research article lipoprotein lipase and ppar alpha gene

Research ArticleLipoprotein Lipase and PPAR Alpha Gene PolymorphismsIncreased Very-Low-Density Lipoprotein Levelsand Decreased High-Density Lipoprotein Levels as RiskMarkers for the Development of Visceral Leishmaniasis byLeishmania infantum

Maacutercia Dias Teixeira Carvalho1 Diego Peres Alonso2 Ceacutelia Maria Vieira Vendrame1

Dorcas Lamounier Costa3 Carlos Henrique Nery Costa3 Guilherme Loureiro Werneck45

Paulo Eduardo Martins Ribolla2 and Hiro Goto16

1 Laboratorio de Soroepidemiologia e Imunobiologia Instituto de Medicina Tropical de Sao Paulo Universidade de Sao PauloAvenida Dr Eneas de Carvalho Aguiar 470 Predio II 4∘ Andar 05403-000 Sao Paulo SP Brazil

2 Departamento de Parasitologia Instituto de Biociencias Universidade Estadual Paulista 18618-970 Botucatu SP Brazil3 Instituto de Doencas Tropicais Natan Portella Universidade Federal do Piauı 64001-450 Teresina PI Brazil4Departamento de Epidemiologia Instituto de Medicina Social Universidade do Estado do Rio de Janeiro20550-900 Rio de Janeiro RJ Brazil

5 Instituto de Estudos em Saude Coletiva Universidade Federal do Rio de Janeiro 21941-598 Rio de Janeiro RJ Brazil6Departamento de Medicina Preventiva Faculdade de Medicina Universidade de Sao Paulo 01246-903 Sao Paulo SP Brazil

Correspondence should be addressed to Hiro Goto hgotouspbr

Received 22 May 2014 Accepted 2 August 2014 Published 27 August 2014

Academic Editor Christophe Chevillard

Copyright copy 2014 Marcia Dias Teixeira Carvalho et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

In visceral leishmaniasis (VL) endemic areas a minority of infected individuals progress to disease since most of them developprotective immunityTherefore we investigated the risk markers of VL within nonimmune sector Analyzing infected symptomaticand asymptomatic and noninfected individuals VL patients presented with reduced high-density lipoprotein cholesterol (HDL-C) elevated triacylglycerol (TAG) and elevated very-low-density lipoprotein cholesterol (VLDL-C) levels A polymorphismanalysis of the lipoprotein lipase (LPL) gene using HindIII restriction digestion (N = 156 samples) (H+ = the presence and Hminus= the absence of mutation) revealed an increased adjusted odds ratio (OR) of VL versus noninfected individuals when the H+H+was compared with the HminusHminus genotype (OR = 213 95 CI = 232ndash33353 P = 0003) The H+H+ genotype and the H+ allelewere associated with elevated VLDL-C and TAG levels (P lt 005) and reduced HDL-C levels (P lt 005) An analysis of the L162Vpolymorphism in the peroxisome proliferator-activated receptor alpha (PPAR120572) gene (n = 248) revealed an increased adjusted ORwhen the LeuVal was compared with the LeuLeu genotype (OR = 877 95 CI = 141ndash7870 P = 0014) High TAG (P = 0021)and VLDL-C (P = 0023) levels were associated with susceptibility to VL whereas low HDL (P = 0006) levels with resistance toinfection The mutated LPL and the PPAR120572 LeuVal genotypes may be considered risk markers for the development of VL

1 IntroductionVisceral leishmaniasis (VL) is a systemic disease that isprevalent in tropical and subtropical regions VL is caused byvarious species of protozoa within the genus Leishmania and

of the Leishmania donovani complex including Leishmaniainfantum (previously referred to as L chagasi) in the newworld [1] In aVL-endemic area themost infected individualsremain asymptomatic Individualswith infectionmaypresent

Hindawi Publishing CorporationMediators of InflammationVolume 2014 Article ID 230129 10 pageshttpdxdoiorg1011552014230129

2 Mediators of Inflammation

with mild symptoms and a minority of infected individualsapproximately 10ndash20 progress to active severe disease thatis characterized by fever hepatosplenomegaly pancytopeniahypergammaglobulinemia and severe weight loss [2 3]However the factors that are associated with susceptibility toinfection progression are not well known

The immune system has been the natural focus ofprevious studies that assessed the risk markers for VLdevelopment Most studies have focused on genes that arerelated to the immune system In studies with approaches thatwere based mainly on family groups with VL infection theSLC11A1 (formerly called as the natural resistance-associatedmacrophage protein-1 NRamp-1) gene on chromosome 2q35[4 5] interleukin-4interleukin-9 [6] the TNF locus [7] alocus on chromosome 22q12 [8 9] IL-18 [10] and IL-1beta[11] polymorphisms have been associated with VL infectionHowever in their 2009 review [12] Blackwell et al suggestedthat most of these polymorphisms had marginal effects oninfection progression Recently the IL-10 819 CT genotypewas associated with susceptibility to VL development in Iran[13] In well-powered population-based studies the CXCR2gene [14] and DLL1 which encode delta-like 1 the ligand forNotch 3 [15] were associated with susceptibility to VL devel-opment but not SLC11A1 in India [16] More recently anotherwell-powered population-based study revealed a consistentcontribution of the HLA-DRB1-HLA-DQA1 HLA class IIregion to VL susceptibility in the Indian subcontinent andBrazil [17] However the role of these genetic determinantsin the pathogenesis of VL was not addressed in these studies

In L infantum infection protective immunity develops inthe majority of individuals who reside in VL-endemic areas[2] During active disease immune derangement occurshowever the immune system remains highly activated [3]Therefore nonspecific factors may play important roles ininfections by parasites that can efficiently evade effectiveimmune responses Furthermore a study has indicated thatfactors not related to acquired cell-mediated immunity mayexplain the progression of VL [18] Therefore in this studywe focused on nonimmune sector that is lipid metabolism

Several studies have reported on lipid alterations inhuman VL cases [19ndash22] In this study we initially analyzedserum-lipid profiles in a large number of patients with clinicalmanifestations of VL We found high triacylglycerol (TAG)levels high very-low-density lipoprotein cholesterol (VLDL-C) levels and low high-density lipoprotein cholesterol (HDL-C) levels in infected symptomatic individuals These alter-ations may have resulted from the inflammatory infectiousprocess however we investigated the possible role of theselipoprotein fractions in susceptibility to L infantum infectionbecause previous results have suggested that amastigotesare largely dependent on protein and lipids as an energysource within host cells [23] In addition to the analysisof lipoprotein fraction alterations we extended the studyto analyze the factors that modulate TAG and HDL levelsThese additional factors included lipoprotein lipase (LPL)which hydrolyzes TAG from TAG-rich lipoprotein particlesin plasma [24] and reduces the uptake of a TAG-rich artificialemulsion [25] apolipoprotein E (apoE) which mediates thebinding of lipoprotein particles to receptors in the liver that

mediate their clearance [26] and peroxisome proliferator-activated receptor alpha (PPAR120572) which is involved inlipid metabolism including roles in oxidation pathways theuptake and transport of fatty acids and lipoprotein synthesisroutes [27] Therefore we focused on LPL apoE and PPAR120572gene polymorphisms as candidates for genetic risk markersfor disease development in L infantum infection LPL genepolymorphisms were searched using HindIII and PVuIIrestriction enzymes known to reveal mutated genes thatcorrelate with TAG level alteration and the former also withHDL level alteration [28 29]We found that theH+H+geno-type and the H+ allele which were identified using HindIIIrestriction in the LPL gene were more frequent in infectedsymptomatic individuals and were notably associated withhigh levels of VLDL-C TAG and HDL-C The apoE gene ispresented in three main isoforms (apoE2 apoE3 and apoE4)that are coded by three common alleles (E2 E3 and E4) [26]We found that the E3E3 genotype and the E3 allele weremore frequent in infected symptomatic individuals than ininfected asymptomatic or noninfected individuals and thesefactors were associated with high levels of VLDL cholesteroland TAG We found that the LeuVal genotype and the Valallele of the L162V polymorphism in the PPAR120572 gene weremore frequent in infected symptomatic individuals than ininfected asymptomatic or noninfected individuals but withno apparent association with alterations in lipoprotein levels

We hypothesized that certain lipid fractions may playa role in the development of L infantum infection In thisstudy we found that high levels of triglycerides and VLDLand low levels of HDL favor the development of activedisease Therefore we suggest that related lipoprotein lipaseand apolipoprotein E genotypes that lead to alterations inthese lipoprotein levels as well as the presence of the PPAR120572L162V allele may be susceptibility factors and markers of VLdevelopment

2 Materials and Methods21 Study Design and Subjects The individuals who wereincluded in this study were from Teresina Piaui StateNortheastern Brazil The inclusion criteria were as followsunrelated individuals who presented with active VL (referredto as infected symptomatic) healthy individuals who hadbeen infected with L infantum as determined by a positiveanti-Leishmania (Montenegro) delayed-type hypersensitivityskin test andor the presence of anti-Leishmania antibodies(referred to as infected asymptomatic) and healthy individ-uals who had no evidence of present or past infection with Linfantum as determined by both a negative Montenegro skintest and the absence of specific antibodies The subjects andmaterials in this study were similar to those in the previousstudy by Alonso et al [30] in which possible ethnic andenvironmental biases were analyzed and deemed not to beconfounding factors

22 Ethics Statement This study was approved by the ethicscommittees of the participating institutions (Comissao deEtica em Pesquisa da Universidade Federal do Piauı andComissao de Etica para Analise de Projetos de Pesquisa da

Mediators of Inflammation 3

Diretoria Clınica do Hospital das Clınicas e da Faculdade deMedicina da Universidade de Sao Paulo) Written informedconsent was obtained from all of the participants or theirparents or guardians Additionally the samples and data weretreated anonymously

23 Analysis of Biochemical Parameters After 12 hours offasting venous blood samples were drawn from 81 indi-viduals to measure lipid parameters The total cholesterol(TC) low-density lipoprotein cholesterol (LDL-C) HDL-Cand TAG concentrations were measured using automatedmethods (Roche Diagnostics Co Indianapolis IN USA)and the VLDL-C concentrations were calculated (TAG con-centration5) using the Friedewald equation

24 Genetic Analysis of the LPL Polymorphism DNA wasextracted from peripheral blood white cells using the GFXGenomic Blood DNA Purification Kit (GE Healthcare Pis-cataway NJ USA) and subjected to amplification by poly-merase chain reaction (PCR) using a Bio-Rad LaboratoriesDNAThermal Cycler (Hercules CA USA)

The variations were confirmed using a restriction frag-ment length polymorphism (RFLP) assay in which the PCR-amplified products were digested by HindIII for 156 individ-uals or by PvuII for 130 individuals Due to the availabilitynot all samples were analyzed using both enzymes Oneprimer set was used to amplify the sequence around aHindIII restriction site in intron 8 (the forward primerwas 51015840-TTTAGGCCTGAAGTTTCCAC-31015840 and the reverseprimer was 51015840-CTCCCTAGAACAGAAGATC-31015840) [31] Theamplified fragment was 13 kb in size The other primerset was obtained from the DNA sequences that flanked aPvuII restriction site in intron 6 (the forward primer was51015840-TAGAGGTTGAGGCACCTGTGC-31015840 and the reverseprimer was 51015840-GTGGGTGAATCACCTGAGGTC-31015840) [32]The amplified fragment was 858 bp longThe amplification ofthe region that flanked the HindIII site was performed over33 cycles at 95∘C for 1min 60∘C for 2min and 72∘C for 2minThe amplification of the sequence that surrounded the PvuIIsite was performed over 32 cycles at 95∘C for 1min 68∘C for2min and 72∘C for 2minThe PCR-amplified products weredigested with HindIII or PvuII HindIII yielded fragmentsthat were 600 bp and 700 bp long and PvuII yielded frag-ments that were 266 bp and 592 bp long [28]

25 Genetic Analysis of the apoE Polymorphism Usingextracted DNA the variations were confirmed by asingle-nucleotide polymorphism (SNP) assay in whichthe PCR-amplified products were analyzed using theSNaPshot Multiplex Kit (Applied Biosystems Foster CityCA USA) for 115 individuals We used one set of primersthat were derived from exon 4 in the apoE gene andPCR was used to amplify a 244 bp region that spannedthe E2 E3 and E4 allelic sites (the forward primer was 51015840-TAAGCTTGGCACGGCTGTCCAAGGA-31015840 and the reverseprimer was 51015840-ACAGAATTCGCCCCGGCCTGGTACAC-31015840) [26] The amplification of the apoE site was performedwith an initial denaturation at 95∘C for 2min whichwas followed by 35 cycles at 95∘C for 1min 60∘C for

1min and 72∘C for 30 s The SNaPshot Multiplex Kit wasused to investigate the presence of the E2 E3 and E4alleles in the amplified products Two specific primersfor apoE SNPs were utilized (the forward primer was 51015840-GCGCGGACATGGAGGACGTG-31015840 and the reverse primerwas 51015840-CCCCCGGCCTGGTACACTGCCAGGC-31015840) TheSNaPshot Multiplex Kit reactions were performed in the ABI377 Automatic Sequencer (Applied Biosystems) followingthe manufacturerrsquos recommendations

26 Genotyping of the PPAR120572 L162V Polymorphism Usingextracted DNA the PPAR120572 gene L162V polymorphism wasgenotyped by allelic discrimination using TaqMan probeson a custom assay-on-demand basis by Applied BiosystemsA pair of oligos was custom-designed to amplify a frag-ment that contained the L162V SNP together with differentallele-specific fluorescent probes using PCR Genotyping wasperformed using a real-time PCR reaction (StepOnePlusApplied Biosystems) according to the following protocol ina 10 120583L reaction 5 120583L of Maxima ProbeROX qPCR MasterMix (2X) (Fermentas) was mixed with 05120583L of TaqManreagents (a 20X master mix that contained the probes andthe pair of oligos) and 45 120583L of autoclaved Milli-Q waterThe reaction was performed following the cycling protocolinitial denaturation for 10min at 95∘C followed by 40 cyclesof denaturation at 95∘C for 15 s and annealing and extensionfor 60 s at 60∘CAfter cycling a graph of allelic discriminationwas generated

27 Statistical Analysis The data were expressed as meansand standard deviations and compared using ANOVA testsThe Student Newman-Keuls posttest was used for post hocstatistical comparisons among the groups and the 119905-test wasused for statistical comparisons between the groups Thefrequencies and comparison distributions of the genotypesand alleles for each polymorphic site were estimated by genecounting Consistency with the Hardy-Weinberg proportionswas tested by the log-likelihood ratio We used the Pearson120594

2 test for trends to analyze differences in the frequency ofthe LPL genotypes and alleles between the outcome groups(infected symptomatic infected asymptomatic and nonin-fected) Associations between levels of lipids and differentgenotypes and the patient outcomes were expressed as oddsratios (OR) and their respective 95 confidence intervals(95 CI) adjusted for age and gender estimated by meansof exact logistic regression models Models compared theinfected (both symptomatic and asymptomatic) individualsto the noninfected individuals The levels of lipids wereanalyzed both as continuous and dichotomous variablesFor the dichotomous analyses the cut-off points used forTC LDL-C HDL-C TAG and VLDL-C were establishedby selecting the point of the ROC curve that maximizedthe sensitivity and specificity for separating noninfected andinfected symptomatic individuals Data were analyzed usingStata statistical software (Stata for Windows 130 StataCorpCollege Station TX) Systat version 10 (Point RichmondRichmond CA) and GraphPad Prism 30 (GraphPad Soft-ware Inc San Diego CA USA) A 119875 value lt005 wasconsidered to be significant


Table 1 Levels of serum lipids in individuals exposed to Leishmania infantum from endemic area

Lipids Infected symptomatic individuals Infected asymptomatic individuals Noninfected individuals119875 value

119899 mgdL 119899 mgdL 119899 mgdLTC 17 1212 plusmn 346 28 1430 plusmn 421 36 1399 plusmn 361 0152LDL-C 17 595 plusmn 308 28 855 plusmn 422 36 771 plusmn 360 0083HDL-C 17 383 plusmn 43lowast 28 390 plusmn 58lowast 36 430 plusmn 72 0012TAG 17 2194 plusmn 849

lowastdagger 28 1691 plusmn 627 36 1579 plusmn 676 0013VLDL-C 17 438 plusmn 169

lowastdagger 28 339 plusmn 124 36 316 plusmn 135 0013TC= total cholesterol LDL-C = low-density lipoprotein cholesterol HDL-C = density lipoprotein cholesterol TAG= triacylglycerol VLDL = very-low-densitylipoprotein cholesterol Lipid values expressed as the mean plusmn SD 119875 value analyzed by ANOVA test with the Student Newman-Keuls posttest lowast119875 lt 005 versusnoninfected individuals dagger119875 lt 005 versus infected asymptomatic individuals 119875 values in bold indicate statistically significant differences

3 Results

In this study using a candidate gene approach to reveal thegenetic traits and the resulting phenotypic expressions thatis lipoprotein levels we aimed to identify the risk markersfor the development of L infantum infection

The assessment of the lipid fraction levels revealed ele-vated triacylglycerol (TAG) levels and elevated very-low-density lipoprotein cholesterol (VLDL-C) levels in all ofthe infected symptomatic individuals compared with theinfected asymptomatic and noninfected individuals (119875 lt005) (Table 1) (according to the III Brazilian Guidelineson Dyslipidemia [33] the optimal TAG level is lt130mgdLfor individuals up to 20 years of age and lt150mgdL inindividuals who are older than 20 years of age) Becausethe ideal age-matched endemic controls for this approachcould not be obtained due to technical and ethical reasonsthe average age (mean plusmn standard deviation) of the infectedsymptomatic individuals (119899 = 17 62 plusmn 76 years ofage) was significantly lower than the average ages of theinfected asymptomatic (119899 = 28 302 plusmn 124 years of age)and noninfected individuals (119899 = 36 279 plusmn 192 yearsof age) To circumvent bias we separately examined theindividuals who were younger than 20 years of age when thedifferences between the groups were still maintained (datanot shown) Additionally we observed reduced high-densitylipoprotein cholesterol (HDL-C) levels in both the infectedsymptomatic and asymptomatic individuals compared withthe noninfected individuals (119875 lt 005) (Table 1)

We analyzed the polymorphisms of the LPL apoE andPPAR120572 genes The genotype distributions for all of thepolymorphisms were in Hardy-Weinberg equilibrium for allof the groups (Table 2) Significant differences were observedin the frequencies of the polymorphic HindIII-restrictedgenotypes (H+H+ versus H+Hminus 119875 lt 005 H+H+ versusHminusHminus 119875 lt 001) and the HindIII-restricted alleles (H+versus Hminus 119875 lt 001) (Table 1) However no differences weredetected between the groups according to the frequenciesof either the genes or the alleles of the PvuII restrictionproducts (Table 2) In the apoE gene the analysis of the threecommon apoE alleles (E2 E3 and E4) that code the threemain apoE isoforms (apoE2 apoE3 and apoE4) indicatedthat the E3E3 genotype was the most frequent followedby the E3E4 E2E3 E2E4 and E4E4 genotypes TheE2E2 genotype was not detected in any of the individuals

In the analysis of the apoE alleles the E3 allele was themost frequent followed by the E4 and E2 alleles Significantdifferences were observed between the frequency of the E4allele and that of the E3 allele (119875 lt 005) (Table 2) Wefound that the E3E3 genotype and the E3 allele were morefrequent in infected symptomatic individuals than in infectedasymptomatic or noninfected individuals (Table 2) In thePPAR120572 gene L162V polymorphism we found significantdifferences in the frequencies of the LeuLeu and LeuVal(119875 lt 005) genotypes and the Leu and Val alleles (119875 lt 001)between the groups (Table 2)

In the analysis of the association between the genotypesand the lipoprotein levels in all of the individuals regardless ofthe outcome the TAG and VLDL-C levels were significantlyhigher in the H+H+ individuals (119875 lt 001) (Table 3)The individuals with the H+H+ genotype had significantlylower HDL-C levels than the individuals with the H+Hminusgenotype (119875 lt 005) The serum-lipid levels were measuredin only two HminusHminus individuals which was insufficient forthe statistical analysis (the means are presented in the legendof Table 3) In the individuals with the P1P1 genotype nosignificant differences in the lipid profiles were observedbetween the P1P2 and P2P2 individuals (Table 3) In theanalysis of the apoE genotypes and alleles we found that theTAG and VLDL-C levels in the individuals with the E2E3genotype were significantly higher than those in the E3E3and E3E4 (119875 lt 005) individuals (Table 3) The serum-lipiddata were available in only one E4E4 individual (all of thevariables are shown in the legend of Table 3)The serum-lipidlevels were not evaluated in the E2E4 and E2E2 individualsFurthermore the TAG and VLDL-C levels were significantlyhigher in the E2 individuals than in the E3 and E4 individuals(119875 lt 005) but the TC levels were higher in the E3 individualsthan in the E2 and E4 individuals (119875 lt 005) (Table 3)The E3allele was associated with higher TC levels than with the E2and E4 alleles however the levels were still optimal [33] Thelipid profiles were not associated with either the genotypesor the alleles of the L162V polymorphism in the PPAR120572 gene(Table 3)

To analyze the association between the genotypic andphenotypic expressions and the outcomes we performed amultivariate analysis to calculate the adjusted odds ratio (OR)because of the significant differences in the ages and genderdistribution among the studied groups The results of this


Table 2 Frequency of genotypes and alleles of LPL apoE and PPAR120572 genes in individuals exposed to Leishmania infantum

Polymorphismssectsect Infected symptomatic individuals Infected asymptomatic individuals Noninfected individuals119875 value

119899 of total 119899 of total 119899 of total

HindIII

H+H+(a) 24 462 17 354 13 232 0040(a vs b)

H+Hminus(b) 26 500 27 563 34 607 0103(b vs c)

HminusHminus(c) 2 38 4 83 9 161 0006(a vs c)

HWEdagger 0116 0138 0099

H+(d) 74 715 61 635 60 536Hminus(e) 30 288 35 365 52 464 0008(d vs e)

PvuII

P1P1(f) 12 286 13 295 16 372 0429(f vs g)

P1P2(g) 25 595 27 614 23 535 0835(g vs h)

P2P2(h) 5 119 4 91 4 93 0507(f vs h)

HWEdagger 0146 0062 0294

P1(i) 49 583 53 602 55 640P2(j) 35 417 35 398 31 360 0452(i vs j)

ApoE

E2E3(k) 3 81 4 105 2 50 0833(k vs l)

E3E3(l) 30 811 24 632 26 650 0116(l vs m)

E3E4(m) 4 108 10 263 10 250 0214(k vs m)

E2E4 0 00 0 00 1 25E4E4 0 00 0 00 1 25

E2(n) 3 41 4 53 3 37 0953(n vs o)

E3(o) 67 905 62 816 64 800 0038(o vs p)

E4(p) 4 54 10 131 13 163 0230(n vs p)

PPPAR120572

LeuLeu(q) 48 800 61 871 92 939 0022(q vs r)

LeuVal(r) 11 183 8 114 6 61ValVal 1 17 1 14 0 00

HWEdagger 0693 0247 0745

Leu(s) 107 892 130 929 190 969Val(t) 13 108 10 71 6 31 0008(s vs t)

sectsect119875 value analyzed by 1205942 test for trend dagger119875 value in Hardy-Weinberg equilibrium (HWE)

Note H+ indicates presence and Hminus indicates absence of the HindIII restriction site P1 indicates absence and P2 indicates presence of the PvuII restrictionsite Apo E polymorphism results in six genotypes (E2E2 E2E3 E2E4 E3E3 E3E4 and E4E4) E2E4 and E4E4 did not have sufficient number to beanalyzed E2E2 was not detected PPAR120572 L162V polymorphism results in three genotypes (LeuLeu LeuVal and ValVal) Bold text indicates statisticallysignificant differences

analysis suggested that the presence of the lipoprotein lipaseH+H+ genotype (adjusted OR = 213 95 CI = 232ndash3353119875 = 0003) the PPAR120572 LeuVal genotype (adjusted OR =877 95 CI = 141ndash787 119875 = 0014) and elevated levels ofVLDL-C (adjusted OR = 108 95 CI = 101ndash117 119875 = 0023)and TAG (adjusted OR = 102 95 CI = 100ndash103 119875 = 0021)were associated with symptomatic L infantum infectionIn contrast the HDL levels were inversely associated withsymptomatic (adjusted OR = 084 95 CI = 071ndash096 119875 =0006) and asymptomatic (adjusted OR = 092 95 CI =084ndash099 119875 = 0027) L infantum infection (Table 4)When the lipoprotein levels were analyzed using the cut-offvalue established by selecting the point on the ROC curvethat maximized the sensitivity and specificity for separatingnoninfected and infected symptomatic individuals the asso-ciation with symptomatic infection became more evident forelevated levels of VLDL-C (cut-off value = 33mgdL adjustedOR = 188 95 CI = 171ndash10144 119875 = 0009) and TAG (cut-off

value = 168mgdL adjusted OR = 188 95 CI = 171ndash10144119875 = 0009)

4 DiscussionDiscovering reliable markers for the assessment of individualand population risks of disease development is a majorchallenge for the scientific community In this study weevaluated potential markers of VL development In areasthat are endemic for VL most infected individuals developa protective immune response Therefore we focused on thenonimmune lipid elements as potential susceptibility factorsbased on the hypothesis that these alterations in VL patientsmay favor parasite growth and disease development Wefurther analyzed the polymorphisms of certain genes thatmay lead to such alterations in lipoprotein levels

In a significant number of patients with clinical manifes-tations of VL we found high TAG levels high VLDL-C levelsand low HDL-C levels in infected symptomatic individuals


Table3Levelsof

lipids(mgdL

)according

togeno

typesa

ndalleleso

fLPL

andof

apoE

inindividu

alse

xposed

toLeish

maniainfantum

Polymorph

isms

Age

Lipids

(mgdL

)TC

aLD

L-Ca

HDL-Ca

TAG

aVLD

L-Ca

HindIII

Genotypes

H+H+(119899=19)

226plusmn154

1508plusmn422

910plusmn44

0381plusmn56

2076plusmn827

415plusmn165

H+Hminus(119899=33)

221plusmn182

1388plusmn348

774plusmn356

419plusmn64

1579plusmn528

316plusmn105

119875value

0914

0274

0228

0037

0011

0010

Alleles

H+(119899=71)

224plusmn166

1453plusmn388

847plusmn40

3399plusmn62

1845plusmn736

369plusmn146

Hminus(119899=37)

227plusmn175

1352plusmn347

744plusmn347

425plusmn63

1545plusmn508

309plusmn101

119875value

0919

0187

0191

004

00029

0029

PvuII

Genotypes

P1P1(119899=15)

224plusmn140

246plusmn167

817plusmn371

400plusmn47

1594plusmn509

319plusmn101

P1P2(119899=27)

246plusmn167

1418plusmn387

843plusmn397

399plusmn63

1730plusmn777

346plusmn155

P2P2(119899=4)

165plusmn178

1328plusmn40

3717plusmn288

415plusmn57

2305plusmn890

460plusmn176

119875valuesect

0622

0878

0828

0878

0217

0220

Alleles

P1(119899=57)

235plusmn151

1428plusmn365

829plusmn377

400plusmn54

1658plusmn64

41658plusmn64

4P2(119899=35)

228plusmn167

1397plusmn381

814plusmn371

403plusmn61

1861plusmn

814

372plusmn162

119875value

0840

0706

0853

0793

0188

0191

apoE

Genotypes

E2E3(119899=4)

237plusmn143

1200plusmn362

657plusmn304

417plusmn78

2753plusmn1135lowastdagger


E3E3(119899=29)

191plusmn148

1521plusmn

401

915plusmn416

392plusmn58

1669plusmn585

334plusmn116

E3E4(119899=6)

355plusmn181

1190plusmn119

657plusmn239

390plusmn30

1773plusmn1004

355plusmn199

119875valuesect

006

80069

0205

0692

0026

002

8

Alleles

E2(119899=4)

237plusmn143

1200plusmn362

657plusmn304

417plusmn78



E3(119899=68)

208plusmn155

1473plusmn394daggerDagger

877plusmn40

2392plusmn57

1742plusmn696

348plusmn138

E4(119899=8)

321plusmn165

1130plusmn150

595plusmn232

387plusmn26

1655plusmn877

331plusmn174

119875valuesect

0152

0029

0099

0661

0029

0031

PPAR120572

Genotypes

LeuLeu(119899=54)

267plusmn176

1379plusmn397

782plusmn388

409plusmn65

1732plusmn812

346plusmn162

LeuVa

l(119899=8)

231plusmn183

1469plusmn326

833plusmn311

403plusmn57

1661plusmn

357

334plusmn69

119875value

0589

0545

0728

0794

0809

0830

Alleles

Leu(119899=116)

265plusmn175

1385plusmn390

786plusmn381

408plusmn64

1727plusmn785

345plusmn156

Val(119899=8)

231plusmn183

1469plusmn326

833plusmn311

403plusmn57

1661plusmn

357

334plusmn69

119875value

0599

0555

0735

0798

0814

0834

Allvalues

aree

xpressed

asthem

eanplusmnSD

inindividu

alse

xposed

toLeish

maniachagasi

a valuesinmgdL

119875valueanalyzed

byStud

ent119905-te

stsect P

valueanalyzed

byANOVA

testwith

theStud

entN

ewman-K

eulspo

sttestIn

relationto

geno

typeslowast

119875lt005versus

E3E3dagger

119875lt005versus

E3E4In

relationto

alleleslowast

119875lt005versus

E3dagger119875lt005versus

E4Dagger119875lt005versus

E2HminusHminusdidno

thaves

ufficientn

umbertobe

analyzed(119899=2)A

ge=280plusmn127T

C=1050plusmn141

LDL-C=500plusmn71H

DL-C

=475plusmn21TA

G=1265plusmn92VLD

L-C=255plusmn21E4

E4didno

thaves

ufficientn

umbertobe

analyzed(119899=1)A

ge=220T

C=950L

DL-C=410H

DL-C=380T

AG=1300VLD

L=260H

+indicates

presence

andHminusindicatesa

bsence

oftheH

indIIIrestr

ictio

nsiteP1

indicatesa

bsence

andP2

indicatesp

resenceo

fthe

PvuIIrestrictio

nsite

NoteAp

oEpo

lymorph

ismresults

insix

geno

typesranking

(E2E2

E2E3

E2E4

E3E3

E3E4

and

E4E4)

andthreea

lleles(E2

E3andE4

)Bo

ldtext

indicatesstatisticallysig

nificantd

ifferences


Table 4 Adjusted odds ratios (OR) and respective 95 confidence intervals for the association between levels of lipids (mgdL) and genotypesof LPL apoE and PPAR120572 and infected symptomatic and asymptomatic L infantum infection

Variable Infected symptomatic infection Infected asymptomatic infectionORlowast 95 CIlowast 119875 value ORlowast 95 CIlowast 119875 value

HindIIIHminusHminus 100 100H+Hminus 637 084ndash834 0083 204 049ndash104 0435H+H+ 213 232ndash3353 0003 348 072ndash204 0143

PvuIIP1P1 100 100P1P2 186 055ndash668 0395 142 052ndash398 0593P2P2 071 010ndash528 1000 120 018ndash781 1000

ApoEE3E4 100 100E3E3 266 052ndash161 0312 091 028ndash293 1000E2E3 431 023ndash1137 0515 190 022 ndash253 0825

PPAR120572LeuLeu 100 100LeuVal 877 141ndash787 0014 276 056ndash179 0272

TC 099 097ndash101 0301 100 099ndash101 0787LDL-C 099 097ndash101 0353 101 099ndash102 0414HDL-C 084 071ndash096 0006 092 084ndash099 0027TAG 102 100ndash103 0021 100 099ndash101 0597VLDL-C 108 101ndash117 0023 101 097ndash105 0580lowastAdjusted for age (le or gt20 years) and gender119875 values in bold indicate statistically significant differences

as previously reported in the literature [19ndash22] LPL apoEand PPAR120572 are known as important components of lipidmetabolismwith roles in the homeostasis of different lipopro-tein fractions and their transport and the polymorphismsof these genes have been associated with cardiovascular dis-eases [28 29 34ndash36] Therefore these gene polymorphismswere analyzed in this study We targeted LPL apoE andPPAR120572 genes with polymorphisms and alleles that wereanalyzed in relation to symptomatic L infantum infectionImportantly an association between these polymorphismsand the expected phenotypes was observed excluding theL162V polymorphism in the PPAR120572 gene This finding is incontrast to most studies on gene polymorphisms in whichthe resulting phenotypes were unidentified In the studiedpopulation the H+H+ genotype and the H+ allele wereassociated with elevated VLDL-C and TAG levels (119875 lt005) and reduced HDL-C levels (119875 lt 005) For the apoEgenes and alleles the genotype and expected phenotypeassociation was unclear because the TAG and VLDL-C levelswere significantly higher in individuals with the E2 allelethan in individuals with the E3 and E4 alleles but not inindividuals with the E3 allele This finding may be due tothe restricted number of samples and to the different rolesof the six apoE allotypes However the association betweenthe E2 allele and the alterations in lipoprotein levels maynot have been uncovered because apoE2 carries significantlyless VLDL cholesterol than apoE4 [35] and apoE2 impairs

the lipolytic conversion of VLDL to LDL through inhibitingLPL activity [34]

The association between the H+H+ genotype and theH+ allele with higher TAG and lower HDL-C levels wasstronger in the infected symptomatic group than in theinfected asymptomatic group PvuII has been associated withvariations in serum lipids in other diseases [29 36] howeverwe did not detect any significant associations between thePvuII-restricted LPL genotypes and the presence of active VLor lipid fraction levels This finding reinforces our suggestionthat theHindIII-restricted LPL genotypes thatwere identifiedin this study are risk markers for the development of symp-tomatic L infantum infection

In the literature high TAG and low HDL-C levels havebeen suggested as parameters for the diagnosis and follow-up of patients with VL [37] However in addition to a singlestudy that suggested a role for LDL in the development of VLthrough tumor necrosis factor-120572 (TNF-120572) production [22]our study is the first to clearly find that the LPL genotypethat leads to a phenotype of high VLDL-C levels high TAGlevels and low HDL-C levels is a risk factor for active vis-ceral leishmaniasis Interestingly we observed reduced high-density lipoprotein cholesterol (HDL-C) levels in both theinfected symptomatic and asymptomatic individuals com-pared with the noninfected individuals which may suggestthat decreased HDL levels initially favor infection WhenTG and VLDL levels increase progression to symptomaticinfection may occur


The biological roles of VLDL and HDL in Leishmaniainfection need to be further analyzed however as an initialapproach we assessed the role of lipoprotein fractions inthe mechanism of cell infection in vitro by examining thephagocytosis of promastigotes by macrophagesWe observed29 more parasites within the macrophages that were incu-bated for six hours with VLDL (119875 lt 005) than in culturewithout the addition of lipoprotein The coaddition of HDLsignificantly reduced (37) the parasite burden comparedwith VLDL (119875 lt 005)

ThePPAR120572L162Vpolymorphismhas been demonstratedto be a risk marker for symptomatic infection however themutated gene was not associated with altered lipoprotein lev-els In a functional study of theVal-mutatedPPAR120572 allele thismutation in the DNA binding domain for PPAR120572 impactedthe function of this transcription factor by preventing itsproper binding to the region of DNA that is under its tran-scriptional control [38] The frequency of this mutated allelewas significantly higher in individuals who presented withsymptomatic infection therefore the inefficient activationof lipid metabolism genes may lead to higher serum levelsof lipid fractions However the results of this study did notindicate a relationship between the polymorphisms and thelipid profiles A possible explanation for this finding is thelow number of individuals who presented with the Val alleleThis variant allele is relatively rare (an observed frequency of108) and the lipid profiles of only 8 individuals who werecarrying this allele were measured Therefore any statisticalevidence for a relationship between this mutation and thelipid profiles would be unlikely Besides the PPAR120572 genecan modulate lipid pathways which may not be reflectedin serum-lipid levels and indirectly affect the outcome ofLeishmania infantum infection In addition PPAR120572 is knownfor its major role in the upregulation of cholesterol traffickingand efflux in macrophages [39] Interestingly in transcrip-tomic studies with Leishmania-infectedmouse macrophagescholesterol accumulation was clearly observed within hostcells whichwas achieved by the upregulation of genes that areimplicated in the uptake of LDL (CD36 and the LDL receptor)and by the downregulation of genes that mediate cholesterolefflux (ABCA1 andCYP27) [40 41] In this scenario a PPAR120572variant allele may contribute to the modulation of infectionby enhancing the accumulation of cholesterol in infectedcells and by shuttling cholesterol to Leishmania parasites Wecannot rule out either the modulation of infection by PPAR120572functions in inflammatory processes [39]

The results of this study and those from two previousstudies [30 42] support the presupposition that the genotypesfor molecules that are not related to the adaptive immunesystem that is LPL PPAR120572 and mannan-binding lectin(MBL) genotypes have a significant correlation with Linfantum infection or disease Notably in addition to the rolesof MBL in the complement system [43] and the autoimmuneprocess [44] MBL levels are affected by the activation ofPPAR120572 [45] which induces LPL expression [46]

These genetic markers and the more easily accessiblelipid levels may be used to evaluate the risk of L infantuminfection and disease in endemic areas and may be used to

follow up patients who present with active-phase infectionFurthermore our results suggest that we should considerstudying lipid-lowering drugs as a complementary treatmentfor VL patients

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Marcia Dias Teixeira Carvalho and Diego Peres Alonso andthe senior authors Paulo Eduardo Martins Ribolla and HiroGoto had respective equal participation in this study

Acknowledgments

The authors would like to acknowledge the ConselhoNacional de Desenvolvimento Cientıfico e Tecnologico(Grant 1545812006-2 research fellowship to H Goto) theFundacao de Amparo a Pesquisa do Estado de Sao Paulo (Fel-lowship 0660006-9 to M Dias Teixeira Carvalho) and theLaboratorio de Investigacao Medica (LIM38 HC-FMUSP)They also would like to thank Valeria Sutti Nunes Mariadas Gracas Prianti Letıcia Amaral Nogueira Alonso andDebora Colombi for providing laboratory assistance andall of the study participants for agreeing to contribute tothis research In addition they would like to thank IsacCastro for the excellent statistical assistance and are gratefulto the Laboratorio de Lıpides (LIM10 HC-FMUSP) for itsassistance

References

[1] I L Mauricio M W Gaunt J R Stothard and M A MilesldquoGenetic typing and phylogeny of the Leishmania donovanicomplex by restriction analysis of PCR amplified gp63 inter-genic regionsrdquo Parasitology vol 122 no 4 pp 393ndash403 2001

[2] R Badaro T C Jones R Lorenco et al ldquoA prospective study ofvisceral leishmaniasis in an endemic area of Brazilrdquo Journal ofInfectious Diseases vol 154 no 4 pp 639ndash649 1986

[3] H Goto and M D G Prianti ldquoImmunoactivation andimmunopathogeny during active visceral leishmaniasisrdquoRevista do Instituto de Medicina Tropical de Sao Paulo vol 51no 5 pp 241ndash246 2009

[4] B Bucheton L Abel M M Kheir et al ldquoGenetic control ofvisceral leishmaniasis in a Sudanese population candidate genetesting indicates a linkage to the NRAMP1 regionrdquo Genes ampImmunity vol 4 no 2 pp 104ndash109 2003

[5] H S Mohamed M E Ibrahim E N Miller et al ldquoSLC11A1(formerlyNRAMP1) and susceptibility to visceral leishmaniasisin the Sudanrdquo European Journal of Human Genetics vol 12 no1 pp 66ndash74 2004

[6] H S Mohamed M E Ibrahim E N Miller et al ldquoGeneticsusceptibility to visceral leishmaniasis in The Sudan linkageand association with IL4 and IFNGR1rdquo Genes and Immunityvol 4 no 5 pp 351ndash355 2003


[7] T M Karplus S M B Jeronimo H Chang et al ldquoAssociationbetween the tumor necrosis factor locus and the clinical out-come of Leishmania chagasi infectionrdquo Infection and Immunityvol 70 no 12 pp 6919ndash6925 2002

[8] B Bucheton L Abel S El-Safi et al ldquoA major susceptibilitylocus on chromosome 22q12 plays a critical role in the controlof kala-azarrdquo American Journal of Human Genetics vol 73 no5 pp 1052ndash1060 2003

[9] B Bucheton L Argiro C Chevillard et al ldquoIdentification of anovel G245R polymorphism in the IL-2 receptor 120573 membraneproximal domain associated with human visceral leishmania-sisrdquo Genes and Immunity vol 8 no 1 pp 79ndash83 2007

[10] A Moravej M Rasouli S Asaei M Kalani and Y MansoorildquoAssociation of interleukin-18 gene variants with susceptibilityto visceral leishmaniasis in Iranian populationrdquo MolecularBiology Reports vol 40 no 6 pp 4009ndash4014 2013

[11] A Moravej M Rasouli M Kalani et al ldquoIL-1120573 (-511TC)gene polymorphismnot IL-1120573 (+3953TC) and LT-120572 (+252AG)gene variants confers susceptibility to visceral leishmaniasisrdquoMolecular Biology Reports vol 39 no 6 pp 6907ndash6914 2012

[12] J M Blackwell M Fakiola M E Ibrahim et al ldquoGenetics andvisceral leishmaniasis Of mice andmanrdquo Parasite Immunologyvol 31 no 5 pp 254ndash266 2009

[13] M Hajilooi K Sardarian M Dadmanesh et al ldquoIs the IL-10 -819 polymorphism associated with visceral leishmaniasisrdquoInflammation pp 1ndash6 2013

[14] S Mehrotra M Fakiola J Oommen et al ldquoGenetic andfunctional evaluation of the role of CXCR1 and CXCR2 insusceptibility to visceral leishmaniasis in North-East IndiardquoBMCMedical Genetics vol 12 article 162 2011

[15] S Mehrotra M Fakiola A Mishra et al ldquoGenetic and func-tional evaluation of the role of DLL1 in susceptibility to visceralleishmaniasis in Indiardquo Infection Genetics and Evolution vol 12no 6 pp 1195ndash1201 2012

[16] S Mehrotra J Oommen A Mishra et al ldquoNo evidence forassociation between SLC11A1 and visceral leishmaniasis inIndiardquo BMCMedical Genetics vol 12 article 71 2011

[17] M Fakiola A Strange H J Cordell et al ldquoCommon variants inthe HLA-DRB1-HLA-DQA1 HLA class II region are associatedwith susceptibility to visceral leishmaniasisrdquo Nature Geneticsvol 45 no 2 pp 208ndash213 2013

[18] C RDavies andA SMGavgani ldquoAge acquired immunity andthe risk of visceral leishmaniasis a prospective study in IranrdquoParasitology vol 119 no 3 pp 247ndash257 1999

[19] E D Bekaert E Dole D Y Dubois et al ldquoAlterations inlipoprotein density classes in infantile visceral Leishmaniasispresence of apolipoprotein SAArdquo European Journal of ClinicalInvestigation vol 22 no 3 pp 190ndash199 1992

[20] E D Bekaert R Kallel M- Bouma et al ldquoPlasma lipoproteinsin infantile visceral Leishmaniasis deficiency of apolipopro-teins A-I and A-IIrdquo Clinica Chimica Acta vol 184 no 2 pp181ndash191 1989

[21] C S Lal A Kumar S Kumar et al ldquoHypocholesterolemiaand increased triglyceride in pediatric visceral leishmaniasisrdquoClinica Chimica Acta vol 382 no 1-2 pp 151ndash153 2007

[22] N M Soares T F Leal M C Fiuza et al ldquoPlasma lipopro-teins in visceral leishmaniasis and their effect on Leishmania-infected macrophagesrdquo Parasite Immunology vol 32 no 4 pp259ndash266 2010

[23] D Rosenzweig D Smith F Opperdoes S Stern RW Olafsonand D Zilberstein ldquoRetooling Leishmania metabolism From

sand fly gut to humanmacrophagerdquoThe FASEB Journal vol 22no 2 pp 590ndash602 2008

[24] H Jansen B Breedveld and K Schoonderwoerd ldquoRole of lipo-protein lipases in postprandial lipid metabolismrdquo Atherosclero-sis vol 141 no 1 pp S31ndashS34 1998

[25] M D T Carvalho L M Harada M Gidlund D F J Ketel-huth P Boschcov and E C R Quintao ldquoMacrophages takeup triacylglycerol-rich emulsions at a faster rate upon co-incubation with native and modified LDL an investigation onthe role of natural chylomicrons in atherosclerosisrdquo Journal ofCellular Biochemistry vol 84 no 2 pp 309ndash323 2001

[26] M Emi L L Wu M A Robertson et al ldquoGenotyping andsequence analysis of apolipoprotein E isoformsrdquo Genomics vol3 no 4 pp 373ndash379 1988

[27] C Duval M Muller and S Kersten ldquoPPARalpha and dyslipi-demiardquo Biochimica et Biophysica Acta vol 1771 no 8 pp 961ndash971 2007

[28] Y I Ahn M I Kamboh R F Hamman S A Cole and RE Ferrell ldquoTwo DNA polymorphisms in the lipoprotein lipasegene and their associations with factors related to cardiovascu-lar diseaserdquo Journal of Lipid Research vol 34 no 3 pp 421ndash4281993

[29] E Socquard A Durlach C Clavel P Nazeyrollas and VDurlach ldquoAssociation of HindIII and PvuII genetic poly-morphisms of lipoprotein lipase with lipid metabolism andmacrovascular events in type 2 diabetic patientsrdquo Diabetes ampMetabolism vol 32 no 3 pp 262ndash269 2006

[30] D P Alonso A F B Ferreira P E M Ribolla et al ldquoGenotypesof themannan-binding lectin gene and susceptibility to visceralleishmaniasis and clinical complicationsrdquo Journal of InfectiousDiseases vol 195 no 8 pp 1212ndash1217 2007

[31] T G Kirchgessner J- Chuat C Heinzmann et al ldquoOrgani-zation of the human lipoprotein lipase gene and evolution ofthe lipase gene familyrdquo Proceedings of the National Academy ofSciences of the United States of America vol 86 no 24 pp 9647ndash9651 1989

[32] K Oka G T Tkalcevic J Stocks D J Galton andW V BrownldquoNucleotide sequence of PvuII polymorphic site at the humanlipoprotein lipase gene locusrdquoNucleic Acids Research vol 17 no16 p 6752 1989

[33] R D Santos ldquoIII Brazilian Guidelines on Dyslipidemias andGuideline of Atherosclerosis Prevention from AtherosclerosisDepartment of Sociedade Brasileira de Cardiologiardquo ArquivosBrasileiros de Cardiologia vol 77 supplement 3 pp 1ndash48 2001

[34] Y Huang X Q Liu S C Rail Jr and R W MahleyldquoApolipoprotein E2 reduces the low density lipoprotein level intransgenicmice by impairing lipoprotein lipase-mediated lipol-ysis of triglyceride-rich lipoproteinsrdquo The Journal of BiologicalChemistry vol 273 no 28 pp 17483ndash17490 1998

[35] L Dong and K H Weisgraber ldquoHuman apolipoprotein E4domain interaction Arginine 61 and glutamic acid 255 interactto direct the preference for very low density lipoproteinsrdquoJournal of Biological Chemistry vol 271 no 32 pp 19053ndash190571996

[36] J L Anderson G J King T L Bair et al ldquoAssociation oflipoprotein lipase gene polymorphisms with coronary arterydiseaserdquo Journal of the American College of Cardiology vol 33no 4 pp 1013ndash1020 1999

[37] G Secmeer A B Cengiz A Gurgey et al ldquoHypertriglyc-eridemia and decreased high-density lipoprotein could be aclue for visceral leishmaniasisrdquo Infectious Diseases in ClinicalPractice vol 14 no 6 pp 401ndash402 2006


[38] A Sapone J M Peters S Sakai et al ldquoThe human per-oxisome proliferator-activated receptor 120572 gene Identificationand functional characterization of two natural allelic variantsrdquoPharmacogenetics vol 10 no 4 pp 321ndash333 2000

[39] E Rigamonti G Chinetti-Gbaguidi and B Staels ldquoRegulationof macrophage functions by PPAR- 120572 PPAR- 120574 and LXRsin mice and menrdquo Arteriosclerosis Thrombosis and VascularBiology vol 28 no 6 pp 1050ndash1059 2008

[40] J Osorio y Fortea E de La Llave B Regnault et al ldquoTranscrip-tional signatures of BALBc mouse macrophages housing mul-tiplying Leishmania amazonensis amastigotesrdquo BMC Genomicsvol 10 article 119 2009

[41] I Rabhi S Rabhi R Ben-Othman et al ldquoTranscriptomic sig-nature of Leishmania infected mice macrophages a metabolicpoint of viewrdquo PLoS Neglected Tropical Diseases vol 6 no 8Article ID e1763 2012

[42] S Hamdi R Ejghal M Idrissi et al ldquoA variant in the promoterof MBL2 is associated with protection against visceral leishma-niasis inMoroccordquo Infection Genetics and Evolution vol 13 no1 pp 162ndash167 2013

[43] W K Eddie Ip K Takahashi R Alan Ezekowitz and LM Stuart ldquoMannose-binding lectin and innate immunityrdquoImmunological Reviews vol 230 no 1 pp 9ndash21 2009

[44] S Saevarsdottir T Vikingsdottir and H Valdimarsson ldquoThepotential role of mannan-binding lectin in the clearance ofself-components including immune complexesrdquo ScandinavianJournal of Immunology vol 60 no 1-2 pp 23ndash29 2004

[45] M Rakhshandehroo R Stienstra N J de Wit et alldquoPlasma mannose-binding lectin is stimulated by PPAR120572 inhumansrdquo The American Journal of PhysiologymdashEndocrinologyand Metabolism vol 302 no 5 pp E595ndashE602 2012

[46] M Panadero C Bocos and E Herrera ldquoRelationship betweenlipoprotein lipase and peroxisome proliferator-activated recep-tor-120572 expression in rat liver during developmentrdquo Journal ofPhysiology and Biochemistry vol 62 no 3 pp 189ndash198 2006

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


with mild symptoms and a minority of infected individualsapproximately 10ndash20 progress to active severe disease thatis characterized by fever hepatosplenomegaly pancytopeniahypergammaglobulinemia and severe weight loss [2 3]However the factors that are associated with susceptibility toinfection progression are not well known

The immune system has been the natural focus ofprevious studies that assessed the risk markers for VLdevelopment Most studies have focused on genes that arerelated to the immune system In studies with approaches thatwere based mainly on family groups with VL infection theSLC11A1 (formerly called as the natural resistance-associatedmacrophage protein-1 NRamp-1) gene on chromosome 2q35[4 5] interleukin-4interleukin-9 [6] the TNF locus [7] alocus on chromosome 22q12 [8 9] IL-18 [10] and IL-1beta[11] polymorphisms have been associated with VL infectionHowever in their 2009 review [12] Blackwell et al suggestedthat most of these polymorphisms had marginal effects oninfection progression Recently the IL-10 819 CT genotypewas associated with susceptibility to VL development in Iran[13] In well-powered population-based studies the CXCR2gene [14] and DLL1 which encode delta-like 1 the ligand forNotch 3 [15] were associated with susceptibility to VL devel-opment but not SLC11A1 in India [16] More recently anotherwell-powered population-based study revealed a consistentcontribution of the HLA-DRB1-HLA-DQA1 HLA class IIregion to VL susceptibility in the Indian subcontinent andBrazil [17] However the role of these genetic determinantsin the pathogenesis of VL was not addressed in these studies

In L infantum infection protective immunity develops inthe majority of individuals who reside in VL-endemic areas[2] During active disease immune derangement occurshowever the immune system remains highly activated [3]Therefore nonspecific factors may play important roles ininfections by parasites that can efficiently evade effectiveimmune responses Furthermore a study has indicated thatfactors not related to acquired cell-mediated immunity mayexplain the progression of VL [18] Therefore in this studywe focused on nonimmune sector that is lipid metabolism

Several studies have reported on lipid alterations inhuman VL cases [19ndash22] In this study we initially analyzedserum-lipid profiles in a large number of patients with clinicalmanifestations of VL We found high triacylglycerol (TAG)levels high very-low-density lipoprotein cholesterol (VLDL-C) levels and low high-density lipoprotein cholesterol (HDL-C) levels in infected symptomatic individuals These alter-ations may have resulted from the inflammatory infectiousprocess however we investigated the possible role of theselipoprotein fractions in susceptibility to L infantum infectionbecause previous results have suggested that amastigotesare largely dependent on protein and lipids as an energysource within host cells [23] In addition to the analysisof lipoprotein fraction alterations we extended the studyto analyze the factors that modulate TAG and HDL levelsThese additional factors included lipoprotein lipase (LPL)which hydrolyzes TAG from TAG-rich lipoprotein particlesin plasma [24] and reduces the uptake of a TAG-rich artificialemulsion [25] apolipoprotein E (apoE) which mediates thebinding of lipoprotein particles to receptors in the liver that

mediate their clearance [26] and peroxisome proliferator-activated receptor alpha (PPAR120572) which is involved inlipid metabolism including roles in oxidation pathways theuptake and transport of fatty acids and lipoprotein synthesisroutes [27] Therefore we focused on LPL apoE and PPAR120572gene polymorphisms as candidates for genetic risk markersfor disease development in L infantum infection LPL genepolymorphisms were searched using HindIII and PVuIIrestriction enzymes known to reveal mutated genes thatcorrelate with TAG level alteration and the former also withHDL level alteration [28 29]We found that theH+H+geno-type and the H+ allele which were identified using HindIIIrestriction in the LPL gene were more frequent in infectedsymptomatic individuals and were notably associated withhigh levels of VLDL-C TAG and HDL-C The apoE gene ispresented in three main isoforms (apoE2 apoE3 and apoE4)that are coded by three common alleles (E2 E3 and E4) [26]We found that the E3E3 genotype and the E3 allele weremore frequent in infected symptomatic individuals than ininfected asymptomatic or noninfected individuals and thesefactors were associated with high levels of VLDL cholesteroland TAG We found that the LeuVal genotype and the Valallele of the L162V polymorphism in the PPAR120572 gene weremore frequent in infected symptomatic individuals than ininfected asymptomatic or noninfected individuals but withno apparent association with alterations in lipoprotein levels

We hypothesized that certain lipid fractions may playa role in the development of L infantum infection In thisstudy we found that high levels of triglycerides and VLDLand low levels of HDL favor the development of activedisease Therefore we suggest that related lipoprotein lipaseand apolipoprotein E genotypes that lead to alterations inthese lipoprotein levels as well as the presence of the PPAR120572L162V allele may be susceptibility factors and markers of VLdevelopment

2 Materials and Methods21 Study Design and Subjects The individuals who wereincluded in this study were from Teresina Piaui StateNortheastern Brazil The inclusion criteria were as followsunrelated individuals who presented with active VL (referredto as infected symptomatic) healthy individuals who hadbeen infected with L infantum as determined by a positiveanti-Leishmania (Montenegro) delayed-type hypersensitivityskin test andor the presence of anti-Leishmania antibodies(referred to as infected asymptomatic) and healthy individ-uals who had no evidence of present or past infection with Linfantum as determined by both a negative Montenegro skintest and the absence of specific antibodies The subjects andmaterials in this study were similar to those in the previousstudy by Alonso et al [30] in which possible ethnic andenvironmental biases were analyzed and deemed not to beconfounding factors

22 Ethics Statement This study was approved by the ethicscommittees of the participating institutions (Comissao deEtica em Pesquisa da Universidade Federal do Piauı andComissao de Etica para Analise de Projetos de Pesquisa da

















3 Results











HindIII

H+H+(a) 24 462 17 354 13 232 0040(a vs b)

H+Hminus(b) 26 500 27 563 34 607 0103(b vs c)


HWEdagger 0116 0138 0099

H+(d) 74 715 61 635 60 536Hminus(e) 30 288 35 365 52 464 0008(d vs e)

PvuII

P1P1(f) 12 286 13 295 16 372 0429(f vs g)

P1P2(g) 25 595 27 614 23 535 0835(g vs h)

P2P2(h) 5 119 4 91 4 93 0507(f vs h)

HWEdagger 0146 0062 0294

P1(i) 49 583 53 602 55 640P2(j) 35 417 35 398 31 360 0452(i vs j)

ApoE

E2E3(k) 3 81 4 105 2 50 0833(k vs l)

E3E3(l) 30 811 24 632 26 650 0116(l vs m)

E3E4(m) 4 108 10 263 10 250 0214(k vs m)

E2E4 0 00 0 00 1 25E4E4 0 00 0 00 1 25

E2(n) 3 41 4 53 3 37 0953(n vs o)

E3(o) 67 905 62 816 64 800 0038(o vs p)

E4(p) 4 54 10 131 13 163 0230(n vs p)

PPPAR120572

LeuLeu(q) 48 800 61 871 92 939 0022(q vs r)

LeuVal(r) 11 183 8 114 6 61ValVal 1 17 1 14 0 00

HWEdagger 0693 0247 0745

Leu(s) 107 892 130 929 190 969Val(t) 13 108 10 71 6 31 0008(s vs t)








Table3Levelsof

lipids(mgdL

)according

togeno

typesa

ndalleleso

fLPL

andof

apoE

inindividu

alse

xposed

toLeish

maniainfantum

Polymorph

isms

Age

Lipids

(mgdL

)TC

aLD

L-Ca

HDL-Ca

TAG

aVLD

L-Ca

HindIII

Genotypes

H+H+(119899=19)

226plusmn154

1508plusmn422

910plusmn44

0381plusmn56

2076plusmn827

415plusmn165

H+Hminus(119899=33)

221plusmn182

1388plusmn348

774plusmn356

419plusmn64

1579plusmn528

316plusmn105

119875value

0914

0274

0228

0037

0011

0010

Alleles

H+(119899=71)

224plusmn166

1453plusmn388

847plusmn40

3399plusmn62

1845plusmn736

369plusmn146

Hminus(119899=37)

227plusmn175

1352plusmn347

744plusmn347

425plusmn63

1545plusmn508

309plusmn101

119875value

0919

0187

0191

004

00029

0029

PvuII

Genotypes

P1P1(119899=15)

224plusmn140

246plusmn167

817plusmn371

400plusmn47

1594plusmn509

319plusmn101

P1P2(119899=27)

246plusmn167

1418plusmn387

843plusmn397

399plusmn63

1730plusmn777

346plusmn155

P2P2(119899=4)

165plusmn178

1328plusmn40

3717plusmn288

415plusmn57

2305plusmn890

460plusmn176

119875valuesect

0622

0878

0828

0878

0217

0220

Alleles

P1(119899=57)

235plusmn151

1428plusmn365

829plusmn377

400plusmn54

1658plusmn64

41658plusmn64

4P2(119899=35)

228plusmn167

1397plusmn381

814plusmn371

403plusmn61

1861plusmn

814

372plusmn162

119875value

0840

0706

0853

0793

0188

0191

apoE

Genotypes

E2E3(119899=4)

237plusmn143

1200plusmn362

657plusmn304

417plusmn78



E3E3(119899=29)

191plusmn148

1521plusmn

401

915plusmn416

392plusmn58

1669plusmn585

334plusmn116

E3E4(119899=6)

355plusmn181

1190plusmn119

657plusmn239

390plusmn30

1773plusmn1004

355plusmn199

119875valuesect

006

80069

0205

0692

0026

002

8

Alleles

E2(119899=4)

237plusmn143

1200plusmn362

657plusmn304

417plusmn78



E3(119899=68)

208plusmn155


877plusmn40

2392plusmn57

1742plusmn696

348plusmn138

E4(119899=8)

321plusmn165

1130plusmn150

595plusmn232

387plusmn26

1655plusmn877

331plusmn174

119875valuesect

0152

0029

0099

0661

0029

0031

PPAR120572

Genotypes

LeuLeu(119899=54)

267plusmn176

1379plusmn397

782plusmn388

409plusmn65

1732plusmn812

346plusmn162

LeuVa

l(119899=8)

231plusmn183

1469plusmn326

833plusmn311

403plusmn57

1661plusmn

357

334plusmn69

119875value

0589

0545

0728

0794

0809

0830

Alleles

Leu(119899=116)

265plusmn175

1385plusmn390

786plusmn381

408plusmn64

1727plusmn785

345plusmn156

Val(119899=8)

231plusmn183

1469plusmn326

833plusmn311

403plusmn57

1661plusmn

357

334plusmn69

119875value

0599

0555

0735

0798

0814

0834

Allvalues

aree

xpressed

asthem

eanplusmnSD

inindividu

alse

xposed

toLeish

maniachagasi

a valuesinmgdL

119875valueanalyzed

byStud

ent119905-te

stsect P

valueanalyzed

byANOVA

testwith

theStud

entN

ewman-K

eulspo

sttestIn

relationto

geno

typeslowast

119875lt005versus

E3E3dagger

119875lt005versus

E3E4In

relationto

alleleslowast

119875lt005versus



E2HminusHminusdidno

thaves

ufficientn

umbertobe

analyzed(119899=2)A

ge=280plusmn127T

C=1050plusmn141

LDL-C=500plusmn71H

DL-C

=475plusmn21TA

G=1265plusmn92VLD

L-C=255plusmn21E4

E4didno

thaves

ufficientn

umbertobe

analyzed(119899=1)A

ge=220T

C=950L

DL-C=410H

DL-C=380T

AG=1300VLD

L=260H

+indicates

presence

andHminusindicatesa

bsence

oftheH

indIIIrestr

ictio

nsiteP1

indicatesa

bsence

andP2

indicatesp

resenceo

fthe

PvuIIrestrictio

nsite

NoteAp

oEpo

lymorph

ismresults

insix

geno

typesranking

(E2E2

E2E3

E2E4

E3E3

E3E4

and

E4E4)

andthreea

lleles(E2

E3andE4

)Bo

ldtext


nificantd

ifferences























Acknowledgments


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
































3 Results











HindIII

H+H+(a) 24 462 17 354 13 232 0040(a vs b)

H+Hminus(b) 26 500 27 563 34 607 0103(b vs c)


HWEdagger 0116 0138 0099

H+(d) 74 715 61 635 60 536Hminus(e) 30 288 35 365 52 464 0008(d vs e)

PvuII

P1P1(f) 12 286 13 295 16 372 0429(f vs g)

P1P2(g) 25 595 27 614 23 535 0835(g vs h)

P2P2(h) 5 119 4 91 4 93 0507(f vs h)

HWEdagger 0146 0062 0294

P1(i) 49 583 53 602 55 640P2(j) 35 417 35 398 31 360 0452(i vs j)

ApoE

E2E3(k) 3 81 4 105 2 50 0833(k vs l)

E3E3(l) 30 811 24 632 26 650 0116(l vs m)

E3E4(m) 4 108 10 263 10 250 0214(k vs m)

E2E4 0 00 0 00 1 25E4E4 0 00 0 00 1 25

E2(n) 3 41 4 53 3 37 0953(n vs o)

E3(o) 67 905 62 816 64 800 0038(o vs p)

E4(p) 4 54 10 131 13 163 0230(n vs p)

PPPAR120572

LeuLeu(q) 48 800 61 871 92 939 0022(q vs r)

LeuVal(r) 11 183 8 114 6 61ValVal 1 17 1 14 0 00

HWEdagger 0693 0247 0745

Leu(s) 107 892 130 929 190 969Val(t) 13 108 10 71 6 31 0008(s vs t)








Table3Levelsof

lipids(mgdL

)according

togeno

typesa

ndalleleso

fLPL

andof

apoE

inindividu

alse

xposed

toLeish

maniainfantum

Polymorph

isms

Age

Lipids

(mgdL

)TC

aLD

L-Ca

HDL-Ca

TAG

aVLD

L-Ca

HindIII

Genotypes

H+H+(119899=19)

226plusmn154

1508plusmn422

910plusmn44

0381plusmn56

2076plusmn827

415plusmn165

H+Hminus(119899=33)

221plusmn182

1388plusmn348

774plusmn356

419plusmn64

1579plusmn528

316plusmn105

119875value

0914

0274

0228

0037

0011

0010

Alleles

H+(119899=71)

224plusmn166

1453plusmn388

847plusmn40

3399plusmn62

1845plusmn736

369plusmn146

Hminus(119899=37)

227plusmn175

1352plusmn347

744plusmn347

425plusmn63

1545plusmn508

309plusmn101

119875value

0919

0187

0191

004

00029

0029

PvuII

Genotypes

P1P1(119899=15)

224plusmn140

246plusmn167

817plusmn371

400plusmn47

1594plusmn509

319plusmn101

P1P2(119899=27)

246plusmn167

1418plusmn387

843plusmn397

399plusmn63

1730plusmn777

346plusmn155

P2P2(119899=4)

165plusmn178

1328plusmn40

3717plusmn288

415plusmn57

2305plusmn890

460plusmn176

119875valuesect

0622

0878

0828

0878

0217

0220

Alleles

P1(119899=57)

235plusmn151

1428plusmn365

829plusmn377

400plusmn54

1658plusmn64

41658plusmn64

4P2(119899=35)

228plusmn167

1397plusmn381

814plusmn371

403plusmn61

1861plusmn

814

372plusmn162

119875value

0840

0706

0853

0793

0188

0191

apoE

Genotypes

E2E3(119899=4)

237plusmn143

1200plusmn362

657plusmn304

417plusmn78



E3E3(119899=29)

191plusmn148

1521plusmn

401

915plusmn416

392plusmn58

1669plusmn585

334plusmn116

E3E4(119899=6)

355plusmn181

1190plusmn119

657plusmn239

390plusmn30

1773plusmn1004

355plusmn199

119875valuesect

006

80069

0205

0692

0026

002

8

Alleles

E2(119899=4)

237plusmn143

1200plusmn362

657plusmn304

417plusmn78



E3(119899=68)

208plusmn155


877plusmn40

2392plusmn57

1742plusmn696

348plusmn138

E4(119899=8)

321plusmn165

1130plusmn150

595plusmn232

387plusmn26

1655plusmn877

331plusmn174

119875valuesect

0152

0029

0099

0661

0029

0031

PPAR120572

Genotypes

LeuLeu(119899=54)

267plusmn176

1379plusmn397

782plusmn388

409plusmn65

1732plusmn812

346plusmn162

LeuVa

l(119899=8)

231plusmn183

1469plusmn326

833plusmn311

403plusmn57

1661plusmn

357

334plusmn69

119875value

0589

0545

0728

0794

0809

0830

Alleles

Leu(119899=116)

265plusmn175

1385plusmn390

786plusmn381

408plusmn64

1727plusmn785

345plusmn156

Val(119899=8)

231plusmn183

1469plusmn326

833plusmn311

403plusmn57

1661plusmn

357

334plusmn69

119875value

0599

0555

0735

0798

0814

0834

Allvalues

aree

xpressed

asthem

eanplusmnSD

inindividu

alse

xposed

toLeish

maniachagasi

a valuesinmgdL

119875valueanalyzed

byStud

ent119905-te

stsect P

valueanalyzed

byANOVA

testwith

theStud

entN

ewman-K

eulspo

sttestIn

relationto

geno

typeslowast

119875lt005versus

E3E3dagger

119875lt005versus

E3E4In

relationto

alleleslowast

119875lt005versus



E2HminusHminusdidno

thaves

ufficientn

umbertobe

analyzed(119899=2)A

ge=280plusmn127T

C=1050plusmn141

LDL-C=500plusmn71H

DL-C

=475plusmn21TA

G=1265plusmn92VLD

L-C=255plusmn21E4

E4didno

thaves

ufficientn

umbertobe

analyzed(119899=1)A

ge=220T

C=950L

DL-C=410H

DL-C=380T

AG=1300VLD

L=260H

+indicates

presence

andHminusindicatesa

bsence

oftheH

indIIIrestr

ictio

nsiteP1

indicatesa

bsence

andP2

indicatesp

resenceo

fthe

PvuIIrestrictio

nsite

NoteAp

oEpo

lymorph

ismresults

insix

geno

typesranking

(E2E2

E2E3

E2E4

E3E3

E3E4

and

E4E4)

andthreea

lleles(E2

E3andE4

)Bo

ldtext


nificantd

ifferences























Acknowledgments


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















HindIII

H+H+(a) 24 462 17 354 13 232 0040(a vs b)

H+Hminus(b) 26 500 27 563 34 607 0103(b vs c)


HWEdagger 0116 0138 0099

H+(d) 74 715 61 635 60 536Hminus(e) 30 288 35 365 52 464 0008(d vs e)

PvuII

P1P1(f) 12 286 13 295 16 372 0429(f vs g)

P1P2(g) 25 595 27 614 23 535 0835(g vs h)

P2P2(h) 5 119 4 91 4 93 0507(f vs h)

HWEdagger 0146 0062 0294

P1(i) 49 583 53 602 55 640P2(j) 35 417 35 398 31 360 0452(i vs j)

ApoE

E2E3(k) 3 81 4 105 2 50 0833(k vs l)

E3E3(l) 30 811 24 632 26 650 0116(l vs m)

E3E4(m) 4 108 10 263 10 250 0214(k vs m)

E2E4 0 00 0 00 1 25E4E4 0 00 0 00 1 25

E2(n) 3 41 4 53 3 37 0953(n vs o)

E3(o) 67 905 62 816 64 800 0038(o vs p)

E4(p) 4 54 10 131 13 163 0230(n vs p)

PPPAR120572

LeuLeu(q) 48 800 61 871 92 939 0022(q vs r)

LeuVal(r) 11 183 8 114 6 61ValVal 1 17 1 14 0 00

HWEdagger 0693 0247 0745

Leu(s) 107 892 130 929 190 969Val(t) 13 108 10 71 6 31 0008(s vs t)








Table3Levelsof

lipids(mgdL

)according

togeno

typesa

ndalleleso

fLPL

andof

apoE

inindividu

alse

xposed

toLeish

maniainfantum

Polymorph

isms

Age

Lipids

(mgdL

)TC

aLD

L-Ca

HDL-Ca

TAG

aVLD

L-Ca

HindIII

Genotypes

H+H+(119899=19)

226plusmn154

1508plusmn422

910plusmn44

0381plusmn56

2076plusmn827

415plusmn165

H+Hminus(119899=33)

221plusmn182

1388plusmn348

774plusmn356

419plusmn64

1579plusmn528

316plusmn105

119875value

0914

0274

0228

0037

0011

0010

Alleles

H+(119899=71)

224plusmn166

1453plusmn388

847plusmn40

3399plusmn62

1845plusmn736

369plusmn146

Hminus(119899=37)

227plusmn175

1352plusmn347

744plusmn347

425plusmn63

1545plusmn508

309plusmn101

119875value

0919

0187

0191

004

00029

0029

PvuII

Genotypes

P1P1(119899=15)

224plusmn140

246plusmn167

817plusmn371

400plusmn47

1594plusmn509

319plusmn101

P1P2(119899=27)

246plusmn167

1418plusmn387

843plusmn397

399plusmn63

1730plusmn777

346plusmn155

P2P2(119899=4)

165plusmn178

1328plusmn40

3717plusmn288

415plusmn57

2305plusmn890

460plusmn176

119875valuesect

0622

0878

0828

0878

0217

0220

Alleles

P1(119899=57)

235plusmn151

1428plusmn365

829plusmn377

400plusmn54

1658plusmn64

41658plusmn64

4P2(119899=35)

228plusmn167

1397plusmn381

814plusmn371

403plusmn61

1861plusmn

814

372plusmn162

119875value

0840

0706

0853

0793

0188

0191

apoE

Genotypes

E2E3(119899=4)

237plusmn143

1200plusmn362

657plusmn304

417plusmn78



E3E3(119899=29)

191plusmn148

1521plusmn

401

915plusmn416

392plusmn58

1669plusmn585

334plusmn116

E3E4(119899=6)

355plusmn181

1190plusmn119

657plusmn239

390plusmn30

1773plusmn1004

355plusmn199

119875valuesect

006

80069

0205

0692

0026

002

8

Alleles

E2(119899=4)

237plusmn143

1200plusmn362

657plusmn304

417plusmn78



E3(119899=68)

208plusmn155


877plusmn40

2392plusmn57

1742plusmn696

348plusmn138

E4(119899=8)

321plusmn165

1130plusmn150

595plusmn232

387plusmn26

1655plusmn877

331plusmn174

119875valuesect

0152

0029

0099

0661

0029

0031

PPAR120572

Genotypes

LeuLeu(119899=54)

267plusmn176

1379plusmn397

782plusmn388

409plusmn65

1732plusmn812

346plusmn162

LeuVa

l(119899=8)

231plusmn183

1469plusmn326

833plusmn311

403plusmn57

1661plusmn

357

334plusmn69

119875value

0589

0545

0728

0794

0809

0830

Alleles

Leu(119899=116)

265plusmn175

1385plusmn390

786plusmn381

408plusmn64

1727plusmn785

345plusmn156

Val(119899=8)

231plusmn183

1469plusmn326

833plusmn311

403plusmn57

1661plusmn

357

334plusmn69

119875value

0599

0555

0735

0798

0814

0834

Allvalues

aree

xpressed

asthem

eanplusmnSD

inindividu

alse

xposed

toLeish

maniachagasi

a valuesinmgdL

119875valueanalyzed

byStud

ent119905-te

stsect P

valueanalyzed

byANOVA

testwith

theStud

entN

ewman-K

eulspo

sttestIn

relationto

geno

typeslowast

119875lt005versus

E3E3dagger

119875lt005versus

E3E4In

relationto

alleleslowast

119875lt005versus



E2HminusHminusdidno

thaves

ufficientn

umbertobe

analyzed(119899=2)A

ge=280plusmn127T

C=1050plusmn141

LDL-C=500plusmn71H

DL-C

=475plusmn21TA

G=1265plusmn92VLD

L-C=255plusmn21E4

E4didno

thaves

ufficientn

umbertobe

analyzed(119899=1)A

ge=220T

C=950L

DL-C=410H

DL-C=380T

AG=1300VLD

L=260H

+indicates

presence

andHminusindicatesa

bsence

oftheH

indIIIrestr

ictio

nsiteP1

indicatesa

bsence

andP2

indicatesp

resenceo

fthe

PvuIIrestrictio

nsite

NoteAp

oEpo

lymorph

ismresults

insix

geno

typesranking

(E2E2

E2E3

E2E4

E3E3

E3E4

and

E4E4)

andthreea

lleles(E2

E3andE4

)Bo

ldtext


nificantd

ifferences























Acknowledgments


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table3Levelsof

lipids(mgdL

)according

togeno

typesa

ndalleleso

fLPL

andof

apoE

inindividu

alse

xposed

toLeish

maniainfantum

Polymorph

isms

Age

Lipids

(mgdL

)TC

aLD

L-Ca

HDL-Ca

TAG

aVLD

L-Ca

HindIII

Genotypes

H+H+(119899=19)

226plusmn154

1508plusmn422

910plusmn44

0381plusmn56

2076plusmn827

415plusmn165

H+Hminus(119899=33)

221plusmn182

1388plusmn348

774plusmn356

419plusmn64

1579plusmn528

316plusmn105

119875value

0914

0274

0228

0037

0011

0010

Alleles

H+(119899=71)

224plusmn166

1453plusmn388

847plusmn40

3399plusmn62

1845plusmn736

369plusmn146

Hminus(119899=37)

227plusmn175

1352plusmn347

744plusmn347

425plusmn63

1545plusmn508

309plusmn101

119875value

0919

0187

0191

004

00029

0029

PvuII

Genotypes

P1P1(119899=15)

224plusmn140

246plusmn167

817plusmn371

400plusmn47

1594plusmn509

319plusmn101

P1P2(119899=27)

246plusmn167

1418plusmn387

843plusmn397

399plusmn63

1730plusmn777

346plusmn155

P2P2(119899=4)

165plusmn178

1328plusmn40

3717plusmn288

415plusmn57

2305plusmn890

460plusmn176

119875valuesect

0622

0878

0828

0878

0217

0220

Alleles

P1(119899=57)

235plusmn151

1428plusmn365

829plusmn377

400plusmn54

1658plusmn64

41658plusmn64

4P2(119899=35)

228plusmn167

1397plusmn381

814plusmn371

403plusmn61

1861plusmn

814

372plusmn162

119875value

0840

0706

0853

0793

0188

0191

apoE

Genotypes

E2E3(119899=4)

237plusmn143

1200plusmn362

657plusmn304

417plusmn78



E3E3(119899=29)

191plusmn148

1521plusmn

401

915plusmn416

392plusmn58

1669plusmn585

334plusmn116

E3E4(119899=6)

355plusmn181

1190plusmn119

657plusmn239

390plusmn30

1773plusmn1004

355plusmn199

119875valuesect

006

80069

0205

0692

0026

002

8

Alleles

E2(119899=4)

237plusmn143

1200plusmn362

657plusmn304

417plusmn78



E3(119899=68)

208plusmn155


877plusmn40

2392plusmn57

1742plusmn696

348plusmn138

E4(119899=8)

321plusmn165

1130plusmn150

595plusmn232

387plusmn26

1655plusmn877

331plusmn174

119875valuesect

0152

0029

0099

0661

0029

0031

PPAR120572

Genotypes

LeuLeu(119899=54)

267plusmn176

1379plusmn397

782plusmn388

409plusmn65

1732plusmn812

346plusmn162

LeuVa

l(119899=8)

231plusmn183

1469plusmn326

833plusmn311

403plusmn57

1661plusmn

357

334plusmn69

119875value

0589

0545

0728

0794

0809

0830

Alleles

Leu(119899=116)

265plusmn175

1385plusmn390

786plusmn381

408plusmn64

1727plusmn785

345plusmn156

Val(119899=8)

231plusmn183

1469plusmn326

833plusmn311

403plusmn57

1661plusmn

357

334plusmn69

119875value

0599

0555

0735

0798

0814

0834

Allvalues

aree

xpressed

asthem

eanplusmnSD

inindividu

alse

xposed

toLeish

maniachagasi

a valuesinmgdL

119875valueanalyzed

byStud

ent119905-te

stsect P

valueanalyzed

byANOVA

testwith

theStud

entN

ewman-K

eulspo

sttestIn

relationto

geno

typeslowast

119875lt005versus

E3E3dagger

119875lt005versus

E3E4In

relationto

alleleslowast

119875lt005versus



E2HminusHminusdidno

thaves

ufficientn

umbertobe

analyzed(119899=2)A

ge=280plusmn127T

C=1050plusmn141

LDL-C=500plusmn71H

DL-C

=475plusmn21TA

G=1265plusmn92VLD

L-C=255plusmn21E4

E4didno

thaves

ufficientn

umbertobe

analyzed(119899=1)A

ge=220T

C=950L

DL-C=410H

DL-C=380T

AG=1300VLD

L=260H

+indicates

presence

andHminusindicatesa

bsence

oftheH

indIIIrestr

ictio

nsiteP1

indicatesa

bsence

andP2

indicatesp

resenceo

fthe

PvuIIrestrictio

nsite

NoteAp

oEpo

lymorph

ismresults

insix

geno

typesranking

(E2E2

E2E3

E2E4

E3E3

E3E4

and

E4E4)

andthreea

lleles(E2

E3andE4

)Bo

ldtext


nificantd

ifferences























Acknowledgments


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






































Acknowledgments


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article lipoprotein lipase and ppar alpha gene

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