Cytokine Gene Therapy in Viral Myocarditis

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- VAD in treatment of Fulminant Myocarditis -

Definition of Myocarditis an INFLAMMATION INFILTRATE and by INJURY to the adjacent myocardial cell that is not typical of INFARCTION Dallas Criteria (1986) inflammatory infiltrate of the myocardium with necrosis and/or degeneration of adjacent myocardium not typical of the ischemic damage associated with coronary artery disease 2

Etiologic Agents of Myocarditis Infectious Agents Virus Coxsackievirus(A,B), HIV Echovirus, Influenza(A,B), Poliovirus, Herpes Simplex, Varicella-Zoster, Rubella, Rubeola, Epstein-Barr,Cytomegalovirus, Mumps, Vaccinia, Hepatitis B, Bacteria : diphtheria, Neisseria... Metazoa : Trichinosis, Echinococcosis Protozoa : Trypanozoma, Toxoplasma Fungus : Aspergillosis, Cadidiasis... Toxic Agents Anthracyclines Catecholamines Interleukin-2 Interferon-alpha2 Hypersensitivity 3

Diagnosis of Viral Myocarditis Serology 4-fold increase of Neutralization Ab titer Virus culture in tissue Viral genomes in tissue by PCR, Hybridization Viral proteins in heart 4

Neutralization Ab Test (MTT Assay) 150 ATCC Ab Serum (2 days) 8A6 Ab Serum (21 days) % of Cell Survival 125 100 75 50 25 0 1:1 1:2 1:4 1:8 Dilution Factor 1:16 1:32 1:64 1:128 JES et al., NEJM 345:179, 2001 5

Detection of enteroviral Genome by in situ Hybridization in CVB2 myocarditis Badorff C. Med Microbiol Immunol (Berl). 2003 Aug 12 6

Detection of enteroviral capsid protein VP1 by immunohistochemistry A : fatal myocarditis B : D-CMP C: Chronic myocarditis D : Negative control with IgG2a x200 : A, B1, C1, D x600 : B2, C2 Circulation 2000;101:231 7

Coxsackievirus Type B3 (CVB3) 7.4 Kb ssrna picornavirus like as polio-, echo-, rhinovirus highly cardiotropic induces myocarditis progress to cardiomyopathy? 8

Diseases Associated with Coxsackievirus 9

Diseases Associated with Coxsackievirus Day 7(Control) Day 7(CVB3-H3) Day 14 (CVB3-H3) Control H3 H3-3140 10

Time Course of Viral Myocarditis N Engl J Med 2000;343:1388 11

Clinical Presentation & Evolution 12

Enteroviral Infection Manifested by Dilated Cardiomyopathy Male / 20

Chest PA 2003/3/21 2003/11/26 2003/07/07 14

Echocardiography and NT-proBNP 3/22 4/4 5/19 10/06 LV 74/81 76/81 74/82 46/62 EF(%) 18 14 19.6 44.9 LA 57 52 54 36 NTproBNP 2254 2089 1680 5.0 15

H & E, x400 IHC, x100 IHC, x100 IHC, x100, control IHC with anti-enteroviral Ab 16

Clinical Presentation & Evolution 17

Unadjusted Transplantation-free Survival According to Clinicopathological Classification N Engl J Med 2000;342:690-5 18

Fulminant Myocarditis with Pancreatitis Male / 15

Case Summary initial presentation A 15-year-old boy was admitted with anterior chest discomfort for three days at a university Hospital. One week earlier, he had had flu-like symptoms. On hospital day 2, ventricular tachycardia and complete RBBB developed. Shock and urine output decreased. CPR was done for 2 times and started hemodialysis. Echocardiography showed low EF (15-20%), moderate TR, MR and moderate pulmonary HT. He was transferred to SMA at hospital day 14. 20

Laboratory Findings - pancreatitis 01/30 02/8 2/16 03/16 Day 1 Day 9 Day 17 OPD LVID s/d 50/59 39/55 40/55 EF 22 45 55 Amylase / Lipase 159 / 938 164 / 739 131 /454 83 / 131 AST / ALT 16 / 102 24 /12 23 / 25 12 / 17 CK-MB 6.41 4.90 NT-proBNP 35000 5805 1991 287.4 21

Endomyocardial biopsy findings done at hospital day 12 (+ 2 weeks) H&E x 200 H&E x 400 MT x 400 MT x 200 Focal mild hypertrophy of muscle cells and enlarged nuclei with focal microcalcifications. Few inflammatory cell infiltration. Minimal interstitial fibrosis. No evidence of viral inclusion 22

Neutralization test with serial sera CVB3 CVB4 23

Fulminant Coxsackieviral B3 Myocarditis First Case Report; Identified CVB3 VP1 proteins in the tissue biopsy from human Fulminant myocarditis Female / 18

A E F G H B C D Case of fulminant Coxsackieviral myocarditis A, C) EKG and Echo at ER B, D) EKG and Echo at day 10 E) Left auricle (H& E stain, x200) F) Left Auricle (Masson s Trichrome stain, x200) G,H) Immunohistochemistry probed by anti-enteroviral VP1 Ab.(G; x100,h; x400) Jeon ES. NEJM 345:179, 2001 25

Ventricular Assistant Device IABP From LA to Aorta 26

Fulminant Myocarditis Treated with EBS Myocarditis with Hepatitis Male / 14

Case Summary initial presentation A 14-year-old boy was admitted with anterior chest pain for a day. Three days earlier, he had had flu-like symptoms. The patient had a positive troponin I (49.97 ng/ml) result and an elevated level of CK-MB (98.27 ng/ml) A coronary angiogram showed no thrombus and no clinically significant stenosis. 28

Case Summary Hospital course His ejection fraction at ER was over 50% Eight hours after admission, complete AV block was developed and IV isoproterenol was started for heart rate control. After then, various ventricular arrhythmias were developed and treated with DC version. Shock and urine output decreased and treated with dopamine and dobutamine.. He was transferred to SMC after temporary pacemaker insertion and with amiodarone infusion (day 0). 29

Case Summary EKG 30

Case Summary Hospital course Ventilation support were started due to hypoxia. At day 1, shock and pulmonary edema progressed, and urine output was decreased and EF was < 20%. Mechanical circulatory support was started with EBS (Terumo, CAPIOX SP101, Tokyo, Japan). Urine output and BP were maintained with EBS and inotropics. After 56 hours of mechanical support with EBS, leftventricular-wall motion was restored and her ejection fraction was 45 % on echocardiography. The EBS was removed and he was discharged at hospital day 17 without any symptoms of heart failure. 31

EBS continuous flow 32

Echocardiography Date Hosp day LVID s/d (mm) EF(%) Other findings Feb. 06 1 pm Feb. 07 6 pm Feb. 07 10 pm 0 1 1 Size were normal range 60 30 20 Mild MR on color Doppler Global hypokinesia (especially anterior & IVS) Hypokinesia Feb. 13 7 29/42 53 (IVS & aneriorior wall) - pericardial effusion Feb. 23 16 32/48 55.5 Normal Wall motion 33

Laboratory Findings - hepatitis 02/07 02/14 02/23 03/08 Day 1 Day 7 Day 16 OPD LVID s/d - 29/42 32/48 - EF 60 20 53 55.5 - AST/ ALT 5730 / 3500 110 / 742 29 / 86 16 / 14 CK-MB 22.43 3.56 0.36 NT-proBNP - 2093 1109 272.6 34

Neutralization test with serial sera CVB3 CVB4 35

Endomyocardial biopsy findings done at hospital day 10 (H&E, MT staining) H&E x 200 MT x 200 H&E x 400 36

Fulminant Coxsackieviral B4 Infection Dystrophin cleaved in infected myocytes in human tissue VAD applied Female / 57

Case Summary initial presentation A 57-year-old woman was admitted with anterior chest pain for a day. Three days earlier, she had had flu-like symptoms. The patient had a positive troponin I (49.97 ng/ml) result and an elevated level of creatinine phosphokinase-mb (98.27 ng/ml) A coronary angiogram showed no thrombus and no clinically significant stenosis. 38

Case Summary Hospital course Eight hours after admission, shock and pulmonary edema developed. Her ejection fraction became less than 15 percent from over 60% at admission. Despite the use of an intra-aortic balloon pump for four hours, her shock and pulmonary edema progressed, and recurrent ventricular tachycardia occurred. Mechanical circulatory support was started with a left ventricular assist device. (550 BIO-Console, Medtronics, Bio-Medicus, ECMO). 39

Case Summary Hospital course After 96 hours of support with the left ventricular assist device (ECMO), left-ventricular-wall motion was restored and her ejection fraction was 56 percent on echocardiography. ECMO was removed after 5 days. She was discharged after 35 days without any symptoms of heart failure. 40

Chest PA 2003/08/08 2003/11/24 41

Neutralization Test for all CVB Serotypes with day 15 serum CVB1 CVB3 CVB5 CVB2 CVB4 CVB6 42

Neutralization Test for CVB4 43

PFU assay from tissue on HeLa Viable virus was isolated from frozen left atrial tissue at day 1. The final concentration of virus was 1.5 x 10 4 PFU/ml 44

At Day 1 H & E, x400 IHC, x100 IHC, x100 IHC, x100, control IHC with anti-enteroviral VP1 Ab 45

Coxsackieviral protease 2A cleaves dystrophin Badorff, Nat Med 5:320, 1999 46

CVB3 protease 2A cleaves dystrophin-sarcoglycan Complexes CVB3, Dystrophin Add Evans blue Evans blue CVB3, α-sarcoglycan CVB3, β-sarcoglycan Badorff, Nat Med 5:320, 1999 47

Anti-dystrophin-DAB at day1 Anti-enteroviral VP1-alkaline P at day 1 x 200 Merged 48

Therapeutic Guideline of Fulminant Myocarditis 52 patients for 3 years (Apr. 1997 Mar. 2000) Circ J 2002;66:133 49

Initial and Cardiac Symptoms Circ J 2002;66:133 50

Guidelines of PCPS for acute fulminate myocarditis (1) Circ J 2002;66:133 51

Ventricular Assistant Device IABP Yes For 1) Bridge to Recovery 2) Bridge to Transplantation REMATCH, NEJM 2001;345:1435 Weaning No No REMATCH study No Survival (%) P=0.001 LV assist device Yes Medical therapy 8% No, NO gas, No. at Risk LV assist device Cardiac Medical therapy & Vascular Center Months Yes / N Engl N J Engl Med J Med 2001;345:1435 52

VAD EBS Artificial Heart 53

Mechanical pumps for Cardiac Support Thermo-Cardiosystems device N Engl J Med 2001;345:1435 54

Development of VAD N Engl J Med 2001;345:1435 55

Indications for Device support 1. Cardiogenic Shock 2. Heart failure dependent on intravenous inotropic support 3. Outpatients with symptomatic heart failure functional class IV 4. Uncontrolled ventricular arrhythmia 5. Cardiac allograft dysfunction and/or cardiac allograft vasculapathy 6. Fulminant myocarditis Consensus Conference Report, JACC 2001;37:340 56

Benefits of mechanical support 1. Decrease cardiac strain and work load 2. Increase subendocardial blood flow 3. Normalize histologic changes fiber orientation Cardiac hypertrophy Decrease myocyte wavy fibers and contraction-band necrosis 4. Decrease chamber size 5. Increase mitochondria energy metabolism 6. Inactivation of neurohumoral factors (RAAS, Sympathetic nervous system) Consensus Conference Report, JACC 2001;37:340 57

Current Status of Mechanical Cardiac Support Devices in USA Consensus Conference Report, JACC 2001;37:340 58

Anticipated Survival According to Severity of Advanced Heart Failure Disease entity Cardiogenic shock Chronic heart failure Heart failure Severity of Heart Failure Chronic HF with exacerbation into critical low output state Acute myocardial infarction Post-cardiotomy shock dependent on intravenous inotropic therapy class IV symptoms on oral therapy Refractory symptoms at rest or minimal exertion Risk factors such as decreasing sodium, increasing creatinine and/or BUN Stabilization as class III refractory ventricular arrhythmias Expected more than 50% Mortality In-hospital 3-6 months 12-24 months less than 12 months less than 12 months more than 24 months Variable, not estimated Chronic severe post-transplant graft dysfunction with allograft vasculopathy less than 12 months Consensus Conference Report, JACC 2001;37:340 59

Guidelines of PCPS for acute fulminate myocarditis (2) Circ J 2002;66:133 60

The end points for critical populations Consensus Conference Report, JACC 2001;37:340 61

Guidelines of PCPS for acute fulminate myocarditis (3) Circ J 2002;66:133 62

Factors Influencing Prognosis Important factors concerning the prognosis were 1) the severity and grade of cardiac and renal dysfunction 2) the adjusted support flow rate to enable recovery from circulatory failure 3) prevention of circulatory disturbances of the legs and multiple organ failure directly associated with PCPS. Long-term prognosis of patients treated with PCPS 1. the readmission rate was 10% 2. the exacerbation rate was 3.3% 3. mortality was 10% during the average follow-up period of 962 days. Optimal management of the mechanical cardiopulmonary support and curative treatment for the myocarditis further improve the outcome of this disease. Circ J 2002;66:133 63

Predictors of Clinical Manifestations and Courses In Patients with Acute Fulminant Coxsackievirus Myocarditis Age/Sex Initial Manifestation EF normalized MCS/ Inotropics CVB type F/25 Dyspnea > 2 year No CVB3 NFM (n=5) M/31 M/46 M/22 Fever Chest pain Dyspnea 9 days 8 days 12 months No No No/yes CVB3 CVB3 CVB3 M/31 Dyspnea 0 No CVB3/4 F/57 Dizziness 6 days VAD CVB4 FM (n=5) M/15 M/14 F/59 Chest pain Dyspnea Chest pain 48 days 8 days 0 No/yes EBS IABP CVB3/4 CVB3 Adeno F/9 Chest pain 5 days No/IABP Adeno 64

Laboratory Markers between FM and NFM NFM FM P value Age 29.2 10.1 30.8 22.3 ns Initial NT pro-bnp 7500 3305 18420 12320 ns Peak TnI 24.8 33.5 889.2 610.8 P<0.05 Peak CK-MB 14.7 21.7 60.1 67.7 P<0.05 WBC 6695 639 8595 5600 P<0.05 Initial ESR 14 0 31.5 17.6 ns Initial CRP 3.29 3.67 6.21 3.05 ns 65

Cytokines between FM and NFM NFM FM P value RVSP by Doppler 44.4 3.2 31.5 2.1 P<0.05 IL-1β IL-6 below detectable range below detectable range ns ns hil-6 (pg/ml) 28.6 9.3 239.7 124.1 P<0.05 TNFRII( pg/ml) 4.2 1.7 26.85 11.9 P<0.05 TNF-α below detectable range ns 66

Predictors of Clinical Manifestations and Courses In Patients with Acute Fulminant Coxsackievirus Myocarditis The clinical courses of acute FM and NFM CVB myocarditis are too different. Among the initial laboratory findings, leukocytosis, initial cardiac enzymes, CK-MB, TnI, and cytokines, hil-6 and TNFRII, may be helpful to predict the course of acute CVB myocarditis Since the patients with FM recover without residual LV dysfunction within one month and had more excellent long-term prognosis, the aggressive hemodynamic support is warranted. 67

University of California, San Diego, USA Kirk Knowlton M.D Neil Berkely Sally Huber, PhD, University of Vermont, USA Andrea Henke, PhD, University of Jena, Germany :,, : : :, 68

Transformation of Myocarditis/Inflammatory cadiomyopathy to idiopathic D-CMP 1 Myocarditis with CMP Virus associated D-CMP 3 Virus Evidence CVB, Adenovirus yes (+)Parvo-19, HCV, CMV - no Recovery 2 Viral myocarditis Autoimmune Myocarditis Virus(-) Virus Negative D-CMP Med Microbiol Immunol 2004;163:61 69

Mechanisms of Virus-induced autoimmune heart disease VIRUS Molecular mimicry Breakdown in suppressor mechnisms Breakthrough of rogue Autoreactive T cell clones Tissue necrosis Insertion of viral epitopes into myocardial cell membrane Cross-reaction Between Anti-viral Ab And Heart antigens Disturbance in the host s Immune response Presentation Of sequestered Myocardial autoantigen Induction of myocardial neoantigen Breakdown of T cell tolerance to myocardial self antigens resulting in autoimmune heart disease Hypothetical mechanism of virus-induced or precipitated autoimmune Heart disease. None of these has been proven in patients with other autoimmune conditions. Med Microbiol Immunol 2004;163:61 70

Neutralization Test for all CVB Serotypes CVB1 CVB3 CVB5 CVB2 CVB4 CVB6 71

Neutralization Test for CVB4 72

Incidence of Viral Genomes in Myocardium MYOCARDITS Bowles/1986 Enterovirus northern 50(%) Kandolf/1991 Enterovirus In situ 20-25 Maisch/1989 CMV In situ 20 Schonian/1991 CMV PCR/In situ 10 Martin/1994 Enterovirus PCR 23 Adenovirus 44 DILATED CARDIOMYOPATHY Kandolf/1991 Enterovirus In situ 20 Schonian/1991 CMV In situ 15 Schonian/1993 CMV PCR <5 Matsumori/1995 HCV PCR 17(%) Maisch, Curr Opi Cardiol, 1996 73

Prevalence of Myocarditis by Biopsy N Engl J Med 2000;343:1388 74

Detection of enteroviral capsid protein VP1 by immunohistochemistry E G F H E) Left auricle, H& E stain, x200 F) Masson s Trichrome stain, x200) G,H) Immunohistochemistry probed by anti-enteroviral VP1 Ab.(G; x100,h; x400) 75

Host Responses after Viral Infection Acute Subacute Chronic Day < 3 days 3-14 days > 14 days Virus titer Viremia Viral Genome Main Cells No cells Macrophage, NK Cytotoxic T Cytokines IFN-γ IL-2, IL-1β,TNF-α IL-1β, TNF-α Mechanism Apoptosis? Necrosis(perforin) Apoptosis? 76

Myocyte Injury in Acute Phase CAR Virus -Induced Direct Cytotoxicity Virus Proliferation Apoptosis Cardiomyocyte 77

Replication-defective CVB3 infection Replication defective Vaccinia-CVB3-dVP0 virus infection can induce myofibril disruption Wessely, Circulation 98, 1998 78

CVB3-dVP0 Transgenic Animal CVB3-dVP0 transgenic animal shows d-cmp phenotype, increased ANF expression and myocardial fibrosis Wessely, J Clin Invest 102, 1998 79

Case Summary HIS and Nt test Serial histological and immunohistochemical analysis of the right atrial appendage, that underwent biopsy at the time of insertion and removal of LVAD, showed the enteroviral capsid protein VP1 (primary antibody, Novocastra Laboratories) over the entire right atrial wall with scanty inflammation infiltrates. Her serum neutralized coxsackievirus B4 (CVB4) in a neutralization test performed with CVB4 (American Type Culture Collection, J.V.B. Benschoten) as a control virus. The titer of neutralizing antibody in her serum at 16 days was more than four times the titer at 5 days and 40 days. 80

Devices for circulatory support currently used in 1. acute circulatory support < 1 month cardiac failure after cardiac operations, myocardial infarction shock or acute cardiomyopathy due to myocarditis or other causes, with a potential likelihood of recovery. 2. more prolonged support from 30 days to <1 year in Waiting for transplantation but deteriorate before a heart becomes available and require mechanical support prior to transplantation. chronic HF regain ventricular function and are able to have the devices removed without requiring transplantation. 3. permanent support as an alternative to transplantation irreversible cardiac failure that might require circulatory support, but they are not good candidates for cardiac transplantation. Therefore, if devices are inserted, they must be considered permanent or destination therapy and are currently investigational. Consensus Conference Report, JACC 2001;37:340 81