part 1 nitric oxide: pathogenic or protective? several malarial anemia

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


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


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


TGF-1

Neutrophil

Lymphocyte

TNF-

Parasitic Products

IL-12

IFN-

Parasitic Products

MIF

NOPGE2


~~~~~~~~~~~~~~~~


Monocyte

Hemozoin

Hemozoin

(1)

(2)

IL-10

pRBCpRBC


TGF-1

Neutrophil

Lymphocyte

TNF-

Parasitic Products

IL-12

IFN-

Parasitic Products

MIF

NOPGE2


~~~~~~~~


Monocyte

Hemozoin

Hemozoin

(1)

(2)

IL-10IL-10IL-10

pRBCpRBC


TGF-1

Neutrophil

Lymphocyte

TNF-

Parasitic Products

IL-12

IFN-

Parasitic Products

MIF

NOPGE2


~~~~~~~~~~~~~~~~


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

part 1 nitric oxide: pathogenic or protective? several malarial anemia

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