1. Part 1 Nitric Oxide: Pathogenic or Protective? Several Malarial Anemia

2. Field Studies in Gabon Lambaréné, Gabon Albert Schweitzer Hospital

3. Community-Based Longitudinal Study Experimental Design Plasma - measure cytokines PBMC -SNAP freeze (In vivo)  NOS Activity -Culture Cells (in vitro) stimulate with cytokines  NOS Activity Isolate Whole Blood Mild Malaria - Hb > 6.0 g/dL -hematocrit > 20% -parasitemia < 50,000/  L AGE: 3-7 TemperatureThick Blood Film Visited Every Two Weeks Severe Malaria - anemia < 6.0 g/dL -hematocrit < 20% -parasitemia > 250,000/  L Compare Prior Mild Malaria Prior Severe Malaria Healthy Children parasite free > 2 mo

4. Increased NOS Activity in Prior Mild Malaria Perkins et al., Infect Immun, 1999; 67:4977-4981 0 10 20 30 40 50 60 NOS enzyme activity (pmol citrulline/mg) Prior Severe Mal (n=15) Prior Mild Mal (n=20) Con * * B IFN-  TNF-  IFN  In Vitro

5. Increased NOS Activity in Prior Mild Malaria 0 100 200 300 400 500 600 NOS enzyme activity (pmol citrulline/mg) * Prior Mild Malaria Prior Severe Malaria (n=20)(n=15) Perkins et al., Infect Immun, 1999; 67:4977-4981 Ex Vivo

6. Potential Explanations Short half-life of blood monocytes suggests: 1) Altered cytokine environment - pro- and anti-inflammatory cytokines - pro- and anti-inflammatory cytokines 2) Host-genetic factors - polymorphisms that regulate disease susceptibility - polymorphisms that regulate disease susceptibility

7. Discovery of a Novel NOS2 Promoter Polymorphism: (G –954C) Kun et al., Lancet, 1998; 351: 265-266 Kun et al., Lancet, 1998; 351: 265-266 Single nucleotide polymorphism in the NOS2 promoter (G-954C)Single nucleotide polymorphism in the NOS2 promoter (G-954C) Associated with less severe forms of malaria in Gabonese childrenAssociated with less severe forms of malaria in Gabonese children G –954C not in a known promoter response elementG –954C not in a known promoter response element  Is the polymorphism associated with increased NO production? Click for larger picture

8. Experimental Design Wild Type Community-Based Longitudinal Study Genotype Children for G -954C PolymorphismIsolate Whole Blood Healthy Controls parasite free > 2 mo Plasma measure cytokines & effector molecules PBMC SNAP freeze (In vivo)  NOS Activity Culture Cells (in vitro) stimulate with cytokines  NOS Activity Compare G -954C Polymorphism

9. NOS2 Promoter Polymorphism Analysis Amplified 680 bp of NOS2 promoter Cut with Bsa I Identifies G-954C Polymorphism 1 2 3 4 5 6 7 U C

10. Higher NOS Activity in G -954C 0 10 20 30 40 50 60 70 NOS Activity (pmol citrulline/mg pro) WT (n = 10) G-C (n = 17) Con IFN-  LPS/IFN-  * * * In Vitro

11. 0 200 400 600 NOS activity (pmol citrulline/mg pro) 1 * * G-CWTUS (WT) n=17n=10n=20 Ex Vivo Higher NOS Activity in G -954C

12. Nuclear Protein Appears to be a Phosphoserine Protein IRF-1 & 2, Stat-1 & 2, c-Jun, E2A, phosphoserine, phosphotyrosine, and phosphothreonine Nuclear proteins NOS2 oligonucleotide probe 32 P + + Antibodies antiphosphoserine A549 Cells Supershift Assay Supershift Assay

13. Increased Binding Affinity of Nuclear Proteins to G –954C Polymorphic Site U937 Cells wt probe G –954C probe NOS2 wt probe 32 P + + NOS2 wt probe NOS2 –954C probe Competition Assay Nuclear proteins

14. Prolonged Time to Re-infection in G -954C Group Curative treatment  4 year follow-up (every two weeks)  no significant association of sickle cell gene with re- infection

15. Conclusions NOS activity in vitro and in vivo is significantly higher in malaria-exposed children who develop mild disease (Cross-sectional)NOS activity in vitro and in vivo is significantly higher in malaria-exposed children who develop mild disease (Cross-sectional) G -954C significantly more frequent in patients with mild malaria (independent of sickle cell genotype)G -954C significantly more frequent in patients with mild malaria (independent of sickle cell genotype) G -954C associated with significant increases in NOS activity in vitro and in vivo (Functional!)G -954C associated with significant increases in NOS activity in vitro and in vivo (Functional!) G -954C significantly associated with “protection”G -954C significantly associated with “protection” - decreased rates of re-infection - prolonged time from one infection to the next

16. Part 2 Association of NOS2 (G –954C) with Cerebral Malaria

17. Decreased Peripheral NO and NOS2 in Cerebral Malaria Anstey et al., J. Exp. Med., 1996 ; 184: 557-567 NOS2 Protein in PBMC NOx in Plasma Click for larger picture

18. Increased NOS2 in Neuronal Cells in Cerebral Malaria Viriyavejakul et al., Histopathology, 200; 37: 269-277 Endothelia l Cells Neurons MicroglialCellsAstrocytes Axons Oligodendroc ytes Macrophages

19. NOS2 (G –954C) Polymorphism in Tanzanian Children with Cerebral Malaria  G –954C not associated with disease severity or NO/NOS2

20. Conclusions Role of NO/NOS2 unclear in cerebral malaria (decreased peripherally but increased centrally)Role of NO/NOS2 unclear in cerebral malaria (decreased peripherally but increased centrally) G-954C polymorphism is not significantly associated with disease severity or NO/NOS2 (peripherally)G-954C polymorphism is not significantly associated with disease severity or NO/NOS2 (peripherally) - Cross sectional study design - Different clinical manifestation of malaria with different measurements of NO/NOS measurements

21. Part 3 NOS2 (G –954C) in a Holoendemic Area of Malaria Transmission Severe Malarial Anemia

22. Field Studies in Kenya Kisumu, Kenya CDC/KEMRI

23. Malaria – HIV – Malaria/HIV Co-infections Pregnancy Enrollment n = 1539 Pregnancy follow-up Delivery Child follow-up n = 1244 Age: 0-5 years OB Hx: Gravidity, Parity, Hx Miscarriage & Hx stillbirth Specimens: Blood film, Hb, plasma & cells Demographics: Age, education, literacy, family size, wealth, village, & house construction Birth Outcomes: Sex, weight, gestational age & birth location Specimens: Placental blood, cord blood & maternal peripheral blood Fortnightly: Signs/symptoms drug use history Monthly: Blood film, Hb, plasma, cells, height & weight

24. Community-Based Longitudinal Study Experimental Design: Kisumu Isolate DNA Protected -% time parasitemia -% time high density < 10,000/  L -% malarial anemia -# of 2 nd line treatment episodes AGE: 0-2 Hemoglobin Thick Blood Film Visited Every Two Weeks Temperature Susceptible -% time parasitemia -% time high density < 10,000/  L -% malarial anemia -# of 2 nd line treatment episodes 1244 children observed over first two years of life Select 100 children from top and bottom ranking parameters Compare frequency of polymorphism

25. NOS2 Polymorphism Frequency Mimics Malaria Endemicity

26. Conclusions NOS2 (G-954C) polymorphism is significantly associated with protection in several malaria anemia (Gabon and Kenya)NOS2 (G-954C) polymorphism is significantly associated with protection in several malaria anemia (Gabon and Kenya) NOS2 (G-954C) polymorphism is significantly associated with increased NOS activity/NO production in severe malarial anemia (Gabon)NOS2 (G-954C) polymorphism is significantly associated with increased NOS activity/NO production in severe malarial anemia (Gabon) NOS2 G-954C polymorphism is not significantly associated with disease severity or NO/NOS2 in cerebral malaria (Tanzania)NOS2 G-954C polymorphism is not significantly associated with disease severity or NO/NOS2 in cerebral malaria (Tanzania)  Underscores the complexity of unraveling disease susceptibility in polygenic diseases

27. Future Directions

28. Collaborators Albert Schweitzer Hospital University of Tuebingen Prof. Dr. Peter G. Kremsner Dr. Doris Luckner Dr. Daniela Schmid Dr. Jürgen Kun Dr. Benjamin Mordmüller Duke University Dr. J. Brice Weinberg Dr. Marc Levesque Mary A. Misukionis CDC / KEMRI Dr. Altaf Lal Dr. Udhayakumar Kumar Dr. Ya Ping Shi University of Pittsburgh Dr. David Finegold Dr. Robert Ferrell Dr. David Peters Christopher Keller Benjamin Nti Jamie Slingluff