pathway signaling governs cellular responses, hypoxia and inflammation including induction of autophagy and cell survival. Spinal cord injury (SCI) is a serious and debilitating health problem that usually causes lifelong disability and leads to neurological dysfunction. While the mTOR signaling cascade has been linked to several neurological disorders, manipulation of the mTOR signaling cascade as a pre‐clinical therapeutic strategy in SCI has not been investigated. We hypothesized that mTOR pathway inhibition would diminish neuroinflammation and prevent neuronal death in a mouse model of SCI.Spinal cord compression injury was performed in adult male mice. SCI was induced in mice by the application of an aneurysm clip at T6‐T7 level. mTOR pathway inhibition was achieved with rapamycin (a mTOR inhibitor; 1mg/kg), or temsirolimus (rapamycin analogues; 0.6mg/kg), or KU0063794 (a dual mTORC1and mTORC2 inhibitor; 8mg/kg) following spinal cord trauma (1 and 6 hours after SCI), and then for 3 subsequent days. Phospho‐activation of the mTOR effectors p70S6kinase and ribosomal S6 protein and expression of infammatory parameters in perilesional area were assayed at 24 hrs, 48 and 72 hrs. Neuronal cell death was evaluated, autophagy was measured using Beclin‐1 and LC3II expression. Iba‐1 labeled, activated microglia were quantified.Neuronal death, and numerous Iba‐1 labeled, activated microglia were evident at 24 and 48h following SCI. Rapamycin or KU0063794 treatment significantly reduced mTOR signaling, neuronal death, and microglial activation, coincident with enhanced expression of Beclin‐1 and LC3II, markers of autophagy induction. KU0063794 was able to blunt the neuroinflammation (evaluated as iNOS, COX2, GFAP and NOS expression) better than rapamycin and temsirolimus.mTOR pathway inhibition prevented neuronal death and diminished neuroinflammation in this model of SCI. Persistent mTOR signaling following SCI suggests a failure of autophagy induction, which may contribute to neuronal death. These results suggest that mTOR signaling may be a novel therapeutic target to reduce neuronal cell death in SCI.Support or Funding InformationNo funding
Memory enhancement induced by PDE2 knockdown in an Alzheimer's disease model of mice
Authors: Xu Ying , Chen Zhuoyou , Pan Jianchun , Zhang Han‐Ting , O'Donnell James M. 
Published: November 2023

PDE2 is the most prevalent of all PDEs expressed in the hippocampus and frontal cortex, regions involved in cognition and especially vulnerable to damage at early stages of AD. Studies suggest that beta‐amyloid 1‐42 (Abeta 1‐42) has a detrimental effect on memory performance that induces the initial and major symptom of Alzheimer's disease (AD) via inhibition of both cAMP/cGMP‐dependent pathway. Microinfusion of Abeta in animal models leads to Abeta deposition, inflammation, neuroapoptosis and memory impairment. However, whether phosphodiesterase 2‐dependent cAMP and/or cGMP pathway is involved in the neuroinflammation and apoptosis that lead to the progression of AD are unclear. The present study found that intraventricular infusion of Abeta1‐42 into bilateral CA1 subregions led to impairment of learning and memory, as evidenced by an increased latency to find the platform and decreased number of platform crossing in the probe trial of the water maze task and decreased 24‐h retention in the step‐down passive avoidance test. The time period that induced the behavioral abnormalities lasted from 2 weeks to 6 months, with the maximal abnormality at 4 months after microinfusion with Abeta 1‐42. These behavioral abnormalities were blocked by microinfusion with lenti‐PDE2‐miRNA at 5′106 tu/ml. Four months after microinfusion with Abeta were also found to increase in the expression of IL‐6, IL‐10, IL‐17 and Bax. The decreased expression of BCl‐2, PKA, PKG and brain‐derived neurotrophic factor (BDNF) in the hippocampus were also significant in these Abeta1‐42‐treated mice. These effects were reversed by treatment with lenti‐PDE2‐miRNA at 5′106 tu/ml. These findings suggest that inhibition of PDE2 would reverse Abeta‐induced learning and memory deficits at least in part by its regulation of neuroinflammatory and apoptotic processes, which are involved in cAMP/cGMP‐BDNF signaling.Support or Funding InformationThis study was supported by 2014–2015 American Association of Colleges of Pharmacy New Investigator Award and Changzhou Sci&Tech Program Grant (No. CJ20130023).
Opicapone improves the effects of L‐DOPA on the MPTP‐induced Parkinson's‐like syndrome in cynomolgus monkeys
Authors: Bonifacio Maria Joao , Medakkar Sonali , Sousa Filipa , Vivian Jeffrey A , Soares‐da‐Silva Patricio 
Published: November 2023

BackgroundOpicapone is a high binding affinity and long‐acting third generation nitrocatechol COMT inhibitor under development for use as adjunctive therapy in levodopa‐treated Parkinson's disease (PD) patients.ObjectivesThe aim of this study was to evaluate the effects of opicapone, as adjunctive therapy to levodopa, in reversing a MPTP‐induced Parkinson's‐like syndrome in cynomolgus monkeys (Macaca Fascicularis).MethodsInduction of a PD‐like syndrome was performed by daily intravenous administrations of MPTP. Characterization of the animals included scoring with the Primate Parkinsonism Motor Rating Scale (PPMRS) and assessment of locomotor activity.ResultsMPTP produced a stable Parkinson's‐like behavioural syndrome as evidenced by tremor, postural changes, rigidity, impaired movements and balance, (i.e., PPMRS scores between 10 and 15) and decreased locomotor activity (13% of their pre‐MPTP values). Opicapone treatment per se, for 14 days, did not change either the PD‐like symptoms or the decreased locomotor behaviour of the subjects. Ascending combinations of levodopa/benserazide dose‐dependently decreased PPMRS reaching statistical significance for the levodopa/benserazide doses of 18/4.5 mg/kg and that effect was further enhanced in opicapone treated subjects. Similarly, levodopa/benserazide dose‐dependently increased locomotor behaviour, reaching significance with levodopa/benserazide doses of 18/4.5 mg/kg. This increase was also potentiated in the presence of opicapone. Opicapone treated subjects as compared to those treated with vehicle, had significantly increased plasma levodopa levels (2‐fold higher AUC) with no changes in Cmax while for 3‐OMD, AUC and Cmax values were both significantly reduced 7–8 fold. Erythrocyte COMT activity was markedly reduced in opicapone treated subjects.ConclusionsOpicapone potentiated the improvements in PD‐like symptoms produced by levodopa/benserazide combinations with a concomitant increase in plasma levodopa exposure, reduction of plasma 3‐OMD levels and erythrocyte COMT activity.
Alpha‐1D‐Adrenoceptors Lacking Their Carboxyl Terminus Inhibit The MAPK/ERK Pathway in Rat‐1 Fibroblasts
Authors: Alfonzo‐Méndez Marco A. , Castillo‐Badillo Jean A. , Romero‐Ávila M. Teresa , García‐Sáinz J. Adolfo 
Published: November 2023

Human alpha‐1D‐adrenoceptors are G protein‐coupled receptors that mediate the actions of adrenaline and noradrenaline participating in the maintenance of homeostasis as well as in the physiopathology of some diseases, such as hypertension and benign prostatic hyperplasia. There is a growing interest in investigating the function of their regulatory domains, such as the carboxyl tail. However, studies focused on the participation of this domain are limited. The objective of this work was to study the role of the human alpha‐1D‐adrenoceptor carboxyl tail in activation of the mitogen‐activated protein kinase pathway. To accomplish this, we assessed changes in extracellular signal‐regulated kinase (ERK) phosphorylation using biochemical and biophysical approaches.We found that Rat‐1 fibroblast expressing amino and carboxyl termini‐truncated (ΔNΔC) alpha‐1D‐adrenoceptor mutants were hardly able to activate the MAP kinase pathway in response to noradrenaline. Furthermore, expression of this mutant also decreased ERK activation induced by activation of other endogenous G protein‐coupled receptors and receptor tyrosine kinases, i. e., this mutant receptor alters downstream step(s) in the mitogen‐activated protein kinase pathway. As part of the mechanism, we showed that alpha‐1D‐adrenoceptor‐mediated ERK activation is PKC‐dependent. Moreover, we identified the inhibition of two key elements in the pathway downstream PKC: Raf‐1 and MEK1. In addition, we found that over‐expression of the alpha‐1D‐adrenoceptor third intracellular loop cannot mimic the effect of the carboxyl tail truncation, but is enough to activate ERK pathway. Our data indicate that both the alpha‐1D‐adrenoceptor carboxyl terminus and the third intracellular loop play roles in ERK signaling.Support or Funding InformationThis research was partially supported by Grants from PAPIIT‐DGAPA‐UNAM and CONACYT.
No Longer “The Dog That Did Not Bark”: Discovery of Xenobiotic Phosphorylation Metabolism of a TNKS‐Wnt Inhibitor When Dosed To Mice or Incubated In Mouse Whole Blood
Authors: Gu Chungang (Chuck) , Doig Peter , Gangl Eric , Johannes Jeffrey W , Wen Shenghua 
Published: November 2023

The phosphorylation of xenobiotics has been deemed impossible and dubbed as “the dog that did not bark” by Andrew Parkinson.1 This was rationalized for a number of reasons, such as limiting consumption of ATP in cells by xenobiotics, avoiding interference with intracellular and intranuclear signaling, and the hypothesis that the phosphorylated metabolites are unlikely to be excreted through the cell membrane if formed in hepatocytes or other cells.A pyrimidinone nicotinamide mimetic (compound 1: (S)‐2‐(4‐(6‐(3,4‐dimethylpiperazin‐1‐yl)‐4‐methylpyridin‐3‐yl)phenyl)‐8‐(hydroxymethyl)quinazolin‐4(3H)‐one) was identified as selective tankyrase and wnt pathway inhibitor.2 When compound 1 was dosed orally in mice, an unexpected phosphorylated metabolite was formed at the benzyl alcohol. This was observed in mouse plasma with AUC at approximately 5–10% of the parent drug. The phosphorylation showed a temporal profile and a dose response that are consistent with metabolite formation. The metabolite was unambiguously characterized by accurate mass measurement and LC‐MS/MS comparison to the authentic synthetic standard in retention time and fragmentation. Numerous control experiments have been done to rule out analytical, sampling, and dosing errors.To further confirm this xenobiotic phosphorylation, as well as our 1st step to identify the mechanism, the phosphorylated metabolite was generated after incubation of 10 μM of compound 1 at 37 °C for 2 h in mouse fresh whole blood, fortified with 5 mM ATP and 10 mM MgCl2. Heparin instead of EDTA was used as the anticoagulant to prevent chelation of the Mg2+. Interestingly, the phosphorylated metabolite was not produced in mouse fresh plasma under exactly the same incubation conditions. In addition, this phophorylated‐metabolite was not detected after incubation in a phosphate‐buffered saline solution (+ATP).
High Fat Diet Feeding Alters Expression of Hepatic Drug‐metabolizing Enzymes in Mice
Authors: Ning Miaoran , Jeong Hyunyoung 
Published: November 2023

Obesity is prevalent in U.S. that more than one‐third of adults are considered to be obese. Medical conditions accompanying obesity (e.g., metabolic diseases) often require drug therapy. However, whether and how obesity alters the expression of drug‐metabolizing enzyme and thus drug pharmacokinetics is poorly defined. Previous studies have shown that high fat diet feeding and subsequent obesity in mice lead to altered expression of transcriptional regulators for cytochrome P450 (CYP) 2D6, including hepatocyte nuclear factor 4α (HNF4α, transcriptional activator of CYP2D6) and small heterodimer partner (SHP, transcriptional repressor of CYP2D6) (1, 2). The objective of this study is to examine whether diet‐induced obesity represses CYP2D6 expression by modulating HNF4α and SHP expression. To this end, male CYP2D6‐humanized transgenic (Tg‐CYP2D6) mice were fed with western diet (WD, TD.88137, 42% kJ from fat) or matching control diet (TD.08485, 13% kJ from fat) for 18 weeks. qRT‐PCR results showed that hepatic expression of CYP2D6 decreased to a small extent (1.5‐ and 1.2‐fold at mRNA and protein levels, respectively). CYP2D6 activity (tested by using a CYP2D6 probe drug debrisoquine) in S9 fractions prepared from the liver tissues did not differ between the groups. While hepatic SHP expression did not differ between the groups, HNF4α mRNA and protein levels decreased 1.5‐fold in WD group. Considering that HNF4α is a global regulator of hepatic genes including other drug‐metabolizing enzyme genes, we examined the expression of mouse endogenous CYPs (Cyp1a2, 2b10, 2c37, 2d40, 2e1, and 3a11). Cyp1a2, 2c37 and 2d40 showed significant decreases in WD group; Cyp2e1 expression did not differ between groups; and Cyp2b10 and 3a11 expression increased significantly. CYP3A activities (in S9 fractions, determined by using triazolam as a substrate) in WD‐fed mice were 2.9‐fold higher as compared with those in control diet group. Notably, the expression of transcription factors that mediate upregulation of Cyp3a11 and Cyp2b10 (pregnane X receptor and constitutive androstane receptor, respectively (3)) did not increase in WD‐fed group, suggesting that increased concentrations in ligands of these transcription factors might be responsible for Cyp3a11 and Cyp2b10 induction in obese mice. Together, these results suggest that obesity represses CYP2D6 expression potentially through decreased HNF4α expression although the magnitude of the change in CYP2D6‐mediated drug metabolism was small. Obesity had differential effects on mouse endogenous CYPs.Support or Funding InformationThis study was supported by NIH (GM112746 and HD065532)