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

Part 1 Nitric Oxide: Pathogenic or Protective? Several Malarial Anemia. Field Studies in Gabon. Lambaréné, Gabon Albert Schweitzer Hospital. AGE: 3-7. Visited Every Two Weeks. Thick Blood Film. Temperature. Mild Malaria - Hb > 6.0 g/dL -hematocrit > 20% -parasitemia < 50,000/ m L.

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

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

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

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

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

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

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

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

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

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

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

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

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

  13. 32P + NOS2 wt probe NOS2 wt probe G –954C probe + NOS2 –954C probe Increased Binding Affinity of Nuclear Proteins to G –954C Polymorphic Site Competition Assay U937 Cells wt probe 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) • 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 significantly associated with “protection” - decreased rates of re-infection - prolonged time from one infection to the next

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

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

  18. Increased NOS2 in Neuronal Cells in Cerebral Malaria Endothelial Cells Neurons Axons Oligodendrocytes Microglial Cells Astrocytes Macrophages Viriyavejakul et al., Histopathology, 200; 37: 269-277

  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) • 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 3NOS2 (G –954C) in a Holoendemic Area of Malaria TransmissionSevere Malarial Anemia

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

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

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

  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 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) • Underscores the complexity of unraveling disease susceptibility in polygenic diseases

  27. Future Directions

  28. Albert Schweitzer Hospital University of Tuebingen Prof. Dr. Peter G. Kremsner Dr. Doris Luckner Dr. Daniela Schmid Dr. Jürgen Kun Dr. Benjamin Mordmüller Collaborators 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 Duke University Dr. J. Brice Weinberg Dr. Marc Levesque Mary A. Misukionis

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