part 1 nitric oxide: pathogenic or protective? several malarial anemia
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Part 1Part 1
Nitric Oxide:Nitric Oxide:Pathogenic or Protective?Pathogenic or Protective?
Several Malarial AnemiaSeveral Malarial Anemia
Field Studies in GabonField Studies in Gabon
Lambaréné, Gabon
Albert Schweitzer Hospital
Community-Based Longitudinal Study
Experimental DesignExperimental Design
Plasma
- measure cytokines
PBMC-SNAP freeze -SNAP freeze (In vivo)(In vivo) NOS Activity -Culture Cells -Culture Cells (in vitro)(in vitro) stimulate with cytokines NOS Activity
Isolate Whole Blood
Mild Malaria- Hb > 6.0 g/dL- Hb > 6.0 g/dL
-hematocrit > 20%-hematocrit > 20%
-parasitemia < -parasitemia < 50,000/50,000/LL
AGE: 3-7
TemperatureThick Blood Film
Visited Every Two Weeks
Severe Malaria- anemia < 6.0 g/dL- anemia < 6.0 g/dL
-hematocrit < 20%-hematocrit < 20%
-parasitemia > 250,000/-parasitemia > 250,000/LL
Compare
Prior Mild Malaria Prior Severe Malaria
Healthy Children
parasite free > 2 mo
Increased NOS Activity in Prior Mild MalariaIncreased NOS Activity in Prior Mild Malaria
Perkins et al., Infect Immun, 1999; 67:4977-4981
0
10
20
30
40
50
60
NO
S e
nzy
me
acti
vity
(p
mo
l cit
rulli
ne/
mg
)
Prior Severe Mal (n=15)
Prior Mild Mal (n=20)
Con
*
*B
IFN- TNF-IFN
In Vitro
Increased NOS Activity in Prior Mild MalariaIncreased NOS Activity in Prior Mild Malaria
0
100
200
300
400
500
600
NO
S e
nzy
me
act
ivit
y (p
mo
l cit
rulli
ne
/mg
)
*
Prior Mild Malaria Prior Severe Malaria(n=20) (n=15)
Perkins et al., Infect Immun, 1999; 67:4977-4981
Ex Vivo
Potential ExplanationsPotential Explanations
Short half-life of blood monocytes suggests:Short half-life of blood monocytes suggests:
1) Altered cytokine environment1) Altered cytokine environment
- pro- and anti-inflammatory cytokines- pro- and anti-inflammatory cytokines
2) Host-genetic factors2) Host-genetic factors
- polymorphisms that regulate disease - polymorphisms that regulate disease
susceptibilitysusceptibility
Discovery of a Novel NOS2 Discovery of a Novel NOS2 Promoter Polymorphism: (G –954C)Promoter Polymorphism: (G –954C)
Kun et al., Kun et al., LancetLancet, 1998; 351: 265-266, 1998; 351: 265-266
• Single nucleotide Single nucleotide
polymorphism in the NOS2 polymorphism in the NOS2
promoter (G-954C)promoter (G-954C)
• Associated with less Associated with less
severe forms of malaria in severe forms of malaria in
Gabonese children Gabonese children
• G –954C not in a known G –954C not in a known
promoter response elementpromoter response element
Is the polymorphism Is the polymorphism
associated with increased associated with increased
NO production?NO production?Click for larger picture
Experimental DesignExperimental Design
Wild Type
Community-Based Longitudinal Study
Genotype Children for G -954C Polymorphism
Isolate Whole Blood
Healthy Controls
parasite free > 2 mo
Plasma
measure cytokines &
effector molecules
PBMC
SNAP freeze SNAP freeze (In vivo)(In vivo)
NOS Activity
Culture Cells Culture Cells (in vitro)(in vitro)
stimulate with cytokines
NOS ActivityCompare
G -954C Polymorphism
NOS2 Promoter NOS2 Promoter Polymorphism AnalysisPolymorphism Analysis
Amplified 680 bp of NOS2 promoter
Cut with Bsa I
Identifies G-954C Polymorphism
1 2 3 4 5 6 7U C U C U C U C U C U C U C
Higher NOS Activity in G -954CHigher NOS Activity in G -954C
0
10
20
30
40
50
60
70N
OS
Act
ivit
y (p
mo
l ci
tru
llin
e/m
g p
ro)
WT (n = 10)
G-C (n = 17)
Con IFN- LPS/IFN-
*
*
*In Vitro
0
200
400
600
NO
S a
ctiv
ity
(pm
ol
citr
ull
ine/
mg
pro
)
1
*
*
G-C WT US (WT)n=17 n=10 n=20
Ex VivoEx Vivo
Higher NOS Activity in G -954CHigher NOS Activity in G -954C
Nuclear Protein Appears to be a Nuclear Protein Appears to be a Phosphoserine ProteinPhosphoserine Protein
IRF-1 & 2, Stat-1 & 2, c-Jun, E2A, IRF-1 & 2, Stat-1 & 2, c-Jun, E2A, phosphoserine,phosphoserine,phosphotyrosine, and phosphothreonine phosphotyrosine, and phosphothreonine
Nuclear Nuclear proteinsproteins
NOS2 oligonucleotide probeNOS2 oligonucleotide probe3232PP++
++AntibodiesAntibodies
antiphosphoserineantiphosphoserine
A549 CellsA549 Cells
Supershift AssaySupershift Assay
Increased Binding Affinity of Increased Binding Affinity of Nuclear Proteins to G –954C Nuclear Proteins to G –954C
Polymorphic Site Polymorphic Site
U937 CellsU937 Cells
wt probewt probe
G –954CG –954Cprobeprobe
NOS2 wt probeNOS2 wt probe3232PP++
++
NOS2 wt probeNOS2 wt probe NOS2 –954C probeNOS2 –954C probe
Competition AssayCompetition Assay
Nuclear Nuclear proteinsproteins
Prolonged Prolonged Time to Time to
Re-infection Re-infection in G -954C in G -954C
Group Group
Curative Curative treatmenttreatment
4 year follow-4 year follow-up (every two up (every two weeks)weeks)
no significant no significant association of association of sickle cell gene sickle cell gene with re-infection with re-infection
ConclusionsConclusions• NOS activityNOS activity in vitro in vitro and and in vivoin vivo is significantly higher in is significantly higher in
malaria-exposed children who develop mild disease (malaria-exposed children who develop mild disease (Cross-Cross-sectionalsectional))
• G -954C significantly more frequent in patients with mild G -954C significantly more frequent in patients with mild malaria (independent of sickle cell genotype)malaria (independent of sickle cell genotype)
• G -954C associated with significant increases in NOS activity G -954C associated with significant increases in NOS activity in vitroin vitro and and in vivo (Functional!)in vivo (Functional!)
• G -954C significantly associated with “protection”G -954C significantly associated with “protection”
- decreased rates of re-infection- decreased rates of re-infection
- prolonged time from one infection to the next- prolonged time from one infection to the next
Part 2Part 2
Association of NOS2 (G –954C) Association of NOS2 (G –954C) with Cerebral Malariawith Cerebral Malaria
Decreased Peripheral NO and NOS2 Decreased Peripheral NO and NOS2 in Cerebral Malariain Cerebral Malaria
Anstey et al., J. Exp. Med., Anstey et al., J. Exp. Med., 1996 ; 184: 557-5671996 ; 184: 557-567
NOS2 Protein in PBMCNOS2 Protein in PBMC
NOx in PlasmaNOx in Plasma
Click for larger picture
Click for larger picture
Increased NOS2 in Neuronal CellsIncreased NOS2 in Neuronal Cellsin Cerebral Malariain Cerebral Malaria
Viriyavejakul et al., Viriyavejakul et al., HistopathologyHistopathology, 200; 37: 269-277, 200; 37: 269-277
EndotheEndothelial Cellslial CellsNeuronsNeurons
MicroglialMicroglialCellsCellsAstrocyteAstrocytess
AxonsAxonsOligodendrOligodendrocytesocytes
MacrophagesMacrophages
NOS2 (G –954C) Polymorphism in NOS2 (G –954C) Polymorphism in Tanzanian Children withTanzanian Children with
Cerebral MalariaCerebral Malaria
G –954C G –954C notnot associated with disease severity or NO/NOS2 associated with disease severity or NO/NOS2
ConclusionsConclusions• Role of NO/NOS2 unclear in cerebral malaria Role of NO/NOS2 unclear in cerebral malaria
(decreased peripherally but increased centrally)(decreased peripherally but increased centrally)
• G-954C polymorphism is not significantly associated G-954C polymorphism is not significantly associated
with disease severity or NO/NOS2 (peripherally)with disease severity or NO/NOS2 (peripherally)
- Cross sectional study design- Cross sectional study design
- Different clinical manifestation of malaria with different - Different clinical manifestation of malaria with different
measurements of NO/NOS measurementsmeasurements of NO/NOS measurements
Part 3Part 3
NOS2 (G –954C) in a Holoendemic NOS2 (G –954C) in a Holoendemic Area of Malaria TransmissionArea of Malaria Transmission
Severe Malarial AnemiaSevere Malarial Anemia
Field Studies in Kenya Field Studies in Kenya
Kisumu, Kenya
CDC/KEMRI
Malaria – HIV – Malaria/HIV Co-infectionsMalaria – HIV – Malaria/HIV Co-infections
PregnancyEnrollment
n = 1539Pregnancyfollow-up
Delivery
Child follow-upn = 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
Community-Based Longitudinal Study
Experimental Design: KisumuExperimental Design: Kisumu
Isolate DNA
Protected-% time parasitemia-% time high density < 10,000/L-% malarial anemia-# of 2nd line treatment episodes
AGE: 0-2
HemoglobinThick Blood Film
Visited Every Two Weeks
Temperature
Susceptible-% time parasitemia% time parasitemia-% time high density < 10,000/% time high density < 10,000/LL-% malarial anemia% malarial anemia-# of 2# of 2ndnd line treatment episodes 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
Region Frequency N
Kenya 14.7 102
Gabon 9.8 193
Senegal 8.9 242
Nigeria 6.8 96
Papua New Guinea 3.6 70
Thailand 2.5 59
USA (African American) 6.7 80
USA (Caucasian) 0 66
Germany (Caucasian) 0 100
Region Frequency N
Kenya 14.7 102
Gabon 9.8 193
Senegal 8.9 242
Nigeria 6.8 96
Papua New Guinea 3.6 70
Thailand 2.5 59
USA (African American) 6.7 80
USA (Caucasian) 0 66
Germany (Caucasian) 0 100
NOS2 NOS2 Polymorphism Polymorphism
FrequencyFrequencyMimics Malaria Mimics Malaria
EndemicityEndemicity
ConclusionsConclusions• NOS2 (G-954C) polymorphism is significantly associated with NOS2 (G-954C) polymorphism is significantly associated with
protection in several malaria anemia (Gabon and Kenya)protection in several malaria anemia (Gabon and Kenya)
• NOS2 (G-954C) polymorphism is significantly associated with NOS2 (G-954C) polymorphism is significantly associated with increased NOS activity/NO production in severe malarial increased NOS activity/NO production in severe malarial anemia (Gabon)anemia (Gabon)
• NOS2 G-954C polymorphism is not significantly associated NOS2 G-954C polymorphism is not significantly associated with disease severity or NO/NOS2 in cerebral malaria with disease severity or NO/NOS2 in cerebral malaria (Tanzania)(Tanzania)
Underscores the complexity of unraveling disease Underscores the complexity of unraveling disease susceptibility in polygenic diseasessusceptibility in polygenic diseases
IL-10
pRBCpRBC
RBCCFU-S BFU-E CFU-E Reticulocyte
TGF-1
Neutrophil
Lymphocyte
TNF-
Parasitic Products
IL-12
IFN-
Parasitic Products
MIF
NOPGE2
MonocyteMonocyteMonocyte
~~~~~~~~
Bone MarrowPeriphery
Monocyte
Hemozoin
Hemozoin
(1)
(2)
IL-10IL-10IL-10
pRBCpRBC
RBCCFU-S BFU-E CFU-E Reticulocyte
TGF-1
Neutrophil
Lymphocyte
TNF-
Parasitic Products
IL-12
IFN-
Parasitic Products
MIF
NOPGE2
MonocyteMonocyteMonocyte
~~~~~~~~~~~~~~~~
Bone MarrowPeriphery
Monocyte
Hemozoin
Hemozoin
(1)
(2)
IL-10
pRBCpRBC
RBCCFU-S BFU-E CFU-E Reticulocyte
TGF-1
Neutrophil
Lymphocyte
TNF-
Parasitic Products
IL-12
IFN-
Parasitic Products
MIF
NOPGE2
MonocyteMonocyteMonocyte
~~~~~~~~
Bone MarrowPeriphery
Monocyte
Hemozoin
Hemozoin
(1)
(2)
IL-10IL-10IL-10
pRBCpRBC
RBCCFU-S BFU-E CFU-E Reticulocyte
TGF-1
Neutrophil
Lymphocyte
TNF-
Parasitic Products
IL-12
IFN-
Parasitic Products
MIF
NOPGE2
MonocyteMonocyteMonocyte
~~~~~~~~~~~~~~~~
Bone MarrowPeriphery
Monocyte
Hemozoin
Hemozoin
(1)
(2)
Future Future DirectionsDirections
CollaboratorsCollaboratorsAlbert Schweitzer HospitalAlbert Schweitzer Hospital
University of TuebingenUniversity of Tuebingen
Prof. Dr. Peter G. KremsnerProf. Dr. Peter G. Kremsner
Dr. Doris LucknerDr. Doris Luckner
Dr. Daniela SchmidDr. Daniela Schmid
Dr. JDr. Jüürgen Kunrgen Kun
Dr. Benjamin MordmDr. Benjamin Mordmüüllerller
Duke UniversityDuke University
Dr. J. Brice WeinbergDr. J. Brice Weinberg
Dr. Marc LevesqueDr. Marc Levesque
Mary A. MisukionisMary A. Misukionis
CDC / KEMRICDC / KEMRI
Dr. Altaf LalDr. Altaf Lal
Dr. Udhayakumar KumarDr. Udhayakumar Kumar
Dr. Ya Ping ShiDr. Ya Ping Shi
University of PittsburghUniversity of Pittsburgh
Dr. David FinegoldDr. David Finegold
Dr. Robert FerrellDr. Robert Ferrell
Dr. David PetersDr. David Peters
Christopher KellerChristopher Keller
Benjamin NtiBenjamin Nti
Jamie SlingluffJamie Slingluff
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