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The Lancet, Volume 360, November 9, 2002

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A new NOS2 promoter polymorphism associated with increased nitric oxide production and protection from severe malaria in Tanzanian and Kenyan children. - PowerPoint PPT Presentation
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A new A new NOS2 NOS2 promoter promoter polymorphism associated polymorphism associated with increased nitric oxide with increased nitric oxide production and protection production and protection from severe malaria in from severe malaria in Tanzanian and Kenyan Tanzanian and Kenyan children children Maurine R Hobbs, Venkatachalam Udhayakumar, Marc C Maurine R Hobbs, Venkatachalam Udhayakumar, Marc C levesque, Jennifer Booth, Jacquelin M Roberts, Ariana levesque, Jennifer Booth, Jacquelin M Roberts, Ariana N Tkachuk, Ann Pole, Hilary Coon, Simon Kariuki, N Tkachuk, Ann Pole, Hilary Coon, Simon Kariuki, Bernard L Nahlen, Esther D Mwaikambo, Altaf L Laf, Bernard L Nahlen, Esther D Mwaikambo, Altaf L Laf, Donald L Granger, Nicholas M Anstey, J Brice Weinberg Donald L Granger, Nicholas M Anstey, J Brice Weinberg The Lancet, Volume 360, November 9, 2002
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Page 1: The Lancet,  Volume 360, November 9, 2002

A new A new NOS2 NOS2 promoter polymorphism promoter polymorphism associated with increased nitric oxide associated with increased nitric oxide

production and protection from production and protection from severe malaria in Tanzanian and severe malaria in Tanzanian and

Kenyan childrenKenyan childrenMaurine R Hobbs, Venkatachalam Udhayakumar, Marc C levesque, Maurine R Hobbs, Venkatachalam Udhayakumar, Marc C levesque,

Jennifer Booth, Jacquelin M Roberts, Ariana N Tkachuk, Ann Pole, Hilary Jennifer Booth, Jacquelin M Roberts, Ariana N Tkachuk, Ann Pole, Hilary Coon, Simon Kariuki, Bernard L Nahlen, Esther D Mwaikambo, Altaf L Coon, Simon Kariuki, Bernard L Nahlen, Esther D Mwaikambo, Altaf L

Laf, Donald L Granger, Nicholas M Anstey, J Brice WeinbergLaf, Donald L Granger, Nicholas M Anstey, J Brice Weinberg

The Lancet, Volume 360, November 9, 2002

Page 2: The Lancet,  Volume 360, November 9, 2002

Plasmodium falciparum Plasmodium falciparum MalariaMalaria

• WHO estimates 1.5-2.7 million deaths WHO estimates 1.5-2.7 million deaths per yearper year

• Most deaths in African children less Most deaths in African children less than 5than 5

• Growing problem of antimalarial drug Growing problem of antimalarial drug resistance with few novel resistance with few novel therapeutics availabletherapeutics available

• Lack of an effective vaccineLack of an effective vaccine

Page 3: The Lancet,  Volume 360, November 9, 2002

Malaria Transmission Malaria Transmission CycleCyclePre-erythrocytic

Erythrocytic

Sporozoites

Sporozoites Merozoites

Male and femalegametocytes

Clinical symptoms

Asymptomatic

Page 4: The Lancet,  Volume 360, November 9, 2002

Clinical Features of Clinical Features of P. falciparumP. falciparum

• P. falciparumP. falciparum can cause severe malaria: can cause severe malaria:-severe anemia-severe anemia-cerebral malaria-cerebral malaria-hypoglycemia-hypoglycemia-respiratory distress-respiratory distress

• Molecular determinants that regulate Molecular determinants that regulate mild versus severe disease largely mild versus severe disease largely unknownunknown

Page 5: The Lancet,  Volume 360, November 9, 2002

Cerebral MalariaCerebral Malaria

• Sequestration of parasitized red blood cells in the post-Sequestration of parasitized red blood cells in the post-capillary venulescapillary venules

• Due to altered immune environment that is contingent on Due to altered immune environment that is contingent on endothelial cells changes and perturbed local endothelial cells changes and perturbed local inflammatory milieuinflammatory milieu

Page 6: The Lancet,  Volume 360, November 9, 2002

Populations at RiskPopulations at Risk• Infants, young children, and Infants, young children, and

pregnant women in malaria pregnant women in malaria endemic regionsendemic regions

– Greater than 3 million deaths Greater than 3 million deaths (primarily in children less than 5 y/o (primarily in children less than 5 y/o due to non-immune status)due to non-immune status)

• Non-immune individuals Non-immune individuals traveling through and/or living traveling through and/or living in malaria endemic regionsin malaria endemic regions

– 35 million non-immune individuals 35 million non-immune individuals travel through malaria endemic travel through malaria endemic regions every yearregions every year

Page 7: The Lancet,  Volume 360, November 9, 2002

Potential SolutionsPotential Solutions• Gain an understanding of the genetic and immunologic basis of Gain an understanding of the genetic and immunologic basis of

protective immunityprotective immunity

• Identify novel targets for therapeutic interventionIdentify novel targets for therapeutic intervention

• Determine reliable markers for measuring protection and Determine reliable markers for measuring protection and pathogenesis for use in pharmacologic and/or vaccine trials pathogenesis for use in pharmacologic and/or vaccine trials

Page 8: The Lancet,  Volume 360, November 9, 2002

Nitric OxideNitric Oxide• Nitric oxide – free radical gas with a half life of 7 secondsNitric oxide – free radical gas with a half life of 7 seconds

• Important molecule in the context of host defense because it Important molecule in the context of host defense because it has antimicrobial propertieshas antimicrobial properties

• Exact mechanism by which NO kills microbes is not well Exact mechanism by which NO kills microbes is not well understoodunderstood

Page 9: The Lancet,  Volume 360, November 9, 2002

Nitric Oxide SynthaseNitric Oxide Synthase

Constitutive ExpressionConstitutive Expression Inducible ExpressionInducible Expression- Ca- Ca2+2+- and Calmodulin-Dependent- and Calmodulin-Dependent - Ca- Ca2+2+- and Calmodulin-Independent- and Calmodulin-Independent

NO Synthesis for Normal NO Synthesis for Normal Physiologic FunctionPhysiologic Function

NO Synthesis in the Setting NO Synthesis in the Setting of Inflammationof Inflammation

eNOS & nNOSeNOS & nNOS NOS3 NOS1NOS3 NOS1

iNOSiNOSNOS2NOS2

Page 10: The Lancet,  Volume 360, November 9, 2002

NOS2 GeneNOS2 Gene

Page 11: The Lancet,  Volume 360, November 9, 2002

Nitric Oxide SynthaseNitric Oxide Synthase• Complex induction and regulationComplex induction and regulation

• Two promoter polymorphisms examined for roles in Two promoter polymorphisms examined for roles in malarial outcomesmalarial outcomes

• -954 G-954 GC associated with mild disease severity in C associated with mild disease severity in GabonGabon

• Long (CCTTT)Long (CCTTT)n n (n≥11) repeats associated with (n≥11) repeats associated with protection from fatal cerebral malaria in Gambian protection from fatal cerebral malaria in Gambian childrenchildren

• No association with cerebral malaria risk or No association with cerebral malaria risk or in vivo in vivo NO production in Tanzanian childrenNO production in Tanzanian children

Page 12: The Lancet,  Volume 360, November 9, 2002

Experimental DesignExperimental Design

• Study examined a novel single Study examined a novel single nucleotide polymorphism in the nucleotide polymorphism in the NOS2 NOS2 promoter and its relation promoter and its relation to malarial disease outcometo malarial disease outcome

• Studied the association between Studied the association between this SNP and this SNP and in vivo in vivo NO NO productionproduction

Page 13: The Lancet,  Volume 360, November 9, 2002

Methods: ParticipantsMethods: Participants

• TanzaniaTanzania Cross sectional study of Cross sectional study of children 6 mo. to 9 years. Healthy children 6 mo. to 9 years. Healthy controls, uncomplicated malaria, controls, uncomplicated malaria, and cerebral malariaand cerebral malaria

• KenyaKenya (ABCP) Mother-child (ABCP) Mother-child pairs. Monthly blood samples to pairs. Monthly blood samples to measure parasitemia and Hbmeasure parasitemia and Hb

Page 14: The Lancet,  Volume 360, November 9, 2002

Methods: ProtocolMethods: Protocol

• To estimate NO production, To estimate NO production, measured fasting plasma and urine measured fasting plasma and urine NONOx x concentrations in Tanzanian concentrations in Tanzanian

controlscontrols

Page 15: The Lancet,  Volume 360, November 9, 2002

Methods: ProtocolMethods: Protocol• Examined Examined NOS2NOS2 promoter (-8 kb to +1 bp) for promoter (-8 kb to +1 bp) for

polymorphisms associated with malaria polymorphisms associated with malaria outcomes in the Tanzanian group outcomes in the Tanzanian group

• Whole genome amplifiedWhole genome amplified

• Initial polymorphism detection screening on 12 Initial polymorphism detection screening on 12 HC’s and 12 CM’s by SSCPHC’s and 12 CM’s by SSCP

• Amplified -1173 CAmplified -1173 CT fragment and analyzed T fragment and analyzed SNP on all controls and patients in the SNP on all controls and patients in the Tanzanian groupTanzanian group

• -954 G-954 GC, (CCTTT)C, (CCTTT)nn, and HbAS previously , and HbAS previously examined in the Tanzanian childrenexamined in the Tanzanian children

Page 16: The Lancet,  Volume 360, November 9, 2002

Methods: ProtocolMethods: Protocol

• Investigated -1173 CInvestigated -1173 CT T polymorphism in the Kenyan cohort polymorphism in the Kenyan cohort by mutagenically separated PCRby mutagenically separated PCR

• Also determined (CCTTT) repeats Also determined (CCTTT) repeats and HbASand HbAS

Page 17: The Lancet,  Volume 360, November 9, 2002

Results: Effect of NOS2 -1173 CResults: Effect of NOS2 -1173 CT T genotype on malarial outcomes in genotype on malarial outcomes in Tanzanian and Kenyan childrenTanzanian and Kenyan children

Page 18: The Lancet,  Volume 360, November 9, 2002

Results: Effect of -1173 CResults: Effect of -1173 CT T genotype on development of SMA genotype on development of SMA and parasitemia in Kenyan childrenand parasitemia in Kenyan children

Page 19: The Lancet,  Volume 360, November 9, 2002

Results: Concentrations of NOResults: Concentrations of NOxx in urine in urine and plasma of fasting Tanzanian and plasma of fasting Tanzanian controls, according to -1173 Ccontrols, according to -1173 CT T

genotypegenotype

Page 20: The Lancet,  Volume 360, November 9, 2002

Results: Effect of (CCTTT)Results: Effect of (CCTTT)nn long vs. long vs. short genotype on development of SMA short genotype on development of SMA

and parasitemia in Kenyan childrenand parasitemia in Kenyan children

Page 21: The Lancet,  Volume 360, November 9, 2002

Results: Effect of -1173 Results: Effect of -1173 CCT/(CCTTT)T/(CCTTT)12-1312-13 genotype on genotype on

development of SMA and parasitemia development of SMA and parasitemia in Kenyan childrenin Kenyan children

Page 22: The Lancet,  Volume 360, November 9, 2002

Results: NOS2 -1173 CResults: NOS2 -1173 CT genotype or T genotype or sickle-cell trait (HbAS) relative to sickle-cell trait (HbAS) relative to

disease category in Tanzanian childrendisease category in Tanzanian children

Page 23: The Lancet,  Volume 360, November 9, 2002

DiscussionDiscussion

• Protective association of -1173 Protective association of -1173 CCT polymorphism in T polymorphism in NOS2NOS2 promoter in two cohortspromoter in two cohorts

• Two populations living in areas of Two populations living in areas of differing malarial endemicity and differing malarial endemicity and distinct disease patternsdistinct disease patterns

Page 24: The Lancet,  Volume 360, November 9, 2002

DiscussionDiscussion

• Associated with protection against both Associated with protection against both cerebral malaria and SMAcerebral malaria and SMA

• Associated with increased NO Associated with increased NO production production in vivo, in vivo, in healthy controlsin healthy controls

• Prevalence of the -1173 CPrevalence of the -1173 CT is similar T is similar to that of HbASto that of HbAS

Page 25: The Lancet,  Volume 360, November 9, 2002

DiscussionDiscussion

• -1173 C-1173 CT may disrupt binding T may disrupt binding site of a transcription repressor site of a transcription repressor (found not to be a new seq. recog. (found not to be a new seq. recog. site for GATA-1 or GATA-2 TF’s)site for GATA-1 or GATA-2 TF’s)

• May be in linkage disequilibrium May be in linkage disequilibrium with another polymorphism in the with another polymorphism in the NOS2 NOS2 genegene

Page 26: The Lancet,  Volume 360, November 9, 2002

Proposed mechanism of the protective Proposed mechanism of the protective effect of the -1173 Ceffect of the -1173 CT NOS2 T NOS2

promoter polymorphism against clinical promoter polymorphism against clinical malariamalaria

Page 27: The Lancet,  Volume 360, November 9, 2002

Proposed protective Proposed protective mechanismsmechanisms

• 1. NO has direct antiparasitic 1. NO has direct antiparasitic effectseffects

• 2. NO decreases expression of 2. NO decreases expression of ICAM-1, VCAM-1, and E-selectinICAM-1, VCAM-1, and E-selectin

• 3. NO potently decreases 3. NO potently decreases macrophage production of TNFmacrophage production of TNF

Page 28: The Lancet,  Volume 360, November 9, 2002

Role of NO in pathogenesis Role of NO in pathogenesis of malariaof malaria

• Pro-inflammatory and detrimental to Pro-inflammatory and detrimental to host survival host survival

• AND/ORAND/OR

• Anti-inflammatory and beneficial to host Anti-inflammatory and beneficial to host survivalsurvival

• Effects may vary with host cell types Effects may vary with host cell types and stage of parasite developmentand stage of parasite development


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