High Dose N-acetylcysteine for H1N1 2009

High Dose N-Acetylcysteine Anti-oxidant therapyA Novel Approach For the Treatment of Novel H1N1 Pneumonia : 

High Dose N-Acetylcysteine Anti-oxidant therapyA Novel Approach For the Treatment of Novel H1N1 Pneumonia Dr. Lai Kang Yiu Consultant Intensive Care Unit Queen Elizabeth Hospital

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The Story Begins in 1997

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Cellular transcriptional profiling in influenza A virus-infected lung epithelial cells: The role of the nonstructural NS1 protein in the evasion of the host innate defense and its potential contribution to pandemic influenza. Geiss GK, Salvatore M, Tumpey TM, Carter VS, Wang X, Basler CF, Taubenberger JK, Bumgarner RE, Palese P, Katze MG, García-Sastre A.Proc. Natl. Acad. Sci. USA 2002 Aug 6;99(16):10736-41. Epub 2002 Jul 29 The makings of a killer Peter Palese, Christopher F. Basler & Adolfo García-Sastre Nature Medicine 8, 927 - 928 (2002) Yuen KY, Chan PKS, Peiris M, Tsang DNC, Que TL, Shortridge KF, Cheung PT, To WK, Ho ETF, Sung R, Cheng AFB, and members of the H5N1 study group. Clinical features and rapid viral diagnosis of human disease associated with avian influenza A H5N1 virus. Lancet 1998; 351(9101):467-471. Attenuation of influenza-like symptomatology and improvement of cell-mediated immunity with long-term N-acetylcysteine treatment S De Flora, C Grassi, and L Carati Eur Respir J 1997; 10:1535-1541 Pandemic with potential to knock out 2/3 of the world population Treatment that can save the world

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H1 Gene Introduced

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U shape Mortality Rate of Seasonal Influenza Vs W shaped Mortality Rate of H1N1 High mortality contributed by NS1 Gene

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The makings of a killer Peter Palese, Christopher F. Basler & Adolfo García-Sastre Nature Medicine  8, 927 - 928 (2002)

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Inhibit viral replication Inhibit viral infection Innate response

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Influenza virus activates the P58IPK-mediated host-cell stress-response pathway, and uses the P58IPK pathway against the host cell by directing P58IPK to block the activation of the protein kinase PKR. The influenza-virus non-structural protein NS1 — a double-stranded (ds)RNA-binding protein — blocks the dsRNA-dependent pathways, including the induction of type I interferon (IFN) expression. In addition, NS1 interacts directly with PKR and inhibits its kinase function. NS1 might also interfere with the function of IFN-regulatory factors (IRFs). eIF-2 , eukaryotic initiation factor 2, -subunit; ISGs, IFN-stimulated genes; JAK, Janus kinase; STAT, signal transducer and activator of transcription. Interplay between the type I IFN pathway and influenza virus Michael G. Katze, Yupeng He & Michael Gale, Jr Nature Reviews Immunology 2, 675-687 (September 2002)

IFN Signaling and NK CellsNATURAL KILLER CELLS IN ANTIVIRAL DEFENSE: Function and Regulation by Innate CytokinesChristine A. Biron, Khuong B. Nguyen, Gary C. Pien, Leslie P. Cousens, and Thais P. Salazar-Mather Annual Review of Immunology Vol. 17: 189-220Positive self regulation of cytotoxicity in human natural killer cells by production of interferon upon exposure to influenza and herpes virusesJ Y Djeu et al JEM vol. 156no. 4 October 1, 1982 1222-1234 : 

IFN Signaling and NK CellsNATURAL KILLER CELLS IN ANTIVIRAL DEFENSE: Function and Regulation by Innate CytokinesChristine A. Biron, Khuong B. Nguyen, Gary C. Pien, Leslie P. Cousens, and Thais P. Salazar-Mather Annual Review of Immunology Vol. 17: 189-220Positive self regulation of cytotoxicity in human natural killer cells by production of interferon upon exposure to influenza and herpes virusesJ Y Djeu et al JEM vol. 156no. 4 October 1, 1982 1222-1234 Activates NK cells  Elimination of virally infected cells Degrade viral RNA Inhibit viral replication Role of interferon in Innate immune response

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The Hong Kong NS1 protein is characterized by the presence of a glutamic acid at position 92. The mere change of this glutamic acid to an aspartic acid, as found in the NS1 of most influenza virus strains, disarmed the virus.

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60% mortality Rate High Mortality Contributed by Suprapotent H5N1 NS1 gene

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High mortality contributed by H5N1 NS1 Gene Cumulative H5N1 Mortality Rate up to 8/2/2010

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NS1 Gene of H5N1 as Sword of Damocles Against Human Race Implication of NS1 Gene of H5N1

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W.H.O. has raised pandemic alert from phase 5 to phase 6 on 11/6/2009

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Viral Load in H1N1 ( From David Hui PWH) NPA is unreliable in confirming diagnosis if patient present late.

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55 22 Pneumococcus 10 Staphylococcus 7 S. Pyogenes 6 HiB 1 In an analysis of lung tissue specimens from 77 confirmed fatal US cases of 2009 H1N1, in which deaths occurred from May 1 to August 20, 2009, bacterial coinfections were present in 22 (29%) cases. Of the 22 cases with bacterial coinfection, 10 were caused by S pneumoniae, 7 by S aureus, 6 by S pyogenes, 2 by S mitis, and 1 by Haemophilus influenzae. In 4 cases, there were multiple pathogens. Median age was 31 years (range, 2 months – 56 years), and half were men. CDC MMWR September 29 2009 Half are vaccine preventable H1N1 death

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In the Denver metropolitan area with active bacterial core surveillance (ABC) program, invasive pneumococcal disease has tripled from an average of 20 to 58 in October 2009. Most of that increase has been in adults under the age of 60. Dr. Anne Schuchat, director of the Centers for Disease Control and Prevention's National Center for Immunization and Respiratory Diseases Nov. 25 2009 20 58

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? Protected by Pneumococal and Haemophilus influenza B Immunization Program with major reduction in mortality 1918 Influenza U.S.A. CDC Volume 12, Number 1, January 2006 1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger* and David M. Morens Very similar Mortality Rate

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W-shaped mortality contributed by NS1 gene

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Critically ill patients with 2009 influenza A(H1N1) infection in Canada. Kumar A JAMA. 302(17):1872-9, 2009 Nov 4. Same W-shaped mortality contributed by NS1 gene

Critically ill patients with 2009 influenza A(H1N1) infection in Canada. Kumar A JAMA. 302(17):1872-9, 2009 Nov 4. : 

Critically ill patients with 2009 influenza A(H1N1) infection in Canada. Kumar A JAMA. 302(17):1872-9, 2009 Nov 4. The spring outbreak of 2009 influenza A(H1N1) infection in Canada affected primarily young, female, and aboriginal patients without major comorbidities, and conferred a 28-day mortality of 14.3% among critically ill patients. The mean (SD) age was 32.3 (21.4) years; 113 patients were female (67.3%), 50 were children (29.8%). The most common comorbidities among critically ill patients were lung disease, obesity, hypertension, and a history of smoking or diabetes, each occurring in 30% to 40% of patients. 1/3 patients required vasopressor support on day 1 following ICU admission. 1/3 patients required advanced ventilatory support and rescue therapies for profound hypoxemic respiratory failure, including high levels of inspired oxygen and PEEP, pressure control, and airway pressure release ventilation, high-frequency oscillatory ventilation, prone positioning ventilation, neuromuscular blockade, inhaled nitric oxide, and extracorporeal membrane oxygenation.

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Extracorporeal Membrane Oxygenation for Nonneonatal Acute Respiratory Failure: The Massachusetts General Hospital Experience From 1990 to 2008 Nehra, Deepika MD Arch Surg Volume 144(5), May 2009, p 427–432 Overall survival 53%

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Burden of ECMO in Australia For the first 2000 patient hospitalized patients for H1N1, 350 required ICU admission and 21 required ECMO. That means that 3.5% patient required ICU admission and 1% of hospitalized patient or 6% of ICU patient required ECMO support. Initial 2000 hospitalized case of H1N1 2009 in Australia 17.5 % 6%

H1N1 patients may be have prolonged virus shedding as opposed to seasonal influenzaThe 49th annual Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC) in San Francisco : 

H1N1 patients may be have prolonged virus shedding as opposed to seasonal influenzaThe 49th annual Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC) in San Francisco Gaston De Serres from Laval University in Quebec and his team, found that eight of 43 patients, or 19%, were still shedding H1N1 virus eight days after they became ill. They found that a week after flu onset, between 45% and 75% of cases still showed evidence of the virus on RT-PCR and that about a third were still shedding replicating virus based on culture findings. David Lye of Tan Tock Seng Hospital in Singaporeassessed the duration of viral shedding in 70 patients treated with oseltamivir (Tamiflu). In Lye's study of 70 patients, 80% of the treated patients were still shedding virus after five days of illness, 47% after seven days and 8% after 10 days. People who didn't get Tamiflu were infectious longer.

H1N1 2009 influenza virus infection during pregnancy in the USADenise J Jamieson The Lancet, 29 July 2009 : 

H1N1 2009 influenza virus infection during pregnancy in the USADenise J Jamieson The Lancet, 29 July 2009 (0·32 per 100 000 pregnant women, 95% CI 0·13—0·52 vs 0·076 per 100 000 population at risk, 95% CI 0·07—0·09). 54.5% mortality rate for hospitalized patient. 17.6% mortality rate

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Maternal Death in Canada and Australia up to 2/1/2010

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Mortality Rate Per Confirmed novel H1N1 Infection 10/4/2010 http://www.flucount.org/

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http://www.flucount.org/ (10/4/2010)

Impact to Countries : 

Impact to Countries Hong Kong Too many tests or poor mitigation ?

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Disease Burden of novel H1N1 2009 to Hong Kong First Wave  Poor Mitigation Beyond Control of Government 4/6/09 1/7/09 HK Book Fair 22/7/09 -28/7/09 21/08 - 24/08 香港動漫電玩節 31/7/09 -4/8/09 Back to school After summer vacation ICU admission closely follow HSI case load except with a phase lag.

Gln136Lys (Q136K) Neuraminidase Mutation : 

2.3% seasonal influenza A (H1N1) viruses in Australasia and Southeast Asia between 2006 and early 2008. No effect on oseltamivir susceptibility but caused approximately a 300-fold and a 70-fold reduction in zanamivir and peramivir susceptibility, respectively. The Q136K mutation did not compromise virus fitness, as the mutant virus grew in vitro at higher rates than those without the mutation, and it was equally transmissible and pathogenic in ferret models. Zanamivir-resistant influenza viruses with a novel neuraminidase mutation. Hurt AC. Holien JK. Parker M. Kelso A. Barr IG. Journal of Virology. 83(20):10366-73, 2009 Oct. Host cell selection of influenza neuraminidase variants: implications for drug resistance monitoring in A(H1N1) viruses. Okomo-Adhiambo M. Nguyen HT. Sleeman K. Sheu TG. Deyde VM. Garten RJ. Xu X. Shaw MW. Klimov AI. Gubareva LV. Antiviral Research. 85(2):381-8, 2010 Feb. Amesh A. Adalja. Zanamivir Resistance in Influenza http://www.flu.org.cn/en/article-7001.html Gln136Lys (Q136K) Neuraminidase Mutation

Human Swine InfluenzaSecond Wave : 

Human Swine InfluenzaSecond Wave Reassortment of NS1 gene from H5N1 H274Y mutation Q136K mutation

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Necrotizing pneumonia during H1N1 2009 pneumonia

Potential Role of High Dose N-Acetylcysteine in Controlling Cytokine Storm of H1N1 Pneumonia : 

Potential Role of High Dose N-Acetylcysteine in Controlling Cytokine Storm of H1N1 Pneumonia Dr. Lai Kang Yiu Intensive Care Unit Queen Elizabeth Hospital

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Pathogenesis of Influenza Virus

Background Information P1 : 

Background Information P1 F/48 BMI 20 Chronic smoker 1ppd for 20 years and Chronic drinker ~ 1/2 glass wine per day Developed URTI since 8/7/2009 with fever, cough yellow sputum and muscle ache after his son who has URTI from 3-6/7/2009. Seen by 3 different GP on 8/7/2009 10/7/2009 and 13/7/2009 and was given symptomatic treatment paracetamol/diclofen/cough mixture/mefenamic acid. Put on azithromycin 250mg b.d. for 3/7 on 13/7/2009 Developed respiratory distress on 15/7/2009 requiring intubation shortly after admission in early morning of 15/7/2009. pH 7.46 PaCO2 4.3 kPa paO2 7.4 kPa HCO3 22 mmol/L BE -0.6 mmol/L SaO2 91% while on 100% non-rebreathing mask before intubation.

ICU Admission : 

ICU Admission Patient was admitted to ICU on 15/7/2009 for ventilatory support Patient has ARDS requiring PEEP 18 cmH2O PCV FIO2 1.0 and NO 20 p.p.m. in order to maintain SpO2 > 90% pH 7.38 PaCO2 5.3 kPa PaO2 8.9 kPa HCO3 23 mmol/L BE – 1.8 mmol/L SaO2 93% (on FIO2 1.0) pH 7.40 PaCO2 5.2 kPa PaO2 13,8 kPa HCO3 25 mmol/L BE 0 mmol/L SaO2 98% on FIO2 1.0 NO 20 p.p.m. Patient has septic shock and was put on inotropic agent (noradrenaline 1ug/Kg/min)

H1N1 Matrix and H1 Gene +ve : 

H1N1 Matrix and H1 Gene +ve NPA (15/7/2009) RSV : Negative Influenza A : Negative Influenza B : Negative Parainfluenza 1,2,3 : Negative PCR for influenza A virus matrix gene positive PCR for human swine influenza A H1 gene positive ==> Human swine influenza positive Sputum (15/7/2009) AFB Smear: Negative (Concentrated method) AFB Culture :- pending TA (16/7/2009) : No growth CSU (16/7/2009): No growth Legionella antigen -ve (15/7/2009) Streptococcus pneumoniae antigen -ve (15/7/2009) Blood culture (15/7/2009): No grwoth HIV Ab –ve (15/7/09)

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15/7/09

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15/7/09

Electrocardiogram : 

Electrocardiogram ECG (15/7/2009) Mild ST depression in inferior and anterolateral leads Echocardiogram (15/7/2009) Thin rim of pericardial effusion but no tamponade effect. Sinus tachycardia while on inotropic agent impaired LVEF 55-60% No definite RWMA No vegetation seen. No valvular thickening Trivial to mild MR/TR No definite VSD/ASD on TEE Echocardiogram (17/7/2009) Thin rim of pericardial effusion but no tamponade effect. Sinus tachycardia. LV function satisfactory. LVEF 60% No RWMA Trivial MR/TR/AR Echocardiogram (23/7/09) No pericardial effusion LVEF 60% No RWMA Trivial TR

Status of Our Patient On Admission : 

Status of Our Patient On Admission

Management in ICU : 

Management in ICU N-Acetylcysteine 100mg/Kg continuous 24 hour infusion 1 tablet of i-CAP B.D. 1 Tablet of B complex B.D.

Anti-oxidant ProtocolTrace Element and Vitamin : 

Anti-oxidant ProtocolTrace Element and Vitamin Each tablet of I-Cap contains Vitamin A 6600 IU Vitamin C 400 mg Vitamin E 150 IU Riboflavin (B2) 10mg Zinc 60mg Selenium 40ug Copper 4mg Manganese 10mg Lutein/Zeaxanthin 4mg Each tablet of B Co contains Thiamine mononitrate 3mg Riboflavin 1.5mg Pyridoxine 0.5mg Calciferol 250 IU Nicotinamide 10mg

Recycling and Redox Cycling of Vitamin E (An Phenolic Antioxidants) : 

Recycling and Redox Cycling of Vitamin E (An Phenolic Antioxidants) GPx- Se I-CAP N-Acetylcysteine B-Co

Progress : 

Progress The patient has rapid and substantial improvement with rapid drop in CRP, decreased oxygen requirement and clearing of lung field during three days infusion of high dose acetylcysteine. N-acetycysteine was reduced to 600mg b.d. after three day of 100mg/Kg/day infusion and steroid was tailed off after short synacthen test result was available and inotropic agent has been weaned off. Patient was put on elastic stocking and pneumatic calf compressor on admission. Prophylactic LMWH was given on 16/7/2009 after her mild thrombocytopenia had improved.

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16/7/09

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16/7/09

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17/7/09

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18/7/09

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19/7/09

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20/7/09 20/7/09

Progress : 

Progress After stopping of N-acetylcysteine infusion, patient has recurrence of fever and slowly increasing oxygen requirement. Initially, it is attributed to nosocomial sepsis. Central line was changed and sepsis workup was performed. All cultures including CMV pp65 were negative. Tazocin was changed to Meronam empirically on 21/7/2009 and vancomycin was stopped on 23/7/09. Patient has substantial deterioration on 23/7/09 requiring PCV and FIO2 0.85. Bronchoscopy was performed and showed only mildly inflammed mucosa but no purulent sputum. Bronchoalveolar lavage only showed scanty growth of candida albican but WBC was negative. Fluconazole was added. β-1,3-D glucan (28/7/09) was negative.

Progress : 

Progress High dose N-acetylcysteine was reinstituted and steroid was reintroduced. Since tracheal aspirate indicated that the H1N1 2009 virus was not eradicated after 8 days of high dose tamiflu therapy, Relenza neubilization 15mg Q6H for 5 days was added for viral control. Daily tracheal aspirate for influenza virus was taken for monitoring of progress. Patient showed rapid improvement with decrease in CRP and oxygen requirement. Viral eradication was achieved after Relenza therapy and there was no relapse after treatment. This time CXR only showed slow improvement. Patient was extubated and discharged to general ward on 4/8/2009. She was afebrile with no leukocytosis and SpO2 99% while on 2L of oxygen on date of discharge.

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Her factor VIII:C level was 159 %.

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22/7/2009

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23/7/09

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23/7/09

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24/7/09

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26/7/09

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27/7/09

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28/7/09

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29/7/09

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5/8/09

Summary I : 

Summary I The protocol of high dose N-acetylcysteine anti-oxidant therapy was associated with rapid control of cytokine storm as evidence by rapid drop in CRP, decreased oxygen requirement and radiological improvement. Symptoms would recur if high dose N-acetylecysteine was stopped if underlying viral infection is not under control. Re-institution of high dose N-acetylcysteine resulted in biochemical and clinical improvement. This can buy time for the anti-viral drug to exert its effect to control the underlying infection. Serial CRP is useful during followed up of patient for early detection of symptomatic deterioration.

Summary II : 

Summary II Control of underlying viral infection is necessary to prevent relapse of disease after anti-oxidant therapy. Daily monitoring of viral load in tracheal aspirate may detect those patient with poor response to Tamiflu therapy. (It is important because resistance to Tamiflu may occur easily by a single amino acid substitution e.g. H274Y mutation). Nosocomial or co-infection should be sought if patients have secondary rise in CRP while on high dose N-acetycysteine therapy since the stimulation of NFkB and IL6 is independent of the reactive oxygen species in bacterial sepsis. Daily monitoring of CRP allow early detection of nosocomial bacterial and fungal infection. Information obtained from our subsequent patients

Summary III : 

Summary III During the course of our patient’s illness, her CRP and fibrinogen were markedly elevated. Her factor VIII:C level was 159 %. This may predispose her to the risk of thromboembolism that was described in novel H1N1 pneumonia. Prophylactic LMWH should be given to prevent DVT and pulmonary embolism unless there is contraindication. CDC MMWR 10/7/2009 Factor VII and fibrinogen levels as risk factors for venous thrombosis: A case-control study of plasma levels and DNA polymorphisms: the Leiden Thrombophilia Study (LETS). Koster T, Rosendaal FR, Reitsma PH, van der Valden PA, Briet E, Vandenbroucke JP. Thromb Haemost. 1994;71:719–722 Excess factor VIII: a common cause of hypercoagulability. Bobrow RS. Journal of the American Board of Family Practice. 18(2):147-9, 2005 Mar-Apr.

Summary IV : 

Summary IV IL6 was one of the major cytokine produced during influenza infection. Symptoms and fever in natural acute influenza correlate with the release of IL-6. Symptom pathogenesis during acute influenza: interleukin-6 and other cytokine responses. Kaiser L. Fritz RS. Straus SE. Gubareva L. Hayden FG. Journal of Medical Virology. 64(3):262-8, 2001 Jul. IL6 can lead to the production of CRP, fibrinogen and factor VIII:C leading to the risk of thromboembolism Pathways by which IL-6 contributes towards haemostasis – an overview Kerr: Br J Haematol, Volume 115(1).October 2001.3-12 The IL6 cytokine pathway may also explain why patients with obesity may have more severe disease and pulmonary embolism because of increased baseline secretion of IL6 induced by adiopokines.

Thrombophilia in H1N1 : 

Thrombophilia in H1N1 Tissue Factor

Pathways by which IL-6 contributes towards haemostasis – an overviewKerr: Br J Haematol, Volume 115(1).October 2001.3-12 : 

Pathways by which IL-6 contributes towards haemostasis – an overviewKerr: Br J Haematol, Volume 115(1).October 2001.3-12 75X IL-1 and IL-2 did not significantly affect factor VIII gene transcription NFķB and C/EBP is necessary for increased factor VIII mRNA transcription 6-9x ↑ Inhibit Inhibit Anti-thrombin III Protein S ＋

Summary V : 

Summary V A prospective randomized control trial should be conducted to test the efficacy of high dose N-acetylcysteine therapy in patient with H1N1 therapy. This has important implication in the treatment of H1N1 second wave if a more malignant strain with high mortality has emerged after gene reassortment. This may even has a role on the potential H5N1 pandemic. N-acetylcysteine can be administered as a oral form that can be given even as an out patient basis. This may have a unique role even in the third world where hopsital or critical facility is very limited. Since N-acetylcysteine is a Category B drug in pregnancy, it may have significant role in reducing the mortality and morbidity of novel H1N1 infection during prengnacy.

Summary VI : 

Summary VI Suboptimal radiological clearing despite clinical improvement after the second course of N-acetylcysteine is expected because influenza virus would also induce IL8 secretion leading to pulmonary fibrosis. Therefore early institution of N-acetylcysteine and more prolonged therapy until viral clearing should be explored by study to prevent such complications.

Expression of IL-6, IL-8, and RANTES on human bronchial epithelial cells, NCI-H292, induced by influenza virus A.Matsukura S, Kokubu F, Noda H, Tokunaga H, Adachi M J Allergy Clin Immunol. 1996 Dec;98(6 Pt 1):1080-7 : 

Expression of IL-6, IL-8, and RANTES on human bronchial epithelial cells, NCI-H292, induced by influenza virus A.Matsukura S, Kokubu F, Noda H, Tokunaga H, Adachi M J Allergy Clin Immunol. 1996 Dec;98(6 Pt 1):1080-7 Chemotactic cytokine CHAK (CC-Chemokine-activated killer) cells ? Pulmonary fibrosis

Latest progress of patient : 

Latest progress of patient Patient was discharged to general ward on 4/8/2009. She was transferred to convalescent hospital for rehabilitation on 14/8/09 and discharged home. CXR 13/8/09

Background Information P2 : 

Background Information P2 M/58 Non-smoker, Non-drinker, BMI 20 Hx of (1) HT (2) Leukocytoclastic vasculitis 1998 (3) NPC T2N2 1998 with RT / brachytherapy (a) First relapse at NP 2000treated with iridium mould (b) Bone secondary to right scapula Rx chemotherapy ( c) Recurrence at neck LN with left RND in KWH Feb, 2004 (4) Hypopituitarism on chronic steroid replacement Admitted on 4/8/2009 because of H1N1pneumonia associated with RSV and MRSA co-infection and relapse of NPC (IgA EBV VCA 1:80) and bulbar palsy. Admitted to ICU on 11/8/2009 because of progressive pulmonary deterioration with respiratory failure despite Tamiflu and antibiotic therapy

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Day of ICU admission F = Fibrinogen (g/L) CRP = C-reactive protein (mg/L); HI = PCR for human swine influenza A H1 gene; Mx= PCR for influenza A virus matrix gene; H1n = H1 from nasopharyngeal aspirate (NPA); Mxn = Mx from NPA

Salient Features of P2 : 

Salient Features of P2 Patient is on chronic steroid therapy with clinical relapse of NPC Low C3 level: C3 is important in priming of T cells and its subsequent migration to lung during influenza infection. Persistent lymphopenia and viral shedding despite high dose Tamiflu and Relenza therapy Patient has high antibody titre to H1N1 but unable to clear the virus because of T cell dysfunction due to low C3, chronic steroid therapy and underlying illness. T cell is necessary for clearing of virus during influenza infection. The patient was treated with similar regime as in patient 1 except a maintenance N-acetylcysteine 50mg/Kg/day was given. We still managed to wean the patient off the ventilator despite active viral infection. The patient was able to be discharged home.

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Natural Antibody and Complement Mediate Neutralization of Influenza Virus in the Absence of Prior Immunity Jerome P. Jayasekera, E. Ashley Moseman, and Michael C. Carroll J Virol. 2007 April; 81(7): 3487–3494. C3, CR2 and IgM are required for the maintenance of protective immunity against influenza virus infection. Complement and natural antibody are required in the long-term memory response to influenza virus. Fernandez Gonzalez S. Jayasekera JP. Carroll MC. Vaccine. 26 Suppl 8:I86-93, 2008 Dec 30.

Summary I : 

Summary I High dose N-acetylcystine anti-oxidant therapy at 100mg/kg IV infusion for 3 days followed by maintenance therapy of N-acetylcysteine at 600mg q.4.h. p.o. after three days is able to control the cytokine storm of novel H1N1 2009 even when there is suboptimal response to anti-viral therapy. A prospective trial should be conducted to test the role of oral N-acetylcysteine on the treatment of novel H1N1 infection because N-acetylcysteine is well absorbed orally and this would reduce the cost of N-acetylcysteine therapy and make is suitable to be used in outpatient basis early in the treatment of novel H1N1 infection.

Summary II : 

Summary II Chronic steroid therapy and low C3 are risk factors for inability to clear the virus and persistent lymphopenia. C3 is important in the development of adaptive T cell response of influenza infection. Adaptive T cell response is important in clearing the virus from influenza infection. Influenza Virus Evades Innate and Adaptive Immunity via the NS1 Protein Ana Fernandez-Sesma, Svetlana Marukian, Barbara J. Ebersole, Dorothy Kaminski, Man-Seong Park, Tony Yuen, Stuart C. Sealfon, Adolfo García-Sastre, and Thomas M. Moran1 Journal of Virology, July 2006, p. 6295-6304, Vol. 80, No. 13

Proposed Management of Severe H1N1 2009 Human Swine Influenza : 

Proposed Management of Severe H1N1 2009 Human Swine Influenza Prophylactic LMWH

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Any Questions ?

Role of High Dose N-Acetylcysteine Anti-oxidant therapy in SuppressingCytokine Storm of H1N1 Pneumonia : 

Role of High Dose N-Acetylcysteine Anti-oxidant therapy in SuppressingCytokine Storm of H1N1 Pneumonia DR. Lai Kang Yiu Intensive Care Unit Queen Elizabeth Hospital

Theory Behind High Dose N-acetylcysteine Therapy for H1N1 Pneumonia : 

Theory Behind High Dose N-acetylcysteine Therapy for H1N1 Pneumonia H1N1 Pneumonia kills by cytokine storm and co-infection NFκβ hold a pivotal role in cytokine activation in influenza infection We can selectively block NFκβ and hence its cytokine activity without affecting the B cell and T cell immunity of influenza virus infection. The activation of NFκβ is induced by reactive oxygen species during influenza infection N-acetylcysteine in doses used for the treatment of acetaminophen (Paracetamol) poisoning can achieve high enough anti-oxidant activity to block reactive oxygen species induced by influenza infection. (hypothesis) "Attenuation of influenza-like symptomatology and improvement of cell-mediated immunity with long-term N-acetylcysteine treatment". S. De Flora, C. Grassi, L. Carati (1997).Eur Respir J: 1535–1541 Falk Nimmerjahn Journal of General Virology (2004), 85, 2347–2356 Active NF-kB signalling is a prerequisite for influenza virus infection Role of oxidants in influenza virus-induced gene expression Katharine Knobil et al Am J Physiol Lung Cell Mol Physiol vol 274: issue 1, L134-L142, 1998 van Klaveren, RJ, Demedts, M, Nemery, B Cellular glutathione turnover in vitro, with emphasis on type II pneumocytes. Eur Respir J 1997;10,1392-1400 Wendl, A, Cikryt, P The level and half life of glutathione in human plasma. FEBS Lett 1980;120,209-211 Effect of N-acetyl cysteine on the concentrations of thiols in plasma, bronchoalveolar lavage fluid, and lung tissue. M M Bridgeman. Thorax 1994;49:670-675 Expression of influenza virus hemagglutinin activates transcription factor NF-kappa B HL Pahl and PA Baeuerle J. Virol., Mar 1995, 1480-1484, Vol 69, No. 3

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Cutting Edge: Influenza A Virus Activates TLR3-Dependent Inflammatory and RIG-I-Dependent Antiviral Responses in Human Lung Epithelial Cells Ronan Le Goffic The Journal of Immunology, 2007, 178: 3368-3372. Cytokine storm can be blocked without affecting anti-viral activity.

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Cytokine Storm TNF-alpha, IL-1beta, IL-6, and IL-8. Expression of influenza virus hemagglutinin activates transcription factor NF-kappa B HL Pahl and PA Baeuerle J. Virol., Mar 1995, 1480-1484, Vol 69, No. 3 The cytokines storm of influenza virus is through the activation of NF-ķB by reactive oxygen species

Theory Behind High Dose N-acetylcysteine Therapy for H1N1 Pneumonia : 

Theory Behind High Dose N-acetylcysteine Therapy for H1N1 Pneumonia When there is bacterial co-infection, the mortality rate of influenza pneumonia increased 10x because of bacterial viral synergy Influenza open up neuraminidase site for attachment of bacteria Bacteria amplify viral proliferation by providing protease for cleavage activation of haemagglutinin. Neuraminidase inhibitor and high dose N-acetylcysteine is synergistic because Neuraminidase can reduce escape of virus and the subsequent exposure of neuraminidase site High dose N-acetycysteine prevent inactivation of anti-protease by reactive oxygen species in epithelial lining fluid and the subsequent cleavage activation of haemoglutinin by bacterial protease. The synergistic action of Tamiflu and high dose N-acetylcysteine in reducing mortality is shown in lethal mice model. Protective effect of n-acetylcysteine in a model of influenza infection in mice Ungheri D, Pisani C, Sanson G, Bertani A, Schioppacassi G, Delgado R, Sironi M, Ghezzi P Int J Immunopathol Pharmacol 2000 Sep-Dec;13(3):123-128. N-acetylcysteine synergizes with oseltamivir in protecting mice from lethal influenza infection Garozzo A, Tempera G, Ungheri D, Timpanaro R, Castro A Int J Immunopathol Pharmacol. 2007 Apr-Jun;20(2):349-54 Synergistic combination of N-acetylcysteine and ribavirin to protect from lethal influenza viral infection in a mouse model. Ghezzi P, Ungheri D. Int J Immunopathol Pharmacol. 2004 Jan-Apr;17(1):99-102.

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Role of Acetylcysteine Role of Tamiflu/Relenza

Slide 118: 

Reactive Oxygen Species Cleavage Activation of Haemagglutinin By protease Anti-protease inactivated Role of anti-oxidant glutathione (GSH) in the epithelial lining fluid (ELF) of the lower respiratory tract GSH X

Slide 119: 

CA-MRSA Role of Antibiotic Therapy

Theory Behind High Dose N-acetylcysteine Therapy for H1N1 Pneumonia : 

Theory Behind High Dose N-acetylcysteine Therapy for H1N1 Pneumonia IL6 was one of the major cytokine produced during influenza infection. Symptoms and fever in natural acute influenza correlate with the release of IL-6. IL6 can lead to the production of CRP, fibrinogen and factor VIII:C. CRP can serve as marker of IL6 level and influenza cytokine activities. Since in bacterial or fungal sepsis, induction of NKκβis not solely dependent on reactive oxygen species, persistent elevation of CRP despite NAC therapy indicate that bacterial co-infection is not under control or there is onset of new nosocomial sepsis. Elevated fibrinogen and factor VIII:C can increase patient’s thromboembolic risk during influenza infection and can explain the increased stroke and myocardial infarction after seasonal influenza and thromboembolism during H1N1 pneumonia. This may also explain why obesity who has a high basal IL-6 level due to adipokines is a risk factor for mortality. Symptom pathogenesis during acute influenza: interleukin-6 and other cytokine responses. Kaiser L. Fritz RS. Straus SE. Gubareva L. Hayden FG. Journal of Medical Virology. 64(3):262-8, 2001 Jul. Pathways by which IL-6 contributes towards haemostasis – an overview Kerr: Br J Haematol, Volume 115(1).October 2001.3-12 Factor VII and fibrinogen levels as risk factors for venous thrombosis: A case-control study of plasma levels and DNA polymorphisms: the Leiden Thrombophilia Study (LETS). Koster T, Rosendaal FR, Reitsma PH, van der Valden PA, Briet E, Vandenbroucke JP. Thromb Haemost. 1994;71:719–722 Excess factor VIII: a common cause of hypercoagulability. Bobrow RS. Journal of the American Board of Family Practice. 18(2):147-9, 2005 Mar-Apr.

Thrombophilia in H1N1 : 

Thrombophilia in H1N1 Tissue Factor

Theory Behind High Dose N-acetylcysteine Therapy for H1N1 Pneumonia : 

Theory Behind High Dose N-acetylcysteine Therapy for H1N1 Pneumonia Adaptive Immune response is necessary for clearing influenza virus infection N-acetylcysteine can improve the adaptive T cell response of influenza infection Maintenance N-acetylcysteine may be beneficial even during the recovery phase of influenza virus infection. "Attenuation of influenza-like symptomatology and improvement of cell-mediated immunity with long-term N-acetylcysteine treatment". S. De Flora, C. Grassi, L. Carati (1997).Eur Respir J: 1535–1541 Influenza Virus Evades Innate and Adaptive Immunity via the NS1 Protein Ana Fernandez-Sesma, Svetlana Marukian, Barbara J. Ebersole, Dorothy Kaminski, Man-Seong Park, Tony Yuen, Stuart C. Sealfon, Adolfo García-Sastre, and Thomas M. Moran1 Journal of Virology, July 2006, p. 6295-6304, Vol. 80, No. 13

Proposed Management of Severe H1N1 2009 Human Swine Influenza : 

Proposed Management of Severe H1N1 2009 Human Swine Influenza Prophylactic LMWH

Slide 126: 

Any Questions ?

Role of High Dose N-Acetylcysteine Anti-oxidant therapy in SuppressingCytokine Storm of H1N1 Pneumonia : 

Role of High Dose N-Acetylcysteine Anti-oxidant therapy in SuppressingCytokine Storm of H1N1 Pneumonia DR. Lai Kang Yiu Intensive Care Unit Queen Elizabeth Hospital

N-acetylcysteine EffectOn Influenza Infection : 

N-acetylcysteine EffectOn Influenza Infection Suppression of cytokine storm Improvement in T cell function No effect on antibody production

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TLR signaling pathways Kiyoshi Takeda, Shizuo Akira Seminars in Immunology 16 (2004) 3–9 Differential Role of TLR- and RLR-Signaling in the Immune Responses to Influenza A Virus Infection and Vaccination Shohei Koyama The Journal of Immunology, 2007, 179, 4711 -4720 Innate & adaptive Immune response Cytokine Storm Cytokine Storm

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Cutting Edge: Influenza A Virus Activates TLR3-Dependent Inflammatory and RIG-I-Dependent Antiviral Responses in Human Lung Epithelial Cells Ronan Le Goffic The Journal of Immunology, 2007, 178: 3368-3372. Cytokine storm can be blocked without affecting anti-viral activity.

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"Attenuation of influenza-like symptomatology and improvement of cell-mediated immunity with long-term N-acetylcysteine treatment". S. De Flora, C. Grassi, L. Carati (1997).Eur Respir J: 1535–1541

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"Attenuation of influenza-like symptomatology and improvement of cell-mediated immunity with long-term N-acetylcysteine treatment". S. De Flora, C. Grassi, L. Carati (1997).Eur Respir J: 1535–1541

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"Attenuation of influenza-like symptomatology and improvement of cell-mediated immunity with long-term N-acetylcysteine treatment". S. De Flora, C. Grassi, L. Carati (1997).Eur Respir J: 1535–1541

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"Attenuation of influenza-like symptomatology and improvement of cell-mediated immunity with long-term N-acetylcysteine treatment". S. De Flora, C. Grassi, L. Carati (1997).Eur Respir J: 1535–1541 N-acetylcysteine 600mg b.d.

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Cytokine Storm TNF-alpha, IL-1beta, IL-6, and IL-8. Se Prevent M1 mutation Se-Px Re-epithelialization of Respiratory Epithelium After Damage The cytokines storm of influenza virus is through the activation of NF-ķB by reactive oxygen species

Nuclear Factor κβ : 

Nuclear Factor κβ Role in cytokine storm Role in Influenza Inhibition by N-acetylcysteine Dr. Lai Kang Yiu Intensive Care Unit Queen Elizabeth Hospital

Slide 138: 

Oxidant Stress, Influenza Virus NFκβusually resides in the cytoplasm bound to inhibitor of NFκβ(I κβα) Upon exposure to oxidant stress, two specific serine residues are rapidly phosphorylated by the IKK1/2 kinase. Once pohsphorylated the Iκβs undergoes degradation via the ubiquitin-proteosome pathway in the main route of NFκβactivation.

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Role of Nuclear Factor κβ in Cytokine Storm

Active NF-kB signalling is a prerequisite for influenza virus infectionFalk Nimmerjahn Journal of General Virology (2004), 85, 2347–2356 : 

Active NF-kB signalling is a prerequisite for influenza virus infectionFalk Nimmerjahn Journal of General Virology (2004), 85, 2347–2356 Cells with low NF-kB activity were resistant to influenza virus infection, but became susceptible upon activation of NF-kB. In addition, blocking of NF-kB activation severely impaired influenza virus infection of otherwise highly susceptible human cells. The requirement of active NF-kB signalling pathway is specific for influenza virus infection. These might be of major importance for both the development of new antiviral therapies and the understanding of influenza virus biology.

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Cytokine Storm TNF-alpha, IL-1beta, IL-6, and IL-8. Expression of influenza virus hemagglutinin activates transcription factor NF-kappa B HL Pahl and PA Baeuerle J. Virol., Mar 1995, 1480-1484, Vol 69, No. 3 The cytokines storm of influenza virus is through the activation of NF-ķB by reactive oxygen species

Slide 142: 

Haemagglutinin Activation of NF-kappa B is caused by the accumulation of proteins in the endoplasmic recticulum membrane, a condition we have called ER overload. Both the release of Ca2+ from the ER and the subsequent production of reactive oxygen intermediates are required for ER-overload-mediated NF-kappa B activation. The ER-overload response: activation of NF-kappa B. Pahl HL. Baeuerle PA. Trends in Biochemical Sciences. 22(2):63-7, 1997 Feb. Signal Transduction From the Endoplasmic Reticulum to the Cell Nucleus Heike L. Pahl Physiological Reviews, Vol. 79, No. 3, July 1999, pp. 683-701 Role of NAC

Expression of IL-6, IL-8, and RANTES on human bronchial epithelial cells, NCI-H292, induced by influenza virus A.Matsukura S, Kokubu F, Noda H, Tokunaga H, Adachi M J Allergy Clin Immunol. 1996 Dec;98(6 Pt 1):1080-7 : 

Expression of IL-6, IL-8, and RANTES on human bronchial epithelial cells, NCI-H292, induced by influenza virus A.Matsukura S, Kokubu F, Noda H, Tokunaga H, Adachi M J Allergy Clin Immunol. 1996 Dec;98(6 Pt 1):1080-7 Chemotactic cytokine CHAK (CC-Chemokine-activated killer) cells ? Pulmonary fibrosis

Oxidants are involved in influenza virus-induced activation of NF- B, in the expression of IL-8 and MnSOD, and in virus-induced cell death. N-acetyl-L-cysteine attenuated virus-induced NF- B activation and interleukin (IL)-8 mRNA induction but did not block induction of MnSOD mRNA. : 

Oxidants are involved in influenza virus-induced activation of NF- B, in the expression of IL-8 and MnSOD, and in virus-induced cell death. N-acetyl-L-cysteine attenuated virus-induced NF- B activation and interleukin (IL)-8 mRNA induction but did not block induction of MnSOD mRNA. Role of oxidants in influenza virus-induced gene expression Katharine Knobil, Augustine M. K. Choi, Gordon W. Weigand, and David B. Jacoby Am J Physiol Lung Cell Mol Physiol 274: L134-L142, 1998;

N-acetylcysteine (NAC) did not affect IV infection-induced increases in MAP kinase phosphorylation, whereas NAC attenuated RANTES production by 18.2%, indicating that reactive oxygen species may act as a second messenger leading to RANTES production via p38 MAP kinase- and JNK-independent pathway. : 

N-acetylcysteine (NAC) did not affect IV infection-induced increases in MAP kinase phosphorylation, whereas NAC attenuated RANTES production by 18.2%, indicating that reactive oxygen species may act as a second messenger leading to RANTES production via p38 MAP kinase- and JNK-independent pathway. p38 Mitogen-Activated Protein Kinase and c-Jun-NH2-Terminal Kinase Regulate RANTES Production by Influenza Virus-Infected Human Bronchial Epithelial Cells Kousei Kujime, Shu Hashimoto2,, Yasuhiro Gon, Kazufumi Shimizu and Takashi Horie The Journal of Immunology, 2000, 164: 3222-3228.

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Indirect Action Action of High Dose NAC Summary of Action of High Dose N-acetylcysteine

Anti-oxidant Vitamin E, Vitamin C, Zinc and N-acetylcysteine have been shown to exert its action on suppressing production of NFκβin ameliorating influenza symptomatology : 

Anti-oxidant Vitamin E, Vitamin C, Zinc and N-acetylcysteine have been shown to exert its action on suppressing production of NFκβin ameliorating influenza symptomatology Only High Dose N-acetylcysteine has been shown in animal study to improvement in survival in lethal mice study

Common Anti-oxidant of the Body : 

Common Anti-oxidant of the Body Regeneration

Slide 149: 

Nucleus Dehydroascorbic acid Ascorbic acid Reactive Oxygen Species Both enzyme inhibited by dehydroascorbic acid but not by ascorbic acid Role of dehydroascorbic acid in inhibition of NFķβ activation NFκB Vitamin C is a kinase inhibitor: Dehydroascorbic acid inhibits I {kappa} B {alpha} kinase {beta}. Carcamo JM, Pedraza A, Borquez-Ojeda O et al. Mol Cell Biol 2004; 24(15):6645-6652.

The Effectiveness of Vitamin C in Preventing and Relieving the Symptoms of Virus-Induced Respiratory InfectionsGorton, J.C. & Jarvis, K. J Manipul Physiol Ther 1999; 22: 530-3 : 

The Effectiveness of Vitamin C in Preventing and Relieving the Symptoms of Virus-Induced Respiratory InfectionsGorton, J.C. & Jarvis, K. J Manipul Physiol Ther 1999; 22: 530-3 Evaluated megadoses of vitamin C (1000mg po tid) in preventing and relieving cold and flu symptoms in subjects aged 18-32. The control group was composed of 463 students while the test study group was composed of 252 students. Vitamin C was also given to the test group after they developed flu symptoms. Results: Vitamin C administered before or after the appearance of the flu relieved symptoms in 85% of test cases compared to controls.

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Nucleus Zn is able to inhibit NF- ķβ activation by blocking the phosphorylation and degradation of the inhibitory proteins IkBs and its multisubunit IkB kinases, essential reactions required for the activation of NFķβ. Role of Zinc in inhibition of NFķβ activation Thiol-reactive metal compounds inhibit NF-[kappa][beta] activation by blocking [IOTA][kappa][beta] kinase. Jeon KI, Jeong JY, Jue DM. J. Immunol. 2000; 164: 5981–9. Inhibitors of NF-KB Activity: Tools for Treatment of Human Ailments Vinay Tergaonkar, Qiutang Li and Inder M. Vennm NFκβ

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Effect of long-term dietary antioxidant supplementation on influenza virus infection Sung Nim Han, Mohsen Meydani, Dayong Wu, Bradley S Bender, et al. The Journals of Gerontology. Oxford: Oct 2000. Vol. 55A, Iss. 10; p. B496 (8 pages)

The Treatment of Pneumonias in Influenza Using AntioxidantsNagibina, M.V. et. al. Terapeuticheskii Arkhiv 1996; 68: 33-35 (Russian) : 

The Treatment of Pneumonias in Influenza Using AntioxidantsNagibina, M.V. et. al. Terapeuticheskii Arkhiv 1996; 68: 33-35 (Russian) Lipid peroxidation occurs in patients with influenza complicated by pneumonia. Parameters of oxidative stress are greatest in the acute phase and in seriously ill patients. Administration of antioxidants, including vitamin E, decreases oxidative stress and is associated with an improved clinical response.

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"Attenuation of influenza-like symptomatology and improvement of cell-mediated immunity with long-term N-acetylcysteine treatment". S. De Flora, C. Grassi, L. Carati (1997).Eur Respir J: 1535–1541

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Role of anti-oxidant N-acetylcysteine in preventing cleavage activation of haemagglutinin epithelial lining fluid (ELF) of the lower respiratory tract

Why High Dose N-Acetylcysteine ?(a) Animal study on survival on mice(b) Elevate GSH level in ELF of LRT : 

Why High Dose N-Acetylcysteine ?(a) Animal study on survival on mice(b) Elevate GSH level in ELF of LRT Supplement GSH in the epithelial lining fluid (ELF) of the lower respiratory tract to prevent inactivation of anti-protease by reactive oxygen species generated by influenza and secondary bacterial infection. The presence of anti-protease can prevent the amplified proteolytic cleavage activation of haemagglutinin of newly released influenza virus in the in the epithelial lining fluid (ELF) of the lower respiratory tract.

The dorminant anti-oxidant molecule both intracellular and in the epithelial lining fluid of the lower respiratory tract is GSH.  Intracellular and extracelluar soluble anti-oxiant : 

The dorminant anti-oxidant molecule both intracellular and in the epithelial lining fluid of the lower respiratory tract is GSH.  Intracellular and extracelluar soluble anti-oxiant In the lung, GSH is present in high concentrations in the epithelial lining fluid (ELF) of the lower respiratory tract, with normal levels in human more than 50- 150 fold greater than in the plasma. Human ELF contain all elements of the redox cycle of the GSH system including glutathione peroxidase and glutathione reductase.

N-acetylcysteine given by mouth is rapidly deacetylated to cysteine, with resulting increases in the concentrations of cysteine in plasma and of reduced glutathione in plasma and the airwaysCysteine and glutathione concentrations in plasma and bronchoalveolar lavage fluid after treatment with N-acetylcysteine Bridgman MME, Marsden M, MacNee W, Flenley DC, Ryle P. Thorax 46:39-42 (1991). : 

N-acetylcysteine given by mouth is rapidly deacetylated to cysteine, with resulting increases in the concentrations of cysteine in plasma and of reduced glutathione in plasma and the airwaysCysteine and glutathione concentrations in plasma and bronchoalveolar lavage fluid after treatment with N-acetylcysteine Bridgman MME, Marsden M, MacNee W, Flenley DC, Ryle P. Thorax 46:39-42 (1991). However when given at 600mg t.d.s, N-acetyl cysteine does not produce a sustained increase in glutathione levels sufficient to increase the antioxidant capacity of the lungs. Effect of N-acetyl cysteine on the concentrations of thiols in plasma, bronchoalveolar lavage fluid, and lung tissue. M M Bridgeman Thorax 1994;49:670-675

: 

Influenza virus hemagglutinin required cleavage into HA1, HA2 during activation HA is synthesised in the rough endoplasmic reticulum, and is transported to the cell surface via the Golgi apparatus. HA is synthesised as a precursor molecule (HA0), which undergoes proteolytic processing into two subunits (HA1 and HA2), which are held together by disulphide bonds) Cleavage of HA0 to HA1 and HA2

If the noninfectious HAO form of the virus is released from cells without being cleaved, extracellular proteases present in pulmonary surfactant can proteolytically cleave this protein (Kido et al. 1993). As a protective mechanism, anti-proteases are present on the surface of alveoli. However, the anti-proteases can be inactivated by ROS. In this regard, it is important to note that during lung inflammation phagocytes increase in number and produce ROS (McCusker 1992). : 

If the noninfectious HAO form of the virus is released from cells without being cleaved, extracellular proteases present in pulmonary surfactant can proteolytically cleave this protein (Kido et al. 1993). As a protective mechanism, anti-proteases are present on the surface of alveoli. However, the anti-proteases can be inactivated by ROS. In this regard, it is important to note that during lung inflammation phagocytes increase in number and produce ROS (McCusker 1992). In vitro study showed that oxidant-treated anti-protease is unable to prevent trypsin from cleaving HAO to HA1/HA2, resulting in a 10,000-fold increase in infectious virus (Hennet et al. 1992a).

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Reactive Oxygen Species Cleavage Activation of Haemagglutinin By protease Anti-protease inactivated Role of anti-oxidant glutathione (GSH) in the epithelial lining fluid (ELF) of the lower respiratory tract GSH

Selenium Supplement is Recommended during High Dose N-acetylcysteine Therapy : 

Selenium Supplement is Recommended during High Dose N-acetylcysteine Therapy Dr. Lai Kang Yiu Intensive Care Unit Queen Elizabeth Hospital

The Important Role of Selenium : 

The Important Role of Selenium Selenium (Se) is required for the activity of GPx (Se-GPx), the enzyme catalysing rate limiting step in the anti-oxidant effect of glutathione and protection against lipoperoxidation. The presence of selenocysteine, rather than cysteine, in the active site of an enzyme increases enzyme activity 100- to 1,000-fold. Selenium deficiency has also been shown to increase the M matrix protein mutation in animal study.

Slide 173: 

Cytokine Storm TNF-alpha, IL-1beta, IL-6, and IL-8. Se Prevent M1 mutation Se-Px Re-epithelialization of Respiratory Epithelium After Damage

Slide 174: 

Selenium deficiency increase mutation in gene for the M1 matrix protein increase pro-inflammatory cytokines/chemokines suppress anti-inflammatory cytokines shift from a TH1 response to a TH2 response

Slide 175: 

GSH GSSH NADP NADPH Dihydroascorbate Ascorbic Acid Glutathione Peroxidase Se-GPx Glutathione Reductase GR Selenium (Se) is required for the activity of GPx (Se-GPx), providing protection against lipoperoxidation. The presence of selenocysteine, rather than cysteine, in the active site of an enzyme increases enzyme activity 100- to 1,000-fold.

Selenium Deficiency and Viral Infection Melinda A. Beck Orville A. Levander and Jean HandySupplement: 11th International Symposium on Trace Elements in Man and Animals The American Society for Nutritional Sciences J. Nutr. 133:1463S-1467S, May 2003 : 

Selenium Deficiency and Viral Infection Melinda A. Beck Orville A. Levander and Jean HandySupplement: 11th International Symposium on Trace Elements in Man and Animals The American Society for Nutritional Sciences J. Nutr. 133:1463S-1467S, May 2003

Slide 178: 

Selenium deficiency increases lung pathology in influenza-infected mice Selenium deficiency induces changes in cellular phenotype of lung infiltrating cells. Selenium deficiency has no effect on viral titers Selenium deficiency has no effect on antibody response Selenium deficiency alters cytokine and chemokine expression it was associated with an increase in the mRNA expression of proinflammatory cytokines and chemokines and a decrease in the expression of antiinflammatory cytokines. the immune response in the infected lung tissue was shifted away from a TH1 response to a TH2 response in the Se-deficient mice. Selenium deficiency increases the pathology of an influenza virus infection MELINDA A. BECK, HEATHER K. NELSON, QING SHI , PETER VAN DAEL , EDUARDO J. SCHIFFRIN , STEPHANIE BLUM , DENIS BARCLAY and ORVILLE A. LEVANDER The FASEB Journal. 2001;15:1481-1483.

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Selenium deficiency increases the pathology of an influenza virus infection MELINDA A. BECK, HEATHER K. NELSON, QING SHI , PETER VAN DAEL , EDUARDO J. SCHIFFRIN , STEPHANIE BLUM , DENIS BARCLAY and ORVILLE A. LEVANDER The FASEB Journal. 2001;15:1481-1483.

T cell FunctionRole in Clearing Influenza VirusCD 95 Programmed Cell Death of Influenza Virus Infection : 

T cell FunctionRole in Clearing Influenza VirusCD 95 Programmed Cell Death of Influenza Virus Infection Dr. Lai Kang Yiu Intensive Care Unit Queen Elizabeth Hospital

Slide 182: 

T4 lymphocytes (helper cells) and T8 lymphocytes (cytotoxic and suppressor cells )

Zinc’s Role in Immune System : 

Zinc’s Role in Immune System Zinc plays a central role in the immune system, and deficiency affects immune function at many levels, both innate and specific. Cell-mediated immunity is profoundly affected in zinc deficiency. Lymphopenia, lymphoid atrophy, defects in specific T- and B-lymphocyte function, and impaired phagocytosis have all been described

Zinc in Innate Immune Response : 

Zinc in Innate Immune Response Zinc induces IFN-[alpha] levels and IL-2 production and is essential to IL-2-mediated T-cell activation. Zinc is a cofactor for calcineurin (an important component of the T cell receptor pathway) and many transcription factors, some of which activate IL-2 promoter. In zinc deficiency, activity of serum thymulin (a thymus specific hormone involved in T cell function) is also decreased, and lytic activity of natural killer cells and the percentage of precursors of cytolytic T cells is decreased. Thymulin promotes T-cell function, including suppressor function and interleukin-2 production. Zinc supplementation in elderly has led to improved cell-mediated immune response due to restoration of thymulin activity.

Zinc and Adaptive Immune Response : 

Zinc and Adaptive Immune Response Zinc deficiency in humans has been associated with the atrophy of lymphoid tissue, reduced antibody response to thymus-dependent antigens, loss of cytotoxic T-lymphocyte responses to tumor cells, and loss of natural killer cell function. A switch from an initial Th1 to Th2 immune response occurs in Zn deficiency and can be reversed when patients are treated with Zn supplements. Zinc and immune function: the biological basis of altered resistance to infection AH Shankar and AS Prasad American Journal of Clinical Nutrition, Vol 68, 447S-463S,

Human Lymphocyte Apoptosis after Exposure to Influenza A Virus[Joan E. Journal of Virology, July 2001, p. 5921-5929, Vol. 75, No. 13] : 

Human Lymphocyte Apoptosis after Exposure to Influenza A Virus[Joan E. Journal of Virology, July 2001, p. 5921-5929, Vol. 75, No. 13] Analysis of lymphocyte subpopulations after exposure to Influenza A virus showed that a portion of CD3+, CD4+, CD8+, and CD19+ lymphocytes became apoptotic (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling positive). The percentage of cells that are infected was shown to be less than the percentage of apoptotic cells, suggesting that direct effects of cell infection by the virus cannot account fully for the high level of cell death. Removal of monocytes-macrophages after IAV exposure reduced the percent of lymphocytes that were apoptotic. Treatment of virus-exposed cultures with anti-tumor necrosis factor alpha did not reduce the percentage of lymphocytes that were apoptotic. In virus-exposed cultures treated with anti-FasL antibody, recombinant soluble human Fas, Ac-DEVD-CHO (caspase-3 inhibitor), or Z-VAD-FMK (general caspase inhibitor), apoptosis and production of the active form of caspase-3 was reduced. The apoptotic cells were Fas-high-density cells while the nonapoptotic cells expressed a low density of Fas. The present studies showed that Fas-FasL signaling plays a major role in the induction of apoptosis in lymphocytes after exposure to IAV.

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Defective neutrophil and monocyte chemotactic, oxidative, and bacterial killing functions Zinc suppresses caspase-3 activation

Influenza A Virus Accelerates Neutrophil Apoptosis and Markedly Potentiates Apoptotic Effects of Bacteria[Maria Luisa Colamussi Blood, Vol. 93 No. 7 (April 1), 1999: pp. 2395-2403] : 

Influenza A Virus Accelerates Neutrophil Apoptosis and Markedly Potentiates Apoptotic Effects of Bacteria[Maria Luisa Colamussi Blood, Vol. 93 No. 7 (April 1), 1999: pp. 2395-2403] Unopsonized E coli accelerates neutrophil apoptosis in a dose-related and time-dependent fashion, and that the degree of neutrophil apoptosis is enhanced when E coli is preincubated with opsonizing antibodies. Potentiation

Slide 191: 

Apoptosis is the orderly process of programmed cell death, by which organisms remodel their tissues. After the nucleus and the cell break down, debris is ingested by white blood cells.

Slide 192: 

Necrotic cells and their organelles are characteristically swollen. There is early membrane damage with eventual loss of plasma membrane integrity and leakage of cytosol into extracellular space. Despite early clumping, the nuclear chromatin undergoes lysis (karyolysis). Cell shrinkage, nuclear condensation, membrane blebbing, fragmentation into membrane bound apoptotic bodies, and membrane changes that eventually lead to phagocytosis of the affected cell

Slide 193: 

Apoptosis can occur via cell-surface-death-receptor- or mitochondrial-dependent pathways. SIV-mediated apoptosis might be regulated by both receptor- and mitochondrial-mediated cell death. Activation of the intrinsic mitochondrial apoptotic pathway in swine influenza virus-mediated cell death Young Ki Choi EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 38, No. 1, 11-17, February 2006

? LDH can serve as a marker of influenza virus induced apoptotic cell degradation : 

? LDH can serve as a marker of influenza virus induced apoptotic cell degradation Lactate Dehydrogenase Leakage as a Marker for Apoptotic Cell Degradation Induced by Influenza Virus Infection in Human Fetal Membrane CellsNoboru Uchide, Kunio Ohyama, Toshio Bessho, Hiroo Toyoda Intervirology 2009;52:164-173

Vitamin E and NAC Role in Preventing CD 95 ligand Programmed Cell Death : 

Vitamin E and NAC Role in Preventing CD 95 ligand Programmed Cell Death Vitamin E inhibits CD95 (APO-1/Fas) ligand (CD95L) expression and protects T cells from activation-induced cell death (AICD). Vitamin E suppresses CD95L promoter activity by downregulation of transcriptional activity of NF-[kappa]B and AP-1 and, consequently, downregulates CD95L mRNA expression and CD95-mediated AICD. The antioxidants N-acetyl-cysteine abolished Fas-induced cell death (International Immunology. 9(1):117-25, 1997 Jan. Deas O. )

Zinc and Apoptosis of Respiratory Epithelial Cell : 

Zinc and Apoptosis of Respiratory Epithelial Cell Zinc suppresses caspase-3 activation and apoptosis in respiratory epithelial cells. The inhibitory effects of Zn in apoptosis is thought to act at multiple sites by: (1) inhibiting endonucleases responsible for DNA fragmentation; (2) inhibiting the activation of caspase-3 and 6, the major executioner caspases; and (3) increasing the increasing the B cell lymphoma-2 (Bcl-2)/Bax ratio(Bcl-2 to Bax ratio), thereby increasing the resistance of cells to apoptosis.. CAD, calcium activated DNase; P21, p21waf1/cip1 protein; Ps flip, phosphatidyl-serine flip.

Re-epithelialization of Respiratory Epithelium After Damage : 

Re-epithelialization of Respiratory Epithelium After Damage After an insult, the respiratory epithelium initiates a tissue-healing process that involves the rapid re-epithelialization of the denuded area. Restoration of the integrity of the epithelium is via the dedifferentiation, spread, and rapid migration of the remaining viable epithelial cells found at the edge of the wound over the denuded basement membrane. This re-epithelization process in the respiratory epithelium is thought to be zinc dependent and was controlled by the upregulation of Zn dependent metalloproteinases (e.g. MMP-9 and MMP-3).

CRP as Biomarker of Influenza Pneumonia and Also Allow Early Detection of Nosocomial Pneumonia : 

CRP as Biomarker of Influenza Pneumonia and Also Allow Early Detection of Nosocomial Pneumonia Dr. Lai Kang Yiu Intensive Care Unit Queen Elizabeth Hospital

Slide 202: 

Cytokine Storm TNF-alpha, IL-1beta, IL-6, and IL-8. Expression of influenza virus hemagglutinin activates transcription factor NF-kappa B HL Pahl and PA Baeuerle J. Virol., Mar 1995, 1480-1484, Vol 69, No. 3 Bacterial or Fungal Sepsis

N-acetylcysteine cannot suppress IL-6 during bacterial sepsis. : 

N-acetylcysteine cannot suppress IL-6 during bacterial sepsis. Administration of N-acetylcysteine results in decreased nuclear factor-kappa B activation in patients with sepsis, associated with decreases in interleukin-8 but not interleukin-6 or soluble intercellular adhesion molecule-1. These pilot data suggest that antioxidant therapy with N-acetylcysteine may be useful in blunting the inflammatory response to sepsis. The effect of N-acetylcysteine on nuclear factor-kappa B activation, interleukin-6, interleukin-8, and intercellular adhesion molecule-1 expression in patients with sepsis. Paterson RL. Galley HF. Webster NR. Critical Care Medicine. 31(11):2574-8, 2003 Nov.

The antioxidants N-acetylcysteine abolished the hypoxic activation of NF-kappa B, TNF-alpha gene transcription, and increases in ROS levels but not LPS activation of NF-kappa B : 

The antioxidants N-acetylcysteine abolished the hypoxic activation of NF-kappa B, TNF-alpha gene transcription, and increases in ROS levels but not LPS activation of NF-kappa B Role of oxidants in NF-kappa B activation and TNF-alpha gene transcription induced by hypoxia and endotoxinNS Chandel, WC Trzyna, DS McClintock, PT Schumacker J. Immunol (2000) 165: 1013-21.

If CRP not suppressed within 3 days by high dose NAC, co-infection inadequately treated. : 

If CRP not suppressed within 3 days by high dose NAC, co-infection inadequately treated. If there is secondary rise in CRP while on high dose N-acetylcysteine therapy, the patient has new nosocomial sepsis not covered by current antibiotic regime. Take Home Message

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The future threat starts in Hong Kong. The future solution starts in Hong Kong H1N1 and H5N1 The problem surfaced in 1997. The solution surfaced in 1997.

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Thank you for your attention