Warren L. Lee Faculty of Medicine, University of Toronto Keenan Research Centre of the Li Ka Shing Knowledge Institute, St. Michael's Hospital
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1 Warren L. Lee Faculty of Medicine, University of Toronto Keenan Research Centre of the Li Ka Shing Knowledge Institute, St. Michael's Hospital
2
3 Influenza one mutant strain away from disaster? The development of a highly transmissible and virulent strain can occur Resistance to existing antivirals may develop rapidly New therapeutic approaches are needed
4 Patient who die from influenza fall into 2 groups Primary viral pneumonia Secondary bacterial pneumonia S. aureus, S. pneumoniae
5 Patient who die from influenza fall into 2 groups Primary viral pneumonia Secondary bacterial pneumonia S. aureus, S. pneumoniae The final common pathway? Acute lung injury
6
7 Dejana. Nature Reviews Molecular Cell Biology (2004)
8
9 Influenza replicates in lung endothelium VIRAL NUCLEIC ACID (RT PCR)
10 IMMUNOSTAIN FOR VIRAL NUCLEOPROTEIN
11 Measurement of Leak Transendothelial Electrical Resistance (TEER) Top electrode Bottom electrode R = 24 Ω cm 2 Dextran flux Measure Fluorescence Add fluorescein-tagged dextran to upper chamber Allow 40 min for dextran flux
12 Measurement of Leak Transendothelial Electrical Resistance (TEER) Top electrode Bottom electrode R = 24 Ω cm 2 Dextran flux Measure Fluorescence Add fluorescein-tagged dextran to upper chamber Allow 40 min for dextran flux
13 Influenza induces endothelial permeability H3N2 (X31) Clinical Isolate (H3N2)
14 Influenza induces endothelial permeability H3N2 (X31) Clinical Isolate (H3N2)
15 Influenza induces endothelial permeability
16 What is the mechanism?
17 Influenza induces apoptosis
18 Blocking apoptosis partially attenuates flu-mediated leak
19 Signaling?
20 Replication-deficient influenza can still infect lung endothelium CONTROL UV FLU Viral protein
21 Fraction of control TEER Replication-deficient influenza induces endothelial permeability Live UV hr 4 hr 8 hr 12 hr 16 hr 20 hr 24 hr Multiplicity of infection = 8
22 Replication-deficient influenza does not induce apoptosis
23 Dejana. Nature Reviews Molecular Cell Biology (2004)
24 Replication-deficient influenza causes a loss of claudin-5
25 No effect on VE-cadherin
26 Flu causes dose-dependent loss of claudin-5
27 Decreased claudin-5 may be due to Decreased production qpcr shows no difference in mrna levels
28 Decreased claudin-5 may be due to Increased degradation
29 Claudin-5 is not degraded in lysosomes
30
31 Claudin-5 is not polyubiquitinated
32 Is Claudin-5 degraded by matrix metalloproteases?
33 Inhibition of matrix metalloproteases prevents the loss of claudin-5
34 Is the loss of claudin-5 required for flu-induced leak?
35 Over-expression of claudin-5 is sufficient to block flu-induced leak
36 camp stimulates claudin-5 synthesis camp
37 camp prevents claudin-5 loss *
38 Formoterol can stimulate claudin-5 synthesis camp Formoterol
39 Formoterol can induce claudin-5 expression
40 Formoterol can block flu-induced claudin-5 loss
41 Formoterol blocks flu-induced leak
42 Flu induces lung injury in mice
43 that is attenuated by formoterol
44 Summary Human influenza can infect and replicate in lung microvascular endothelium Infection increases lung endothelial permeability Endothelial apoptosis Loss of claudin-5 (apoptosis-independent) Enhancement of endothelial barrier integrity may represent a novel therapeutic approach for severe influenza Plos One (10):e47323
45 Ongoing Work Patient who die from influenza fall into 2 groups Primary viral pneumonia (20%) Secondary bacterial pneumonia (75%) S. aureus, S. pneumoniae
46 Superinfection after influenza Autopsy data from 1918 found bacterial overgrowth An era of no antibiotics An animal study of S. aureus superinfection found that bacterial overgrowth did not explain mortality (Iverson et al., JID 2011)
47 Does influenza predispose to acute lung injury from Staphylococcus aureus? Stay tuned
48 Acknowledgements Lee Lab Susan Armstrong Changsen Wang Asela Gamage Jayesh Tigdi Carlo Dumpit Yongjun Wang Steffany Charles Dr. Samira Mubareka (Sunnybrook) Dr. Theo Moraes (Sickkids) Xiaoe Si Funding Ontario Thoracic Society PSI Foundation McLaughlin Centre Early Researcher Award (Ontario)
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