Part 1 Nitric Oxide: Pathogenic or Protective Several Malarial Anemia

1
Part 1Nitric OxidePathogenic or
Protective?Several Malarial Anemia
2
Field Studies in Gabon
Lambaréné, Gabon Albert Schweitzer Hospital
3
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
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 674977-4981
5
Increased NOS Activity in Prior Mild Malaria
Ex Vivo
Perkins et al., Infect Immun, 1999 674977-4981
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
Experimental Design
Community-Based Longitudinal Study
Healthy Controls parasite free gt 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
Higher NOS Activity in G -954C
In Vitro
11
Higher NOS Activity in G -954C
Ex Vivo


G-C
WT
US (WT)
n17
n10
n20
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
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
Experimental Design Kisumu
Community-Based Longitudinal Study
AGE 0-2

• Protected
• time parasitemia
• time high density lt 10,000/mL
• malarial anemia
• of 2nd line treatment episodes

• Susceptible
• time parasitemia
• time high density lt 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
Collaborators
CDC / KEMRI Dr. Altaf Lal Dr. Udhayakumar
Kumar Dr. Ya Ping Shi

• Albert Schweitzer Hospital
• University of Tuebingen
• Prof. Dr. Peter G. Kremsner
• Dr. Doris Luckner
• Dr. Daniela Schmid
• Dr. Jürgen Kun
• Dr. Benjamin Mordmüller

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