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Principal Investigator: PINGNIAN HE
Organization: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
Fiscal Year: 2022
Award: $324,720
Funding agency: National Institute of Diabetes and Digestive and Kidney Diseases
A. Provide supporting data showing that diabetic MPs contribute to the increases in the propagation of the virus
compared to non-diabetic MP.
A1. Demonstrate higher number of cellular internalizations of S-protein-bound DB MPs than that of S-protein-bound
normal MPs. Experiments will be conducted in microvessels developed in microfluidics. Our preliminary data showed
no significant differences in ACE2 expression and ACE2-mediated MP interaction with S-protein between normal and
DB MPs. The main factors that contribute to DB MP-mediated increases in virus entry are their much higher quantity
and more adhesive surface due to their largely externalized phosphatidylserine (PS) than that of normal MPs. GFPtagged
S-protein will be incubated with MPs isolated from normal and DB plasma and flow cytometry will be used to
confirm the S-protein-bound MPs. To demonstrate the quality differences between normal and DB MPs, equal amount
of S-protein-bound DB and normal MPs will be perfused into the in vitro microvessels. Confocal images will be collected
to compare the internalized S-protein-bound DB MPs with that of normal MPs by quantification of GFP fluorescence. To
demonstrate the role of externalized PS on DB MPs in the MP adhesion and internalization, the S-protein-bound DB MPs
will be pre-coated with a lipid binding protein, Annexin V, before perfusion into microvessels. We predict that precoating
of DB MPs with Annexin V will prevent the DB MP adhesion to ECs and reduce their cellular internalizations.
A2. Demonstrate DB MP-mediated increases in HCoV-NL63 infection and propagation when compared to normal MPs.
HCoV-NL63 (BEI Resources) will be propagated in Caco-2 cells to produce a working virus stock. Cultured ECs will be
incubated with HCoV-NL63 with serial dilutions (1:10-1000) in the presence of DB and normal plasma or equal number
of isolated DB and normal MPs, respectively. The incubation of HCoV-NL63 alone serves as viral infection control. To
demonstrate the specific role of DB MPs in facilitating viral infection from that of other components in DB plasma such
as inflammatory mediators and cytokines, cultured ECs will be incubated with HCoV-NL63 in the presence of MP-free
DB plasma and the results will be compared with DB plasma containing increased MPs. For rigor, efficiency of HCoVNL63
infection will be determined using three distinct methods. Total virus will be harvested from cells and supernatant
at 96 hpi and quantified by both plaque assay and 50% tissue culture infectious dose (TCID 50) assay as previously
published (PMID 19014487). Infection will also be quantified using a one-step, real-time, quantitative reverse
transcription PCR assay on RNA harvested from cells at 96 hpi using a commercially available kit (Liferiver). Supernatant
will be collected daily to examine the release of MPs and cytokines, serving as indicators of viral infection-induced EC
activation. Results will be compared among groups (3-4 replicates per group for all proposed preliminary studies).
B. Provide preliminary data to support that HCoV-NL63 virus could produce the illness that is similar to SARS-CoV-2.
HCoV-NL63 has been reported to cause upper respiratory tract infections in children, and also to cause pneumonia, as
well as airway inflammation in K18-ACE2 mice. In this preliminary study, we will develop a hamster model of HCoVNL63
infection. Golden Syrian hamsters (8-10 weeks old, Charles River) will be used for this study. Normal and STZinduced
diabetic hamsters will be intranasally inoculated with serial dilutions of HCoV-NL63 (5 x 104, 5 x 105, or 5 x 106
TCID50) in 200 μL PBS to establish the ID50 in vivo. Oral and rectal swabs will be collected every 24 hours starting 1-day
post-inoculation (dpi) for up to five days. Body weight and clinical signs of illness as indicated by labored breathing,
hunched posture and immobility, and temperature will be monitored twice a day. Hamsters will be euthanized on 3
and 5 dpi to harvest lungs and peripheral tissues. Remaining animals are sacrificed at 7 dpi (n = 3 per group). Hamster
lungs will be assessed for histology and viral replication (measure viral RNA levels by qPCR). Cytokines will be measured
in bronchoalveolar lavage and lung homogenate. Plasma will be collected for MP and cytokine analysis (details see
application experimental designs C4B2). Results will be compared among groups. We predict that HCoV-NL63-infected
DB hamsters have more severe respiratory inflammatory responses than HCoV-NL63-infected normal hamsters. Our
previous study showed that cross-transfusion of DB plasma to normal rats caused immediate vascular inflammation in
the recipient rat, indicating a role of DB MPs in mediating propagation of inflammation in the vasculature. To evaluate
the specific role of DB MPs in facilitation and propagation of HCoV-NL63 infection, 40% plasma volume of a HCoV-NL63-
infected normal hamster will be replaced by MP-rich and MP-free DB plasma (normal plasma as sham control) or vice
versa and the viral infection-induced illness and vascular pathology will be compared between groups. In case HCoVNL63
infection alone causes very mild clinical symptoms in hamsters, the alternative is to coinfect hamster with human
influenza H1N1 and HCoV-NL63. Hamster is an established animal model for the study of human influenza infection and
coinfection of SRAS-CoV-2 and influenza has been shown to enhance the severity of pneumonia in hamsters.
C. Provide evidence for a novel therapeutic target to alleviate the adverse outcomes of COVID-19 patients with diabetes.
The focus of this application is to reveal a novel mechanism by which diabetes exacerbates the COVID-19 outcomes.
The proposed mechanistic studies will lead to the identification of a novel target for therapeutic development. Our
previous study showed that carotid injection of Annexin V (1 ml, 100 μg/Kg) to a DB rat reduced plasma MPs from 465 x
104/μL to 22 x 104/μL 1h after the injection. We will apply the same approach to HCoV-NL63 infected DB hamsters, and
the plasma MPs and viral infection outcomes will be evaluated accordingly. The drug development is out of scope of
this application. However, these preliminary studies will provide mechanistic evidence supporting the development of
a Annexin V-based therapeutic product in the future studies.
Terms: <0-11 years old><2019 novel corona virus><2019 novel coronavirus><2019-nCoV><2019-nCoV S protein><2019-nCoV spike glycoprotein><2019-nCoV spike protein><ACE Inhibitors><ACE2><ANX5><ANX5 Gene><ANXA5><ANXA5 gene><Accounting><Adhesions><Adhesives><Anchorin CII><Anchorin CII Gene><AngII><Angiotensin I-Converting Enzyme Inhibitors><Angiotensin II><Angiotensin Receptor><Angiotensin-Converting Enzyme Antagonists><Angiotensin-Converting Enzyme Inhibitors><Animal Model><Animal Models and Related Studies><Animals><Annexin A5 Gene><Annexin A5 Protein><Annexin V><Area><Assay><Binding><Binding Proteins><Binding Sites><Bioassay><Biologic Assays><Biological Assay><Blood Cells><Blood Circulation><Blood Plasma><Blood Plasma Volume><Blood Sample><Blood Vessels><Blood leukocyte><Blood specimen><Bloodstream><Body Tissues><Body Weight><Breathing><Bronchioalveolar Lavage><Bronchoalveolar Lavage><Bronchopulmonary Lavage><CBP-I><COVID associated morbidity><COVID induced morbidity><COVID infected patient><COVID morbidity><COVID patient><COVID positive patient><COVID related morbidity><COVID-19><COVID-19 S protein><COVID-19 associated morbidity><COVID-19 induced morbidity><COVID-19 infected patient><COVID-19 infection><COVID-19 morbidity><COVID-19 pathogenesis><COVID-19 patient><COVID-19 positive patient><COVID-19 related morbidity><COVID-19 spike glycoprotein><COVID-19 spike protein><COVID-19 virus><COVID19><COVID19 S protein><COVID19 infection><COVID19 pathogenesis><COVID19 patient><COVID19 positive patient><COVID19 spike glycoprotein><COVID19 spike protein><COVID19 virus><CV-19><CV19><CaCo2><Caco-2 Cells><Calphobindin I><Cardiovascular Diseases><Cell Body><Cell membrane><Cells><Characteristics><Child><Child Youth><Children (0-21)><Circulation><Clinical><CoV-2><CoV2><Coagulants><Combining Site><Common Rat Strains><Communicable Diseases><Conflict><Conflict (Psychology)><Confocal Microscopy><Coronaviridae><Coronavirus><Cricetinae><Crowns><Cytoplasmic Membrane><Data><Dental crowns><Development><Diabetes Mellitus><Disease><Disorder><Dose><Down-Regulation><Downregulation><Drug Targeting><Dysfunction><ENX2><ENX2 Gene><Economics><Electron Microscopy><Endocytosis><Endonexin II><Endonexin II Gene><Endothelial Cells><Endothelium><Enzyme Gene><Enzymes><Experimental Designs><Flow Cytofluorometries><Flow Cytofluorometry><Flow Cytometry><Flow Microfluorimetry><Flow Microfluorometry><Fluorescence><Functional disorder><Future><Golden Hamsters><Golden Syrian Hamsters><Grippe><H1N1><H1N1 Virus><HCoV><Hamsters><Hamsters Mammals><Harvest><Histology><Hour><Human><Immunoblotting><In Vitro><Incubated><Individual><Infection><Infection Control><Infectious Disease Pathway><Infectious Diseases><Infectious Disorder><Inflammation><Inflammation Mediators><Inflammatory><Inflammatory Response><Influenza><Influenza A Virus, H1N1 Subtype><Injections><K-18><K-18 conjugate><K18><K18 combination><Kininase II Antagonists><Kininase II Inhibitors><Laboratories><Leukocytes><Leukocytes Reticuloendothelial System><Ligand Binding Protein><Ligand Binding Protein Gene><Lipid Binding><Lipocortin V Gene><Lipocortin-V><Lung><Lung Lavage><Lung Respiratory System><Marrow leukocyte><Measures><Mediating><Mesocricetus auratus><Meta-Analysis><Methods><Mice><Mice Mammals><Microfluidic Device><Microfluidic Lab-On-A-Chip><Microfluidic Microchips><Microfluidics><Microvascular Dysfunction><Modeling><Modern Man><Molecular Interaction><Monitor><Murine><Mus><Non-Polyadenylated RNA><Oral><Organ><Organelles><Outcome><PAP-I><PP4 Gene><Pathogenesis><Pathology><Patients><Perfusion><Peripheral><Peripheral Blood Cell><Persons><Phenotype><Phosphatidylserines><Physiopathology><Placental Anticoagulant Protein I><Placental Protein 4><Plaque Assay><Plasma><Plasma Membrane><Plasma Serum><Plasma Volume><Pneumonia><Posture><Protein Binding><Proteins><Publishing><RNA><RNA Gene Products><Rat><Rats Mammals><Rattus><Reactive Site><Receptor Protein><Renin-Angiotensin-Aldosterone System><Reporting><Research Resources><Resolution><Resources><Respiratory Aspiration><Respiratory Inspiration><Reticuloendothelial System, Serum, Plasma><Reverse Transcription><Ribonucleic Acid><Rivers><Role><SARS corona virus 2><SARS-CO-V2><SARS-COVID-2><SARS-CoV-2><SARS-CoV-2 S protein><SARS-CoV-2 infected patient><SARS-CoV-2 infection><SARS-CoV-2 pathogenesis><SARS-CoV-2 patient><SARS-CoV-2 positive patient><SARS-CoV-2 spike glycoprotein><SARS-CoV-2 spike protein><SARS-CoV2><SARS-CoV2 S protein><SARS-CoV2 infection><SARS-CoV2 spike glycoprotein><SARS-CoV2 spike protein><SARS-associated corona virus 2><SARS-associated coronavirus 2><SARS-coronavirus-2><SARS-related corona virus 2><SARS-related coronavirus 2><SARSCoV2><STZ><Serine Phosphoglycerides><Severe Acute Respiratory Coronavirus 2><Severe Acute Respiratory Distress Syndrome CoV 2><Severe Acute Respiratory Distress Syndrome Corona Virus 2><Severe Acute Respiratory Distress Syndrome Coronavirus 2><Severe Acute Respiratory Syndrome CoV 2><Severe Acute Respiratory Syndrome-associated coronavirus 2><Severe Acute Respiratory Syndrome-related coronavirus 2><Severe acute respiratory syndrome associated corona virus 2><Severe acute respiratory syndrome corona virus 2><Severe acute respiratory syndrome coronavirus 2><Severe acute respiratory syndrome coronavirus 2 S protein><Severe acute respiratory syndrome coronavirus 2 infection><Severe acute respiratory syndrome coronavirus 2 spike glycoprotein><Severe acute respiratory syndrome coronavirus 2 spike protein><Severe acute respiratory syndrome related corona virus 2><Severities><Streptozocin><Streptozotocin><Study models><Surface><Swab><Symptoms><Syrian Hamsters><Techniques><Temperature><Testing><Therapeutic><Thromboplastin Inhibitor><Time><Tissues><Transfusion><Up-Regulation><Upper Respiratory Infections><Upper Respiratory Tract Infection><Upregulation><VAC-Alpha><Vascular Anticoagulant-Alpha><Viral Diseases><Virulent><Virus><Virus Diseases><Virus Replication><Virus-like particle><Western Blotting><Western Immunoblotting><White Blood Cells><White Cell><Wuhan coronavirus><Zanosar><adverse consequence><adverse outcome><airway epithelium inflammation><airway inflammation><angiotensin converting enzyme 2><angiotensin converting enzyme II><annexin A5><base><bound protein><bronchopulmonary lavage therapy><cardiovascular disorder><cell type><co-infection><co-morbid><co-morbidity><coinfection><comorbidity><confocal imaging><corona virus><corona virus disease 2019><coronavirus disease 2019><coronavirus disease 2019 S protein><coronavirus disease 2019 associated morbidity><coronavirus disease 2019 induced morbidity><coronavirus disease 2019 infected patient><coronavirus disease 2019 infection><coronavirus disease 2019 morbidity><coronavirus disease 2019 pathogenesis><coronavirus disease 2019 patient><coronavirus disease 2019 positive patient><coronavirus disease 2019 related morbidity><coronavirus disease 2019 spike glycoprotein><coronavirus disease 2019 spike 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influenza virus><infected with severe acute respiratory syndrome coronavirus 2><inflammatory mediator><influenza infection><influenza virus infection><injury to organs><inspiration><life-threatening COVID><life-threatening COVID-19><life-threatening SARS-CoV-2><life-threatening coronavirus disease><life-threatening coronavirus disease 2019><life-threatening severe acute respiratory syndrome coronavirus 2><lipid bound><microfluidic chip><microvascular complications><microvascular disease><model of animal><model organism><mortality><mortality risk><nCoV2><new drug target><new druggable target><new pharmacotherapy target><new therapeutic target><new therapy target><non-diabetic><nondiabetic><novel><novel drug target><novel druggable target><novel pharmacotherapy target><novel therapeutic target><novel therapy target><organ injury><pandemic><pandemic disease><pathophysiology><patient infected with COVID><patient infected with COVID-19><patient infected with SARS-CoV-2><patient infected with coronavirus disease><patient infected with coronavirus disease 2019><patient infected with severe acute respiratory syndrome coronavirus 2><patient with COVID><patient with COVID-19><patient with COVID19><patient with SARS-CoV-2><patient with coronavirus disease><patient with coronavirus disease 2019><patient with severe acute respiratory distress syndrome coronavirus 2><plasmalemma><prevent><preventing><protein blotting><pulmonary><receptor><rectal><respiratory><respiratory inflammation><respiratory tract inflammation><response><serious COVID><serious COVID-19><serious SARS-CoV-2><serious coronavirus disease><serious coronavirus disease 2019><serious severe acute respiratory syndrome coronavirus 2><severe COVID><severe COVID-19><severe COVID19><severe SARS-CoV-2><severe acute respiratory syndrome coronavirus 2 infected patient><severe acute respiratory syndrome coronavirus 2 pathogenesis><severe acute respiratory syndrome coronavirus 2 patient><severe acute respiratory syndrome 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