bluuebunny/crossref_metadata_2025 huggingface dataset:
Anatomical risk factors for internal carotid artery dissection: a novel metric (LB50)
Authors: Irizarry Rachel , Hartley Benjamin , Secko Michael , Marquez Samuel
Published: June 2021

Internal carotid artery (ICA) dissections are a major cause of ischemic stroke in young adult population, with 70% occurring in persons aged 35‐50. Past studies investigated vascular pathology within the layers of the vessel, such as atherosclerosis, fibromuscular dysplasia, and connective tissue disease (CTD). Yet, these risk factors are uncommon in young patients, and CTD accounts for only 1‐5% of spontaneous arterial dissections. A putative mechanism for these events involves aberrant anatomical variants in the exposed cervical portion of the ICA; 75% of spontaneous dissections occur in this region, usually 2cm from the ICA/ECA bifurcation. In this study, we measured external diameter, bifurcating angle, and extracranial vessel length in a mixed sample of cadaveric dissections from SUNY Downstate anatomy laboratory (n=24). Measures were used to calculate a novel metric of vascular resistance using Poiseuille’s Law, which we termed the “relative vascular resistance” (RVR), at the instance of bifurcation. Results showed measures significantly non‐normally distributed (p<0.05), with RVR varying by many orders of magnitude. These results corroborated with 3‐D radiological reconstructions of this region, as well as 2‐dimensional Doppler ultrasound in live patients. We propose that the RVR at bifurcation be further studied as a predictor of ICA dissection risk in young adults.
Dexmedetomidine reduced astrocyte connexin 43 and attenuated neuropathic pain induced by partial sciatic nerve ligation in rats (LB503)
Authors: Wang lingzhi , Huang huansen , Xia weiyi , He yanbing , Cheung Chi‐Wai , Xia zhengyuan
Published: June 2021

Dexmedetomidine(DEX) has been shown to attenuate neuropathic pain (NP), but the mechanism is unclear. Astrocytes connexin 43(Cx43) is increased in subjects with NP and plays an important role in the development and maintenance of NP (Glia. 2012;60(11):1660‐70). We thus investigated the role of astroglia Cx43 in DEX‐induced anti‐nociceptive effects in rats with NP. Peripheral NP was induced by partial sciatic nerve ligation (PSNL) in SD rats. After 10 days of inducing NP, DEX was intrathecally administrated for 5 days and ipsilateral mechanical allodynia and thermal hyperalgesia were examined by Von Frey Test and Hargreaves Test. Compared with control rats, PSNL induced significant ipsilateral mechanical allodynia and thermal hyperalgesia (P<0.05) that were coincident with enhanced Cx43 and increased astroglia activation in ipsilateral lumbosacral dorsal spinal cord. DEX inhibited astroglia Cx43 expression and attenuated NP (P<0.05 vs. control). Cx43 gene knock‐down did not significantly further enhance DEX mediated attenuation of NP. We concluded that astroglia Cx43 plays a critical role in DEX mediated anti‐nociceptive effects in rats with NP.

Grant Funding Source: Supported by Departmental development fund Supported by Science and Technology Planning Project of Guangdong Province(2010B060900089). The authors are grateful to Pro.Huang for critical design and revise. huanghs5480@163.com
Imbalance of M1/M2 alveolar macrophages phenotype in bronchial asthma (LB506)
Authors: Malyshev Igor , Lyamina Svetlana
Published: June 2021

This study evaluated imbalance of M1/M2 alveolar macrophages (AM) phenotype in BA of various state and healthy volunteers (HV). Methods: In bronchoalveolar lavage fluid (BALF) and culture medium of AM in patients with intermittent BA (iBA) (n=15, 46,56±4,18 y.o.), persistent BA (pBA) (n=15, 48,24±3,27 y.o) and HV (n=10, 51,83±3,52 y.o.) AM phenotype was assessed by flow cytometry (Beckman Coulter FC500) by production of proinflammatory M1 cytokines: IL‐1β, IL‐8, IL‐12p70, IFN‐γ, TNF‐α and TNF‐β; anti‐inflammatory М2 cytokines: IL‐4, IL‐5 and IL‐10, and bivalent М1/М2 cytokines: IL‐2 and IL‐6. Results: Analysis of BALF cytokine profile in iBA and pBA identified evident imbalance of pulmonary immune response towards M2 phenotype as compared to HV. Generalized M1/M2 indices in iBA and pBA were comparable ‐ 0.84 and 0.88. The value of M1/M2 index in healthy persons was assumed equal to 1. Genuine AM phenotype assessed by cytokine production in culture medium revealed significant changes in BA regardless of the disease state. In both iBA and pBA M1/M2 indices changed towards M1 vs. HV (4.68 and 1.59, respectively). Conclusions: Analysis of cytokine pattern in bronchoalveolar lavage fluid and culture medium of alveolar macrophages in bronchial asthma revealed imbalance of M1/M2 alveolar macrophages phenotype and local pulmonary immune response. Shift of genuine alveolar macrophages phenotype towards M1, probably, reflect common proinflammatory component of the disease and contribute to asthma exacerbation. Drift of cytokine pattern in BALF towards M2 phenotype most likely indicate compensatory reaction of alveolar macrophages and of the innate immune response.
IL‐6 production in human bronchial epithelial cells in response to house dust mite allergen (LB507)
Authors: Lewis Christina , Fischer Horst
Published: June 2021

Airway epithelial cells lie at the interface between a host and its environment, representing the first line of defense against pathogens. In addition to its role as a protective barrier, the airway epithelium can activate underlying dendritic cells, thereby initiating and prolonging airway inflammation. Furthermore, alterations in epithelial ion transport mechanisms can modify many aspects of epithelial signaling, mucus properties, bacterial defenses, and induction of immune responses. IL‐6 is a proinflammatory cytokine produced mainly by cells of the innate immune system and is increased in a variety of pulmonary diseases, though its role(s) remain unclear. The objective of our research is to determine the mechanism(s) by which the airway epithelium drives the immunopathology of allergic asthma and to clarify the relationship(s) between the epithelium and IL‐6 in the pathogenesis of asthma. Normal Human Bronchial Epithelial (NHBE)(Lonza Walkersville) were grown on collagen‐coated transwell inserts and cultured at air‐liquid interface (ALI) for ~20d to achieve full differentiation. Transepithelial resistance (TER) was monitored, and upon reaching 350‐500 Ω, the cells were treated with house dust mite allergen (HDM, 50 or 100 ug/mL; Greer Laboratories) for 24 h. IL‐6 cytokine production was measured via ELISA (R&D Systems). IL‐6 production increased in a dose‐dependent manner in HDM‐treated cultures (50 ug/ml HDM: 878+/‐235 pg/mL, or 142% increase, and for 100 ug/mL of HDM: 1325+/‐18.7 pg/mL, or 215% increase, p=0.04) as compared to sham treatment groups (618+/‐433 pg/mL). Our data indicate that bronchial epithelial cells may drive allergic asthma via production of IL‐6. Our ongoing studies seek to determine the relationship(s) between the airway epithelium, IL‐6 production, and other aspects of epithelial dysfunction in asthma pathogenesis, including dysregulated mucus production and epithelial ion transport.
Extraintestinal helminth infection reduces the development of colitis‐associated colorectal cancer (LB518)
Authors: Terrazas Luis , Callejas Blanca , León‐Cabrera Sonia , Perez‐Plasencia Carlos , Gutiérrez‐Cirlos Emma Bertha , Chirino Yolanda , Ledesma‐Soto Yadira , Avila‐Moreno Federico , Rodriguez‐Sosa Miriam
Published: June 2021

Whereas a causal association between viral and bacterial infections with tumor development has been well established in animal and epidemiological studies, the impact of parasitic infections in cancer development is almost unknown. Evidences indicate that cancer can be triggered by chronic or deregulated inflammatory processes, such as colitis‐associated colorectal cancer (CAC). Using a helminth infection model, we have analyzed the role of helminth‐induced anti‐inflammatory response in determining the outcome of CAC. Following Taenia crassiceps infection and development of CAC, mice displayed inhibited colonic inflammatory responses and reduced circulating and colonic CD11b+Ly6C+CCR2+ monocytes. Instead they increased the expression of IL‐4 and alternatively activated macrophages (AAMs) markers at the colonic tissue. Strikingly, T. crassiceps‐infected mice developed few tumors and reduced pathology. In contrast, uninfected mice mounted strong inflammatory responses, did not express AAMs markers, developed severe pathology and displayed multiple tumors. Thus, immunomodulatory activities induced by helminth infections may have a role in the resistance and progression of CAC.

Grant Funding Source: Supported by 167799 CONACYT (Mexico), PAPIIT‐UNAM IN213512, PAPCA‐2013 FES‐Iztacala 16, 19, 28
An IL‐33 immunotherapy that blocks tumor growth by establishing innate lymphoid cells. (LB52)
Authors: KIM JUYANG
Published: June 2021

An IL‐33 immunotherapy that blocks tumor growth by establishing innate lymphoid cells.

Wonyoung Kim1, Juyang Kim1, Hyun Ju Kim2, U‐jeong Moon2, So Yeon Jung2, Hye‐Jeong Choi3, H. R. Cho1,4, B. Kwon1,2

1Biomedical Research Center, Ulsan University Hospital, School of Medicine, University of Ulsan, Ulsan, Korea

2School of Biological Sciences, University of Ulsan, Ulsan, Korea

3Department of Pathology, Ulsan University Hospital, School of Medicine, University of Ulsan, Ulsan, Korea

4Department of Surgery, Ulsan University Hospital, School of Medicine, University of Ulsan, Ulsan, Korea

Abstract A long‐standing question in the field of tumor immunotherapy is how type 2 T helper cell (Th2) cytokines can block tumor growth. The anti‐tumor effects of Th2 cytokines are particularly prominent when they are manipulated to be continuously secreted in tumor mass. This observation suggests that Th2 cytokines may create a tumor microenvironment unfavorable for tumor growth independently of adaptive immunity. We show here that sustained intratumoral expression of IL‐33 establishes a high numbers of innate lymphoid cells secreting high levels of CXCL1/2 which are induced scenescence of tumor cells. IL‐33 increased intratumoral hypoxia which is induced high levels of CXCR2 and down‐regulation of HIF‐1α in tumor. CXCR2 signaling strongly induces apoptosis of tumor cells, thereby preventing normal angiogenesis in tumor mass. Our results identify a previously unrecognized function of ILC2s which mediate type‐2 innate immunity leading to antitumor activity.

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Histamine blockade reduces femoral blood flow with no apparent change in vascular permeability after muscle damaging exercise (LB523)
Authors: Ely Matthew , Romero Steven , Gilbert‐Gard Kacy , Sieck Dylan , Buck Tahisha , Halliwill John
Published: June 2021

Blood flow to previously exercised muscle remains elevated for up to two hrs after exercise and is mediated by activation of histamine receptors. Activation of histamine receptors could lead to increased vascular permeability, an initial stage of inflammation, associated with muscle damage. Therefore, the purpose of this experiment was to determine if histamine receptors contribute to the increased permeability in a model of exercise‐induced muscle damage. Muscle damage was induced in seven volunteers (2F, 5M) by 45‐min of downhill running (‐10% grade) at a heart rate of ~150 BPM. Volunteers were randomly assigned to histamine blockade (540 mg fexofenadine; an H1 blocker and 300 mg ranitidine; an H2 blocker) or control (no drug). Prior to and for 120 min following exercise, femoral blood flow was measured using Doppler ultrasound, while vascular permeability was measured as a change in thigh and calf circumference during four min of supravenous pressure occlusion. The rise in femoral blood flow after exercise was 52 ± 12% (Mean ± SE) lower with histamine blockade vs control (p<0.05). Additionally, thigh and calf vascular permeability tended to be higher with histamine blockade (109 ± 92%; 42 ± 21%) vs control (73 ± 58%; 34 ± 20 %)(p>0.05). Thus, it appears that post‐exercise histamine receptor activation may not contribute to the initial stages of an inflammatory response for muscle damaging exercise.

Grant Funding Source: NIH grant HL115027
Biochemical and pharmacological rationale for the use of newer oral anticoagulants in the long term management of heparin induced thrombocytopenia (LB526)
Authors: Fareed Jawed , Hoppensteadt Debra , Walenga Jeanine , Jeske Walter , Lewis Bruce
Published: June 2021

Introduction: Heparin induced thromboycytopenia (HIT) is a catastrophic complication of heparin therapy. While parenteral antithrombin agents can be used for acute/intravenous management of this syndrome there is a need for long term outpatient antithrombotic management of HIT patients. The purpose of this study is to investigate the use of newer oral anticoagulant agents (NOACs) as substitute anticoagulants for HIT patients.

Material and Methods: Individual (25) and pooled (5 )HIT sera were used to investigate their effects on platelet aggregation, PF4 release and thrombin generation. The effect of rivaroxaban, apixaban and dabigatran was compared with unfractionated heparin (UFH) in platelet rich plasmas normal donors. The relative immunogenic potential of each of these agents was also compared in a rabbit model.

Results: In contrast to UFH none of the NOACs produced any aggregation of platelets (7+3) vs (64+9%) While heparin resulted in a release of PF4 from platelets (179 + 11) the NOACs produced marginal release (<30ng/ml). In the thrombin generation assays variable results were obtained with NOACs. The relative immunogenicity of NOACs was marginal in comparison to heparin in rabbits.

Conclusion: These results suggest that the NOACs do not interact or release PF4 from patients. Moreover, none of these agents produce activation or aggregation of platelets. These observations warrant bridged clinical trials with parenteral anticoagulant agents to ensure the efficacy and safety of NOACs in HIT.
Genomic Annotation of Drosophila biarmipes Dot Chromosome Contig 19 (LB531)
Authors: McCullough Austin , Greuel Brian
Published: June 2021

Comparative analysis of the genome of Drosophila melanogaster with other Drosophila species at various evolutionary distances can reveal information about differences in euchromatic and heterochromatic gene structure, and how evolutionary processes have changed the expression of genes located in these regions. The goal of this project, performed in conjunction with the Genomics Education Partnership at Washington University, was to annotate all the major features of Drosophila biarmipes dot chromosome Contig 19 ‐ including genes, pseudogenes, and repetitive sequences ‐ to generate a high quality section of the genome of this species. Basic Local Alignment Search Tool (BLAST) was used along with Gene Record Finder to compare the protein sequence of D. melanogaster exons to the D. biarmipes contig in order to locate the placement of the homologous exons in the latter species. Gene predicting programs were used to examine regions of the genome not immediately shown to contain genes. This information was entered into Gene Model Checker to compile a comprehensive map of the contig. Contig 19 is on the fourth (dot) chromosome of Drosophila biarmipes and it contains three genes, Syt7, Rad23 and Zip102B. Both the Syt7 and Rad23 genes showed high sequence conservation with their orthologs in D. melanogaster and are present in the same orientation. The Zip102B gene showed evidence of greater sequence divergence. Consistent RNAseq data and agreement between gene prediction programs led to the conclusion that this sequence divergence was due to the accumulation of point mutations relative to a common ancestral species. The contig displayed a repetitive element percentage of 40.32 percent, which is consistent with a heterochromatic DNA classification. No pseudogenes were identified in this contig.

Grant Funding Source: Supported by HHMI grant # 52005780 to SCR Elgin at Washington University (St. Louis).
Reducing leukocyte‐endothelial interactions through FPR2/ALX in microglial instigated stroke (LB543)
Authors: Smith Helen , Gavins Felicity
Published: June 2021

Members of the formyl peptide receptor family (in particular FPR2/ALX; Fpr2/3 in mouse) act as endogenous anti‐inflammatory/pro‐resolving agents. We investigated the mechanism through which an inflammatory response was generated in a model of global stroke, as well as the pharmacological potential of FPR ligands in modulating this inflammation.

Mice underwent bilateral common carotid artery occlusion (BCCAO, 5min) and reperfusion (40min or 2h). FPR agonists (Annexin A1 peptide Ac2‐26 (AnxA1Ac2‐26) and 15‐epi‐lipoxin A4 (15‐epi‐LXA4)) were administered at the start of reperfusion, and resulting cerebral leukocyte‐endothelial (L‐E) interactions viewed using intravital microscopy. Leukocyte rolling and adhesion were then quantified in each mouse.

At 40 min reperfusion, 15‐epi‐LXA4 (0.5μg and 4μg/mouse) reduced inflammation vs. vehicle treatment, although this reduction was not sustained at 2h with 0.5μg/mouse. AnxA1Ac2‐26 treatment decreased leukocyte adhesion at 40min and 2h vs. vehicle. Combined treatment of AnxA1Ac2‐26 and FPR antagonists Boc2 or WRW4 (FPR2/ALX‐selective) abrogated the effects of AnxA1Ac2‐26 at 40min, suggesting an Fpr2/3 mechanism. Abrogation was not sustained at to the same extent at 2h. Immunogold‐labeling showed co‐localization of endogenous AnxA1 with Fpr2/3 in the brain in treatment groups, further indicating a protective Fpr2/3 mechanism in the model.

Further work identified microglial activity as a potential mechanism for increased L‐E interactions in vehicle‐treated groups. Increased numbers of microglia were observed following BCCAO and 2h reperfusion in immunostained brain slices. In addition, pre‐treatment with minocycline (an inhibitor of microglial activity) abrogated the increased L‐E interactions observed through intravital microscopy.

To conclude, 15‐epi‐LXA4 and AnxA1Ac2‐26 reduced L‐E interactions (which were at least in part instigated through microglial activity) in this model of stroke, in a dose and time‐dependent manner, and likely via FPR2/ALX. These novel data indicate that that modulation of FPR2/ALX could produce significant protective effects in clinical stroke.

Grant Funding Source: Supported by BBSRC
Reenergizing aged mitochondria to combat retinal degeneration (LB555)
Authors: Mills William , Alam Nazia , Prusky Glen , Szeto Hazel
Published: June 2021

Bioenergetic failure is the major risk factor for Age‐Related Macular Degeneration (AMD) and Diabetic Retinopathy (DR). Within the eye, the delicate microvasculature of the choroid and the metabolically active retinal pigment epithelium (RPE) are especially sensitive to the stresses of age and diabetes. Using mouse models of diabetes and advanced age, we demonstrated diabetes/aging‐induced abnormalities of the mitochondria in the choroid and RPE, alterations to the choroidal microvasculature, disruption to the RPE, and an increase in inflammation markers. Treatment with the novel cardiolipin‐targeted antioxidant tetrapeptide SS‐31, after onset of diabetes or in aged mice, restored proper RPE integrity and mitochondrial morphology, as well as reduced the intrusion of neovascular tufts from the choroid through the RPE and the level of inflammation markers. Moreover, SS‐31 administration restored visual function as demonstrated by optokinetic tracking. Thus we are the first to pioneer a mitochondrial‐targeted approach for resolving feed‐forward mitochondrial damage and restoring bioenergetic capacity in the choroid and RPE, thereby preventing disease progression and restoring visual function in early AMD and DR.

Grant Funding Source: Supported by the Tri‐Institutional Training Program in Vision Research (5T32EY007138‐20)
Phenotyping age‐sependent changes in the 5xFAD Alzheimer’s Disease Model mice (LB556)
Authors: Yang Charles , Feng Ruiben , Yang Ling , Jiang Shujuan , Liu Na , Yan Li , Liu yi , Yang Cecilia , Wang KeWei
Published: June 2021

5XFAD transgenic (Tg) mice express 5 human familial Alzheimer’s Disease (AD) gene mutations ‐ Amyloid Precursor Protein mutations from the Swedish, Florida and London families, along with 2 mutations in presenilin‐1. Their early onset brain pathology, accelerated Aβ42 amyloid deposition, age‐dependent neurodegeneration and cognitive decline, make these mice an attractive translatable AD model.

We used behavioral, biochemical and electrophysiological analyses to track age‐dependent changes in 5XFAD mice, compared with their age‐matched wild‐type mice. With sensitive NanoPro Western, 3 month old 5XFAD mice showed a significant increase of p‐T181‐Tau in the cerebral spinal fluid. Their hippocampal ELISA Aβ40 and 42 levels increased progressively from 2,3,6 months. Under confocal microscopy, the secondary and tertiary dendritic spine counts in Golgi‐stained 9 month prefrontal and hippocampal pyramidal neurons showed a significant loss. Hippocampal slice electrophysiology using the multielectrode MED64 system is used to assess synaptic plasticity, i.e. long‐term potentiation.

Using the Laboras vibration‐sensitive platforms, we discovered that there was a significant dark phase hyperactivity and bi‐directional circling that resembled striatal degeneration, and hyperlocomotion at 9, 12, but not 2 month, matching the chronic EEG recordings of a significant reduction in NREM and REM sleep at 9 month. The onset of cognitive decline started at 4 months (working memory, Y‐maze spontaneous alteration), 5 month (episodic memory, Novel Object Recognition), and 6 month (spatial memory, Morris Water Maze). Sub‐chronic treatment in 4 months old 5XFAD mice by: a) Donepezil + Memantine (2 weeks), or b) Traditional Chinese herbal formula (1 month), led to a significantly improvement in Y‐maze spontaneous alternation performance.

Findings from these studies guide us to select the right ages of 5XFAD mice to test for pharmacological changes in AD biomarkers and behaviors in AD research.
Improving repair and regeneration after spinal cord injury through combinatorial therapy (LB558)
Authors: Torres Luisa , Robinson John , Tsirka Stella
Published: June 2021

Spinal cord injury (SCI) results in the death of neurons, disruption of neuronal connections, demyelination, and inflammation. There are three phases of SCI: acute (from the time of impact to the first few days post‐injury), secondary (hours to weeks), and chronic (months to years). Most therapeutic interventions have focused on the secondary and chronic phases to promote healing and prevent further damage. However, little can be done to modify the acute phase. We show that the small molecule inhibitor Pifithrin‐µ (PFTμ), which prevents the association of p53 with Bcl/Bax without affecting p53’s transcriptional activity, significantly reduces lesion volume and modestly improves motor function in injured mice if applied immediately after injury. Similarly, application of microglia inhibitory factor (MIF/TKP) significantly reduces lesion volume and decreases microglial activation and secondary death. Combinatory use of PFTμ and MIF further improves motor function in mice. Given that PFTμ and MIF function via different mechanisms we anticipate that combining both approaches will result in improved histopathological outcomes in addition to improved motor coordination after SCI.
Effect of scutellarin on vascular impairment by hypoxia reoxygenation in isolated coronary artery from rat (LB562)
Authors: Yang Weimin , Chen Yajuan , Wang Lei , Li Yang , Chen Chen , Qing Chen
Published: June 2021

Scutellarin (SCU), a flavonoid from a traditional Chinese medicinal plant, is clinically used to treat cardiovascular diseases. In this study, we investigated the vasoprotective effects of SCU against hypoxia reoxygenation (HR) in isolated rat coronary artery (CA). HR treatment significantly impaired acetylcholine (ACh,0.001‐100 µM)‐induced endothelium‐dependent relaxation in isolated CA rings and these effects were reversed by pre‐incubation with SCU (500 µM). SCU pre‐incubation did not affect ACh response in normal CA without HR treatment. HR treatment did not significantly altered nitroglycerin (NTG, 0.001‐10 µM) induced endothelium‐independent vasodilation in CA and SCU (500 µM) had no significant effect on NTG response in CA with or without HR treatment. The PKG inhibitor Rp‐8‐Br‐cGMPS (4 µM) blocked both the vasorelaxative effects of SCU in CA rings without HR treatment and the vasoprotective effects of SCU in HR‐treated CA. These results demonstrate that SCU repairs HR‐induced vascular impairment in endothelium‐dependent vasodilation and might be via activation of the PKG pathway.

Grant Funding Source: Supported by grants from the National Natural Science Foundation of China (Nos. 30960450 and 81173110) and Yunnan Provincial Science and Technology Department (Nos. 2011FA022, 2012BC012, 2008CD054, and 2008ZC111M).
Activation of G‐protein Coupled Estrogen Receptor 1 Protects the Heart Against Ischemia/Reperfusion Injury by Inhibiting the mPTP Opening Via MEK/ERK/GSK‐3β Pathway (LB564)
Authors: Harris Tanoya , Singh Harpreet , Olde Bjorn , Leeb‐Lundberg Fredrik , Toro Ligia , Stefani Enrico , Bopassa Jean Chrisostome
Published: June 2021

Introduction: Estrogen effect can be mediated by three receptors: Classical estrogen receptors: alpha (ERα) and beta (ERβ), and recently identified G protein‐coupled estrogen receptor1 (GPER1).

Hypothesis: We investigated the role of ERα, ERβ and GPER1 in mediating rapid estrogen‐induced cardioprotection in male mice hearts subjected to ischemia/reperfusion using wild type (WT) and gene specific knockout animals.

Methods: Isolated hearts from wild type (WT: C57BL/6NCrL), ERα‐/‐, ERβ‐/‐ and GPER1‐/‐ were perfused using Langendorff apparatus with Krebs Henseleit buffer (control) or with the addition of estrogen (40 nM). Hearts were subjected to 18 min global ischemia followed by 60 min reperfusion. Cardiac function was recorded during the entire experiment and myocardial infarct size measured by TTC staining at the end of the reperfusion. Mitochondria calcium retention capacity (CRC) required to induce the mitochondrial permeability transition pore (mPTP) opening was assessed after 10 min reperfusion. Protein levels were measured by Western Blot in whole heart lysates after 5 min treatment just before ischemia, and after 10 min reperfusion. LY294002 and U0126 were used as inhibitor of PI‐3K/Akt, and MAPK/ERK translocation, respectively.

Results: In WT, ERα‐/‐ and ERβ‐/‐, estrogen treatment significantly improved cardiac functional recovery, reduced infarct size and improved mitochondrial CRC. However, estrogen effects were completely absent in GPER1‐/‐. Estrogen treatment during 5 min before ischemia induced up‐regulation of Akt, GSK‐3β, and ERK1/2 phosphorylation in WT mouse as compared with control but not in GPER1‐/‐. U126 abolished estrogen effect on mitochondrial CRC while LY294002 could not prevent estrogen effect on GSK‐3β observed in WT. P<0.05 and n=3‐6.

Conclusion: Rapid activation of GPER1 induces cardioprotection effect against ischemia/ reperfusion injury. Estrogen effects through GPER1 are associated with phosphorylation of GSK‐3β and ERK1/2, and inhibition of the mPTP opening.

Grant Funding Source: American Heart Association, NIH
Active hexose correlated compound moderates lps‐induced gut injury in rats (LB576)
Authors: Doursout Marie‐Francoise , Segal Graeme , Ahn Stephanie , Devakottai Sundar , Liang Yangyan , Wakame Koji , Sundaresan Alamelu , Kulkarni Anil
Published: June 2021

Introduction: We hypothesize that Active Hexose Correlated Compound (AHCC), a fermented mushroom extract, restores immune function in lipopolysaccharide (LPS) induced inflammation in the gut, especially when endogenous nitric oxide (NO) signaling pathway is impaired. We identified the molecular mechanisms in the gut in rats subjected to LPS‐induced immune dysfunction, treated and non‐treated with AHCC, a potent promoter of immune support.

Method: Six rats were instrumented under anesthesia to record mean arterial blood pressure (MAP) and heart rate (HR) and for drug administrations. Following recovery, conscious animals were pretreated with AHCC in drinking water (6%) prior to be subjected to LPS; 20 mg/kg; IV to induce inflammation. MAP and HR were continuously recorded for 3 hrs. following LPS administration. Simultaneously to hemodynamic measurements, blood samples were collected for NO and cytokines production. Animals were sacrificed at 3 hrs. and gut harvested to assess histology.

Results and Conclusions: Ourpreliminary data have shown that AHCC induced reductions in LPS‐induced hypotension by 20% and gut edema by 30%. Cytokines e.g. TNF‐α, TGF β and IL‐1 were reduced by 40% in LPS rats pre‐treated with AHCC. Administration of AHCC prior to LPS challenge ameliorates LPS‐induced microvilli disruption in the gut. In summary, our data show that AHCC restored immune function in rats subjected to inflammation and specifically decreased excessive inflammation/oxidative stress in the gut.

Grant Funding Source: None
A Humanized Anti‐Cocaine Monoclonal Antibody Decreases the Urinary Excretion of Cocaine from Mice (LB588)
Authors: Gooden Felicia , Tabet Michael , Nieman Michelle , Ball William , Norman Andrew
Published: June 2021

The recombinant humanized monoclonal antibody (mAb) h2E2 is a lead candidate for immunotherapy of cocaine abuse. This anti‐cocaine mAb has been shown in vivo to inhibit cocaine’s distribution to mouse brain by 19‐fold compared to controls, with an equivalent increase in plasma cocaine concentrations. Despite binding to h2E2 in plasma, cocaine appears to be eliminated more rapidly than in controls. However, in vitro studies using pH‐buffered (1:1) mouse plasma demonstrated that h2E2 protects cocaine from metabolism to the major inactive metabolites of cocaine, ecgonine methyl ester (EME) and benzoylecgonine (BE). As cocaine and its metabolites are also excreted renally, tests for abuse measure urine concentrations of cocaine and BE. It is hypothesized ‐ based upon h2E2’s high affinity and effects on cocaine’s distribution and metabolism ‐ that the presence of h2E2 would decrease urinary excretion of cocaine, EME, and BE. This hypothesis was tested in male Swiss‐Webster mice pre‐treated with an i.v. infusion of vehicle or h2E2 (1.6 µmol/kg) 1 hour prior to i.v. bolus injection of an equimolar dose of cocaine HCl. Excreted urine was collected 17‐20 minutes after cocaine administration and cocaine, EME, and BE concentrations were determined using GC/MS. The mAb h2E2 caused dramatic 8‐fold (88%), 7.7‐fold (87%), and 3.6‐fold (72%) decreases in urinary cocaine, BE, and EME concentrations, respectively, as compared to levels in the absence of h2E2. If similar effects occur in humans, caution should be used when interpreting efficacy measures of anti‐cocaine vaccines and mAbs that are based on urine cocaine and metabolite concentrations.

Grant Funding Source: Supported by NIH grant DP1DAP31386.
Genetic variation in aldo‐keto reductase 1D1 (AKR1D1) affects expression and activity of multiple cytochrome P450s (LB595)
Authors: Chaudhry Amarjit , Thirumaran Ranjit , Yasuda Kazuto , Yang Xia , Fan Yiping , Strom Stephen , Schuetz Erin
Published: June 2021

Human liver gene regulatory network analysis identified a cytochrome P450 subnetwork with AKR1D1 as its key regulatory driver. This study assessed the biologic importance of AKR1D1 [key enzyme in synthesis of bile acids, ligand activators of FXR, PXR and CAR, known P450 regulators] to hepatic P450 expression. Overexpression of AKR1D1 in primary human hepatocytes led to increased expression of CYP3A4, 2C8, 2C9, 2C19, and 2B6. Conversely, AKR1D1 knockdown decreased expression of these P450s. Resequencing AKR1D1 from donor livers identified a 3'‐UTR (rs1872930) SNP that was significantly associated with higher AKR1D1 mRNA. AKR1D1 3'‐UTR‐luciferase reporter studies showed that variant allele resulted in higher luciferase activity, suggesting that the SNP increases AKR1D1 mRNA stability and/or translation efficiency. AKR1D1 3'‐UTR SNP was significantly associated with increased hepatic mRNA of multiple P450s (CYP3A4, 2C8, 2C9, 2C19, and 2B6) and CYP3A4, 2C8, 2C19, and 2B6 activities. After adjusting for multiple testing, association remained significant for AKR1D1, CYP2C9, and CYP2C8 mRNA and CYP2C8 activity. These results provide new insights into the variation in expression and activity of P450s that can account for interindividual differences in drug metabolism/efficacy. In conclusion, we provide the first experimental evidence supporting a role for AKR1D1 as a key genetic regulator of P450 network.
Modeling P450 SItes of Metabolism (LB596)
Authors: Swamidass S. Joshua
Published: June 2021

Our group has been working on ways to improve the design and use of P450 modeling technologies. This is important because a large proportion of drugs are metabolized by P450 enzymes, and variants in P450s commonly cause patients to respond differently to medicines. We are pursuing two innovations in this area. First, we have developed P450 models that can predict what atoms are metabolized on molecules based on their structure. Our approach uses training data, molecules where the sites of metabolism are known, to build a predictive model that can accurately predict how new molecules are metabolized by 9 separate P450 isozymes. Knowing which atoms are modified‐‐‐the sites of P450 metabolism‐‐‐enables chemists to modify molecules to improve their metabolic profiles. Second, we developed a strategy for that can make use of region‐resolution site‐of‐metabolism data, instead of exclusively relying on the atom‐resolution data to which prior methods are confined. This unique capability can be leveraged to improve the accuracy of prediction of sites of metabolism on molecule, until now, impossible to predict. Our hope is to extend this technology to model how P450 variants metabolize drugs differently from one another. Effective modeling of metabolism can help overcome the central challenges in modern drug discovery and development: reducing the cost and risk of developing new drugs by ensuring the right medicines are given to the right patients.
Role of glucocorticoid receptor and pregnane X receptor in dexamethasone induction of rat hepatic aryl hydrocarbon receptor nuclear translocator and NADPH‐cytochrome P450 oxidoreductase (LB597)
Authors: Riddick David , Hunter Sarah , Vonk Alex
Published: June 2021

The aryl hydrocarbon receptor (AHR) nuclear translocator (ARNT), as the AHR’s heterodimerization partner, and NADPH‐cytochrome P450 oxidoreductase (POR), as the key electron donor for all microsomal P450s, are important players in the adaptive and toxic responses to polycyclic and halogenated aromatic hydrocarbons. Expression of both ARNT and POR in rat liver is induced by dexamethasone (DEX), a synthetic glucocorticoid known to activate both the glucocorticoid receptor (GR) and the pregnane X receptor (PXR). To study the role of GR in ARNT and POR regulation, we treated male rats with a low DEX dose (0.5 mg/kg), shown to activate GR selectively, and a high DEX dose (50 mg/kg), shown to activate both GR and PXR, in the absence and presence of the GR antagonist RU486 (50 mg/kg). The role of PXR was assessed by comparing the responses to similar low (1 mg/kg) and high (50 mg/kg) DEX doses in wild‐type and PXR‐knockout rats. In both studies, rats were euthanized at 6 and 24 h after DEX dosing. The induction of ARNT mRNA levels by low‐dose DEX and the increase in ARNT protein levels by high‐dose DEX were prevented by RU486, whereas the induced levels of ARNT mRNA and protein did not differ between wild‐type and PXR‐knockout rats. RU486 did not inhibit the induction by DEX of POR expression at the mRNA, protein, and catalytic activity levels. The induction of POR mRNA levels by low‐ and high‐dose DEX was significantly attenuated in PXR‐knockout rats and the increase in POR protein levels and catalytic activity by high‐dose DEX was completely absent in PXR‐knockout rats. These results are consistent with important roles for GR in the DEX induction of rat hepatic ARNT and PXR in the DEX induction of rat hepatic POR.

Grant Funding Source: Supported by the Canadian Institutes of Health Research
The Pharmacokinetics/Pharmacodynamics of Resveratrol and Mechanism‐based PK‐PD Modeling Research (LB598)
Authors: Chen Hui , Liu Xinxia , Wan Heng , Zeng Can
Published: June 2021

Objective: The aim of this research was to develop a mechanism‐based pharmacokinetic‐pharmacodynamic(PK‐PD) model that could quantitatively characterize the time‐dependent relationship between the PK and PD of Resveratrol(Res) in melanoma tumor‐bearing mice for elucidation of its functional mechanisms and molecular targets. Methods: The plasma concentration and AUC of Res and its main metabolites (Res‐glucuronide,Rse‐G; Res‐sulfate,Res‐S) were respectively fitted to the activities, mRNA and protein levels of tyrosinase and COX‐2 for screening of PK input and PD endpoint. Results: The administration of Res could reduce the growth speed of tumor, inhibit the activities of tyrosinase and COX‐2, and down regulate the mRNA and protein expression of COX‐2 in tumor‐bearing mice. Sigmoid Emax model combined with exponential equation was fitted to the PK‐PD modeling for the effects of Res treatment on tumor volume, simple Emax model was fitted to the PK‐PD modeling for the effects on enzyme activity, mRNA and protein expression of COX‐2, while sigmoid Emax model was fitted to the effects on activities of tyrosinase. Conclusion: Res‐G and Res‐S may play roles in the in vivo activities of Res, which could be attributed to their own activities or the deconjugation reactions of metabolite in tumor tissues. PK‐PD models were developed for the effects of Res treatment on enzyme activity of tyrosinase and COX‐2, as well as mRNA and protein levels of COX‐2, which could quantitatively describe the correlation between the PK and PD of Res, Res‐G and Res‐S.

Grant Funding Source: Supported by National Natural Science Foundation of China (30973586)
Effects of Diabetes‐Like Altered Glycemia on BBB Gating Functions: Impact on Efflux Transporters (LB600)
Authors: Sajja Ravi , Cucullo Luca
Published: June 2021

Altered glycemic conditions, such as those observed in diabetic patients, impair blood‐brain barrier (BBB) endothelial physiology and serve as potential risk factor for the onset and progression of major CNS disorders (such as Alzheimer’s disease). In this study, we assessed the impact of diabetes‐like hyper and hypoglycemic conditions on the expression and functionality of major BBB efflux transporters (P‐glycoprotein and MRP4). Confluent monocultures of hCMEC/D3 endothelial cells (a well characterized human BBB endothelial cell line) were exposed to normal (5.5mM), hypo (2.2mM) ‐ or hyper (35mM)‐glycemic conditions of D‐glucose for 3, 6, 12 and 24 h. Following treatment, cells were either lysed or fixed for subsequent protein expression and distribution analysis by western blotting and immunofluorescence. Further, parallel hCMEC/D3 cultures were chronically exposed to identical experimental conditions, but for a period of 3 days, and profiled for genome‐wide transcriptional changes using Illumina® DNA microarray. Our results indicate an up‐regulation of P‐glycoprotein (P‐gp) and MRP4 protein following 12 ‐24 h acute exposure to hypoglycemia (~1.1 fold increase, vs. control), while no differences were observed at 3 h. Hyperglycemic exposure resulted in a time‐dependent alteration of P‐glycoprotein and MRP4 expression with an initial increase at 3 h followed by restoration to normal (MRP4) or down‐regulation (P‐glycoprotein). These results were further confirmed by using L‐glucose as an osmotic control. Gene chip analysis revealed a moderate increase of P‐gp and MRP4 gene expression following repeated exposures to hyperglycemia. In contrast, chronic exposure to cycles of hyper ‐ hypoglycemia significantly decreased P‐gp gene expression (≍ 50% vs. control) while the MRP4 expression was mostly unaffected. In summary, our data demonstrate that acute or recurrent exposures to hypo‐ and hyperglycemia potentially alter the protein/gene expression of major BBB efflux transporters that would impact the BBB integrity and gating function as well as brain drug disposition. Additional studies are currently in progress to determine the changes in the functionality of these transporters in relation to their altered expression.

Grant Funding Source: Supported by A.R.D.F and in part by NIH/NIDA R01‐DA029121‐01A1 to LC
Targeted temperature management with novel adenosine agonist (LB604)
Authors: Bailey Isaac , Bogren Lori , Drew Kelly
Published: June 2021

The decreased metabolism and body temperature (Tb) of hibernating mammals may increase resistance to loss of bone density during prolonged periods of disuse and ischemia/reperfusion injury through targeted temperature management. The A1 adenosine receptor (A1AR) agonist N6‐cyclohexyladenosine (CHA) induces a torpor‐like state in rats, however, CHA has not been tested in humans and full A1AR agonists have cardiovascular risks. Capadenoson (CAP) is a partial A1AR agonist designed to minimize cardiovascular risk and is currently approved for use in humans. The study objective was to test the hypothesis that CAP would decrease Tb in rats in a manner similar to CHA with less cardiovascular risk as a potential means to decrease Tb and metabolism through non‐invasive measures.

Procedures were approved by UAF IACUC. Adult, male Sprague‐Dawley rats (n=10), instrumented with IP iButton dataloggers (Maxim Integrated, San Jose, CA) were housed at 23°C on 12L:12D and fed ad lib. Rats were moved to a Ta of 17°C 20‐24h prior to treatments then moved back to Ta of 23°C 24h after treatments with no additional supplemental warming. Tb was monitored at 10min intervals, HR was monitored every 2h using a Littmann© Model 4000 electronic stethoscope (3M, St. Paul, MN) and Beat Counter app (http://vetapps.co.uk). Treatments (vehicles, 1mg/kg CHA; 1 and 2 mg/kg CAP) were administered IP per a balanced block design with 1 week between tests.

CHA decreased Tb by 1°C to 17°C, and HR by 12 to 80% from baseline; the time to return to baseline varied. CAP gave more consistent but modest responses with Tb and HR drops of 1.3 and 1.6°C and 24 and 30% for 1 and 2mg/kg doses. All rats returned to baseline levels without evidence of stress indicated by porphyrin accumulation around the eyes.

In contrast to expectations, full and partial A1AR agonists differed significantly in effects on Tb and HR in adult rats.

Grant Funding Source: Supported by NIH R15NS070779, Alaska Space Grant Program, Undergraduate research fellowship
Estrogen sulfotransferase(est/sult1e1) promotes human adipogenesis (LB606)
Authors: Ihunnah Chibueze , Philips Brian , Ravuri Sudheer , Gibbs Robert , Kirisci Levent , Rubin J , Marra Kacey , Xie Wen
Published: June 2021

The estrogen sulfotransferase (EST/SULT1E1) is known to catalyze the sulfoconjugation and deactivation of estrogens. The goal of this study is to determine whether and how EST plays a role in human adipogenesis. By using human primary adipose derived stem cells (ASCs) and whole fat tissues from the abdominal fat of obese and non‐obese subjects, we showed that the expression of EST was low in pre‐adipocytes but increased upon differentiation. Overexpression and knockdown of EST in ASCs promoted and inhibited differentiation, respectively. The pro‐adipogenic activity of EST in humans was opposite to the anti‐adipogenic effect of the same enzyme in rodents. Mechanistically, EST promoted adipogenesis by deactivating estrogens. The pro‐adipogenic effect of EST can be recapitulated by using an estrogen receptor (ER) antagonist or ERα knockdown. In contrast, activation of ER in ASCs inhibited adipogenesis by decreasing the recruitment of the adipogenic peroxisome proliferator activated receptor(PPARγ) onto its target gene promoters, whereas ER antagonism increased the recruitment of PPARγ to its target gene promoters. Linear regression analysis revealed a positive correlation between the expression of EST and body mass index (BMI)as well as a negative correlation between the ERα expression and BMI. We conclude that EST is a pro‐adipogenic factor, which may serve as a druggable target to inhibit the turnover and accumulation of adipocytes in obese patients.

Grant Funding Source: Supported by NIH Ruth L. Kirschstein National Research Service Awards for Individual Predoctoral Fellowships to Promote Diversity in Health‐Related Research
Cellular effects of Vpr in astrocytes and neurons (LB61)
Authors: Santiago Ismael , Torres Lilith , Noel Richard
Published: June 2021

Combined Antiretroviral Therapy (cART) reduces viral loads of HIV by preventing virus replication to slow down the progression to AIDS. Therapy does not prevent all viral gene expression and so morbidities continue to present in HIV infected people. HIV associated neurocognitive disorders (HAND) affect approximately 50% of HIV‐1 positive individuals. Viral protein expression in astrocytes may contribute to HAND. Viral protein R (Vpr) is an important factor in HIV replication and a neurotoxin. We hypothesized Vpr can be toxic to astrocytes or neurons. To test our hypothesis we measured cell markers of apoptosis, viability and toxicity using fluorescent assays in co‐cultures of Vpr‐transfected SVGA astrocytes and SH‐SY5Y neurons. Vpr treated cultures exhibited modestly higher cytotoxicity and unchanged viability or apoptosis in neurons. In conclusion, the results suggest that Vpr has sub‐lethal effects and may cause milder toxicity, such as changes in morphology. Future experiments will be directed at testing for actin changes caused by Vpr expression in astrocytes.
Crotalphine: fluorescent analogues, structural analysis and its effect on co‐cultures of peritoneal macrophages and DRG neurons (LB615)
Authors: Remuzgo Cesar , Sampaio Sandra , Picolo Gisele , Machini Maria , Cury Yara
Published: June 2021

Crotalphine (CRP) is a 14‐mer peptide isolated from the venom of C. durissus terrificus “rattlesnake” that triggers long‐lasting antinociception (3‐5 days). This effect is mediated by activation of peripheral CB2 cannabinoid receptors, the subsequent release of dynorphin A and activation of opioid receptors. In inflamed tissues, granulocytes and macrophages are the main source of opioid peptides, which play a substantial role in the modulation of pain via interactions with opioid receptors located in peripheral sensory nerve fibers. In this work, we synthesized CRP and fluorescent analogues, analyzed its structure and investigated its effect on peritoneal macrophages and co‐cultures of dorsal root ganglia (DRG) neurons and peritoneal macrophages. All procedures were approved by ethics committee of the Butantan Institute (CEUAIB: 946/12) and performed in accordance to the International Association for the Study of Pain guidelines. CRP and the obtained analogues [Glu1]‐CRP, carboxyfluorescein (CF)‐[Glu1]‐CRP, CF‐[Gln1]‐CRP were able to induce antinociception. Circular dichroism spectra of CRP and [Glu1]‐CRP have shown bands indicative of the presence of random and helix structure and has allowed the construction of a 3D‐structure model of CRP. Fluorescence microscopy and mass spectra analysis of peritoneal resident or LPS‐activated macrophages treated with CF‐[Gln1]‐CRP (1 µM) showed that this peptide internalizes and is distributed in the cytoplasm of the cells, releasing dynorphin A (1‐13) and Met‐enkephalin to extracellular medium. Internalization of κ‐opioid receptors into small DRG neurons in co‐cultures of peritoneal macrophages and DRG neurons treated with bradykinin (10 μM) and CRP (1 μM), was also observed. On the other hand, we do not observed internalization of κ‐opioid receptors into macrophages. In conclusion, the data suggest a functional crosstalk between macrophages and DRG neurons in the presence of CRP. This phenomenon could contribute to the in vivo antinociceptive effect of this molecule.

Grant Funding Source: Supported by FAPESP (2011/09333‐7), CeTICs/CEPID/FAPESP (2013/07467‐1) and INCTTox/CNPq (150848/2010‐2)