Individual variation in levels of haptoglobin-related protein in children from Gabon.
ABSTRACT Haptoglobin related protein (Hpr) is a key component of trypanosome lytic factors (TLF), a subset of high-density lipoproteins (HDL) that form the first line of human defence against African trypanosomes. Hpr, like haptoglobin (Hp) can bind to hemoglobin (Hb) and it is the Hpr-Hb complexes which bind to these parasites allowing uptake of TLF. This unique form of innate immunity is primate-specific. To date, there have been no population studies of plasma levels of Hpr, particularly in relation to hemolysis and a high prevalence of ahaptoglobinemia as found in malaria endemic areas.
We developed a specific enzyme-linked immunosorbent assay to measure levels of plasma Hpr in Gabonese children sampled during a period of seasonal malaria transmission when acute phase responses (APR), malaria infection and associated hemolysis were prevalent. Median Hpr concentration was 0.28 mg/ml (range 0.03-1.1). This was 5-fold higher than that found in Caucasian children (0.049 mg/ml, range 0.002-0.26) with no evidence of an APR. A general linear model was used to investigate associations between Hpr levels, host polymorphisms, parasitological factors and the acute phase proteins, Hp, C-reactive protein (CRP) and albumin. Levels of Hpr were associated with Hp genotype, decreased with age and were higher in females. Hpr concentration was strongly correlated with that of Hp, but not CRP.
Individual variation in Hpr levels was related to Hp level, Hp genotype, demographics, malaria status and the APR. The strong correlations between plasma levels of Hp and Hpr suggest that they are regulated by similar mechanisms. These population-based observations indicate that a more dynamic view of the relative roles of Hpr and Hpr-Hb complexes needs to be considered in understanding innate immunity to African trypanosomes and possibly other pathogens including the newly discovered Plasmodium spp of humans and primates.
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ABSTRACT: Human innate immunity to non-pathogenic species of African trypanosomes is provided by human high density lipoprotein (HDL) particles. Here we show that native human HDLs containing haptoglobin-related protein (Hpr), apolipoprotein L-I (apoL-I) and apolipoprotein A-I (apoA-I) are the principle antimicrobial molecules providing protection from trypanosome infection. Other HDL subclasses containing either apoA-I and apoL-I or apoA-I and Hpr have reduced trypanolytic activity, whereas HDL subclasses lacking apoL-I and Hpr are non-toxic to trypanosomes. Highly purified, lipid-free Hpr and apoL-I were both toxic to Trypanosoma brucei brucei but with specific activities at least 500-fold less than those of native HDLs, suggesting that association of these apolipoproteins within the HDL particle was necessary for optimal cytotoxicity. These studies show that HDLs can serve as platforms for the assembly of multiple synergistic proteins and that these assemblies may play a critical role in the evolution of primate-specific innate immunity to trypanosome infection.Journal of Biological Chemistry 10/2005; 280(38):32578-85. · 4.60 Impact Factor
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ABSTRACT: Most clinical conditions are accompanied by corresponding changes in serum levels of some, if not all, of the acute phase proteins. While conditions that affect the acute phase proteins are usually inflammatory in nature, non-inflammatory conditions also can cause changes (e.g., malnutrition, some malignancies without secondary inflammation, and genetic polymorphism). Only after the confounding effects of non-inflammatory conditions are taken into account can these measurements be used to detect and stage the inflammatory process and to evaluate the impact of treatment. In this third article in a series, reference ranges for serum levels for three of the acute phase proteins that increase during inflammation are examined: alpha1-acid glycoprotein (orosomucoid), alpha-antitrypsin, and haptoglobin. The study is based on a cohort of 55,199 Caucasian individuals from northern New England, tested in our laboratory between 1994 and 1999. Measurements were standardized against CRM 470 (RPPHS) and analyzed using a previously described statistical approach. Individuals with unequivocal laboratory evidence of inflammation (C-reactive protein of 10 mg/l or higher) were excluded. Levels of a,-acid glycoprotein changed little during life and between the sexes. Levels of alpha1-antitrypsin varied somewhat by age, rising slightly beyond age 55; males followed a pattern similar to that for females. For this protein, it was necessary to apply two equations to describe the lower levels associated with certain phenotypes. Haptoglobin levels fell significantly during the first decade of life for both males and females and climbed thereafter. Males and females displayed a similar pattern. When values were expressed as multiples of the age- and gender-specific median levels, the resulting distributions fitted a log-Gaussian distribution well over a broad range. When patient data are normalized in this manner, the distribution parameters can be used to assign a centile corresponding to an individual's measurement, thus simplifying interpretation.Journal of Clinical Laboratory Analysis 02/2000; 14(6):284-92. · 1.14 Impact Factor
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ABSTRACT: Thirty-three kilobase pairs (kb) of human DNA containing the haptoglobin (Hp) and haptoglobin-related (Hpr) gene pair were cloned, and the nucleotide sequence of 21-kb DNA was determined. The two genes are closely linked, with Hpr being 2.2 kb downstream of Hp. Six hundred nucleotides of DNA occur between the two genes that are not found either 5' to the Hp gene or 3' to the Hpr gene. After the duplication event, the first intron of the Hpr gene acquired a 9-kb insert consisting mainly of a retrovirus-like element with a potential primer-binding site homologous to a mouse isoleucine tRNA. The element forms a repeated family in the human genome that I name RTVL-I (retrovirus-like element-isoleucine). In the coding region of the Hpr gene, there are no frameshift or nonsense mutations and its exon-intron splicing sites, 5' flanking and 3' flanking sequences do not show any obvious defects. There are 28 amino acid differences between the decoded amino acid sequences of the Hpr and Hp genes. Sixteen of these differences occur in the hpr beta chain, and all appear to be located on the surface of the molecule in places not thought to be involved in the hemoglobin binding function of haptoglobin. The structure of the Hpr gene suggests that the gene may be expressed and give rise to a functional product.Journal of Biological Chemistry 07/1985; 260(11):6698-709. · 4.60 Impact Factor
Individual Variation in Levels of Haptoglobin-Related
Protein in Children from Gabon
Heather J. Imrie1.¤a, Freya J. I. Fowkes1,2.¤b, Florence Migot-Nabias3,4, Adrian J. F. Luty3,4,
Philippe Deloron3,4, Stephen L. Hajduk5, Karen P. Day2*
1Peter Medawar Building for Pathogen Research and Department of Zoology, University of Oxford, Oxford, United Kingdom, 2Department of Microbiology, Division of
Parasitology, New York University School of Medicine, New York, New York, United States of America, 3Institut de Recherche pour le De ´veloppement, UMR 216 Me `re et
Enfant Face aux Infections Tropicales, Paris, France, 4Faculte ´ de Pharmacie, Universite ´ Paris Descartes, Sorbonne Paris Cite ´, France, 5Department of Biochemistry and
Molecular Biology, University of Georgia, Athens, Georgia, United States of America
Background: Haptoglobin related protein (Hpr) is a key component of trypanosome lytic factors (TLF), a subset of high-
density lipoproteins (HDL) that form the first line of human defence against African trypanosomes. Hpr, like haptoglobin
(Hp) can bind to hemoglobin (Hb) and it is the Hpr-Hb complexes which bind to these parasites allowing uptake of TLF. This
unique form of innate immunity is primate-specific. To date, there have been no population studies of plasma levels of Hpr,
particularly in relation to hemolysis and a high prevalence of ahaptoglobinemia as found in malaria endemic areas.
Methods and Principal Findings: We developed a specific enzyme-linked immunosorbent assay to measure levels of
plasma Hpr in Gabonese children sampled during a period of seasonal malaria transmission when acute phase responses
(APR), malaria infection and associated hemolysis were prevalent. Median Hpr concentration was 0.28 mg/ml (range 0.03–
1.1). This was 5-fold higher than that found in Caucasian children (0.049 mg/ml, range 0.002–0.26) with no evidence of an
APR. A general linear model was used to investigate associations between Hpr levels, host polymorphisms, parasitological
factors and the acute phase proteins, Hp, C-reactive protein (CRP) and albumin. Levels of Hpr were associated with Hp
genotype, decreased with age and were higher in females. Hpr concentration was strongly correlated with that of Hp, but
Conclusions/Significance: Individual variation in Hpr levels was related to Hp level, Hp genotype, demographics, malaria
status and the APR. The strong correlations between plasma levels of Hp and Hpr suggest that they are regulated by similar
mechanisms. These population-based observations indicate that a more dynamic view of the relative roles of Hpr and Hpr-
Hb complexes needs to be considered in understanding innate immunity to African trypanosomes and possibly other
pathogens including the newly discovered Plasmodium spp of humans and primates.
Citation: Imrie HJ, Fowkes FJI, Migot-Nabias F, Luty AJF, Deloron P, et al. (2012) Individual Variation in Levels of Haptoglobin-Related Protein in Children from
Gabon. PLoS ONE 7(11): e49816. doi:10.1371/journal.pone.0049816
Editor: Kevin K. A. Tetteh, London School of Hygiene and Tropical Medicine, United Kingdom
Received June 18, 2012; Accepted October 17, 2012; Published November 20, 2012
Copyright: ? 2012 Imrie et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was funded by the European Community (E.C. Grant No. IC18-CT98-0359) and the Wellcome Trust. Professor Day was supported by
a Research Leave Fellowship from the Wellcome Trust (047069/Z/96/Z). Dr. Imrie and Dr. Fowkes were supported by Programme Grant funding awarded to
Professor Day from the Wellcome Trust (041354). The studies on TLF in the Hajduk laboratory were supported by a grant from the National Institutes of Health
(AI039099). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: Karen.Day@nyumc.org
¤a Current address: Nottingham Trent University, Nottingham, United Kingdom
¤b Current address: Centre for Population Health, Burnet Institute, Melbourne, Australia
. These authors contributed equally to this work.
Haptoglobin-related protein (Hpr) is found within trypanosome
lytic factor (TLF), the component of human plasma (and that of
some old world primates) which kills T. b. brucei . Thus
T. b. brucei is not infective for humans, although it causes a chronic
wasting disease, nagana, in cattle [1,2]. TLF is present in plasma
in two forms. TLF-1 occurs within the high-density lipoprotein
(HDL) fraction of plasma and, like other HDL molecules, its
structure is complex, comprising a cholesterol ester core and an
outer layer of phospholipid, neutral lipid and the integrated
lipoproteins, Hpr and apolipoprotein L-I (apoL-I) . A pro-
portion of Hpr and apoL-I, together with apoA-1 and IgM is also
present in lipid-poor complexes, namely TLF-2 .
Hpr is found predominantly as a 45 kD heterodimer consisting
of one a- and one b-chain covalently bound together to form (ab)-
dimers . A small proportion is found as an (a2b2)-tetramer .
The sequence of Hpr is .90% identical with that of the acute
phase protein haptoglobin (Hp) . Hp is comprised of a- and b-
chains forming tetramers (phenotype Hp1-1) and polymers
(phenotypes Hp2-1 and Hp2-2) . The Hp a-chain is encoded
by two co-dominant alleles, Hp1(of which there are two suballeles;
Hp1Sand Hp1F), and Hp2[7,8]. The a2-chain is almost twice as
long as the a1-chain, having evolved via a duplication event .
PLOS ONE | www.plosone.org1November 2012 | Volume 7 | Issue 11 | e49816
Phenotypic variation between Hp 1F and Hp1S, where F indicates
fast and S slow migration during electrophoresis, is dependent
upon minor amino acid differences .The primary function of
Hp is to bind hemoglobin (Hb), the b-chain of Hp binding to one
Hb (ab) subunit . This enables the safe removal of Hb via
CD163-mediated internalisation in macrophages and results in
rapid clearance of Hp [5,10]. T. b. brucei also takes up Hp-Hb
complexes, but via an unrelated surface receptor (TbHpHbR),
which facilitates endocytosis and lysosomal localization, the heme
moiety being liberated and incorporated into hemoproteins .
The Hb-binding region is conserved within Hpr facilitating high
affinity Hb binding (Kd.1029M) [5,11]. The T. b. brucei surface
receptor binds Hpr-Hb complexes within TLF, thus internalizing
the toxic HDL leading to lysosomal membrane disruption and
parasite lysis [12,13]. In contrast to Hp-Hb, Hpr-Hb does not bind
to mammalian CD163 and the mechanism whereby Hpr-Hb
complexes are cleared from plasma, in the absence of trypano-
somes, is unknown .
Hpr and Hp are primarily expressed in the liver, however, Hpr
transcript levels were determined to be only 6% of those of Hp due
to the presence of a retroviral-like element in intron 1 . Levels
of Hpr have been found to be 10-fold lower than those of Hp
(0.03–0.04 mg/ml and 0.27–1.39 mg/ml respectively) [15,16]. Hp
is an acute phase protein i.e. one whose concentration changes by
at least 25% during inflammatory disorders . During an acute
phase response (APR), interleukin-6 (IL-6) results in up-regulation
of Hp production, levels increasing 2-4-fold . The Hp and Hpr
genes are only 2.2 kbp apart and have similar promoter regions
. IL-6 responsive elements (IL-6RE) have been identified in
both the Hp and Hpr promoters . Whether Hpr levels change
in response to IL-6, particularly during an APR, is yet to be
Hpr levels have not been studied at a population level and the
association between Hpr, Hp and the APR is unknown,
particularly in relation to malaria-induced hemolysis. Consequent-
ly, we developed a specific ELISA that distinguishes between Hp
and Hpr. Hpr levels were measured, together with Hp and the
acute phase proteins, C-reactive protein (CRP) and albumin, in
a large cohort of children living in a malaria-endemic area of
Gabon. We investigated the association of Hpr levels in relation to
these acute phase proteins as well as Hp genotype, demographics
and malariometric indices. As a comparison, levels of Hpr were
also measured in healthy children from the United Kingdom who
do not experience such a chronic burden of infectious disease and
were not exposed to malaria.
Details of the Gabon study design, population and laboratory
methods have been published previously [20,21]. Briefly, the study
was undertaken in two villages (Dienga and Bakoumba) in South-
East Gabon near the Congo border. Malaria is highly endemic
with peaks of transmission at the end of the rainy seasons from
September-December and in March-June. . The samples were
collected May-June when malaria transmission is intense. There
are no tsetse flies in Dienga and Bakoumba, but trypanosomiasis is
present in cattle and man in other areas of Gabon [23,24]. A cross-
sectional survey was conducted in May 2000 in a cohort of 741
asymptomatic children aged 1–12 years. Plasma samples were
available for determination of Hpr levels in 553 children.
Ethical clearance was obtained from the ‘‘Comite ´ d’Ethique
Pour La Recherche En Me ´decine Humaine’’ as well as the
Ministry of Public Health and the Governor of the Province. After
consultation with the villages’ authorities regarding an acceptable
procedure, a village-wide information meeting was held. In
Bakoumba, a public convocation was addressed to all inhabitants
where those present included medical authorities (Director of the
Hospital, Head of the medical laboratory), local authorities (Sous-
Pre ´fet, school headmaster, chief of SODEPAL: Socie ´te ´ D’Exploi-
tation du Parc de la Le ´ke ´di and traditional leaders) as well as
leaders of the project at CIRMF (Centre International de
Recherches Me ´dicales de Franceville). The research project was
presented orally and discussed. Additional information was
provided through the director of SODEPAL (Jean Bourgeais)
and the medical staff of the hospital. In Dienga, information was
provided orally by the staff of the health center (a nurse and
a laboratory worker recruited among villagers). People understood
that samples would be used for research on malaria, in relation to
red blood cell polymorphisms. The verbal consent of parents/
guardians was sought and was given, after allowing time for
appropriate consultation, via the representatives of parent
associations. Parents that were unwilling for their children to
participate were identified and their children subsequently
excluded, without prejudice, from study surveys. The Comite ´
d’Ethique Pour La Recherche En Me ´decine Humaine was
informed that an individual verbal consent was obtained and
agreed to this, literacy levels being low. Ethical approval was also
granted by the Institutional Review Board of the New York
University School of Medicine.
Samples taken from 3–4 year old Caucasian children allowed
comparison of Hpr levels in two different populations. These
serum samples were obtained from stored serum previously
collected from healthy children as part of studies conducted by
the Oxford Vaccine Group. The use of such samples for further
studies was approved by the Oxford Clinical Research Ethics
Committee as C02.013 ‘Use of stored serum samples held by the
Oxford Vaccine Group to further vaccine related research’.
Demographic, Parasitological and Genetic Variables
Age and sex were recorded for each child. Parasite densities of
Plasmodium species were counted on Giemsa stained thick blood
smears, and were recorded as the number of parasites/ml of blood,
assuming an average leucocyte count of 8000/ml . Hp
genotype (including 1F and 1S subtypes) was determined by
PCR, using the method of Yano et al (1998) with modifications
Plasma Protein Levels
a capture enzyme-linked immunosorbent assay (ELISA) which
was developed using a specific anti-Hpr monoclonal antibody and
polyclonal anti-Hp which cross-reacts with Hpr . The
monoclonal antibody, SF14.11, was prepared by hyper-immuni-
zation of mice with purified TLF-1 and was identified by screening
hybridoma culture for neutralization of TLF killing of T. b. brucei
. This antibody is specific for Hpr and reacts with native
human Hpr in immunoprecipitation and non-reducing western
blot assays. Reactivity of the antibody with Hpr is abolished by
reduction with either dithiothreitol or b-mercaptoethanol. On
western blots the SF14.11 reacts predominantly with the 45 kDa
Hpr heterodimer consisting of one a- and one b-chain and weakly
with the 92 kDa Hpr heterotetramer containing two a- and two b-
chains . It was found to be necessary to purify the antibody
Hpr was detected using
Levels of HPR in Gabon
PLOS ONE | www.plosone.org2November 2012 | Volume 7 | Issue 11 | e49816
from ascites fluid by passing over a protein G column, otherwise at
all concentrations used, very slow development and low optical
density (OD) values were obtained with all ELISA methods
Initially various concentrations of polyclonal anti-Hp were used
as capture antibody, followed by varying concentrations of pooled
plasma (containing Hp and Hpr) (neat to 1 in 1,000,000 dilutions
in 3%BSA/PBS) or purified Hp1-1 (Sigma) (100 mg/ml to
0.1 ng/ml), then anti-Hpr (diluted to 10 mg/ml PBS) and finally
anti-mouse-conjugate. If polyclonal anti-Hp was used as capture
antibody, the ELISA was non-specific, as cross-reaction with Hp
occurred. Attempts to remove Hp by means of Hb bound to 1 mm
aliphatic amine latex beads (Interfacial Dynamics Corporation,
USA) were unsuccessful, only removing just over half of Hp.
Alternative methods were attempted using anti-apolipoprotein A-I
as capture antibody and anti-Hpr as detector antibody and vice
versa but no significant OD values were obtained.
The method which was found to be sensitive and relatively
specific was to use the monoclonal anti-Hpr as capture antibody
and the polyclonal anti-Hp as detection antibody. The anti-Hpr
antibody was diluted in PBS (10 mg/ml) and 100 ml aliquots used
to coat 96-well plates (Nunc, Hereford, UK) overnight at 4uC.
Following washes (PBS/0.05% Tween 20) and blocking (3% BSA
in PBS), the samples, diluted 1:1000 in 3%BSA/PBS, were added
and the plate incubated at room temperature for 60 minutes.
Detection was by rabbit anti-human Hp (Sigma, Poole, UK),
diluted to 3.33 mg/mL, followed by goat anti-rabbit IgG alkaline
phosphatase conjugate (Sigma, Poole, UK), diluted to 46 ng/mL.
The substrate was p-nitrophenyl phosphate (Sigma, Poole, UK) at
1 mg/ml in 10% diethanolamine, containing 0.5 mM MgCl2,
pH 9.8. The reaction was allowed to proceed for 1 hour and the
OD at 405 nm was measured. The ELISA was calibrated using
purified Hpr as a standard prepared by detergent solubilization of
human HDL followed by affinity chromatography using a matrix
of anti-Hpr (SF14.11) coupled to Affi-gel 10 beads (Bio-Rad) .
The total protein in the preparation was measured by the Lowry
method and the proportion corresponding to Hpr was determined
by densitometry; the component proteins in the preparation were
separated on SDS-PAGE gel, stained with Coomassie blue and the
proportion within each band was measured using a Fluor-S
densitometer (Bio-Rad, Hemel Hempstead, UK). It was found that
56% by weight of the total protein corresponded to the Hpr ab-
dimer and 24% to the tetramer, the remainder being albumin.
Thus, 80% of the total protein was Hpr, which corresponded to
218 mg/ml. Further dilutions of this preparation were used to
make standards for the ELISA.
The reproducibility of the ELISA was investigated by taking 10
plasma samples from random UK blood-donors (courtesy of John
Radcliff Infirmary) and running the samples on the same and
different ELISA plates. The intra-plate coefficient of variation
(CV) was found to be 4.2%. The inter-plate CV was 21%, but for
the majority of samples the CV was ,15% with only a few
samples with low Hpr concentrations giving higher CV values.
The similarity in structure between Hpr and Hp might have
caused cross-reactivity within the ELISA, particularly since it has
previously been shown that 0.9% of plasma Hp associates with
HDL . This will be a constant factor that will be detected by
the reporter anti-Hp polyclonal antibody. Nevertheless, an ELISA
that used monoclonal anti-Hpr as capture antibody and rabbit
polyclonal anti-Hp as detector antibody was specific for capturing
Hpr only. This assay could detect a protein in plasma and in
a purified HDL fraction but not in a preparation of purified Hp1-1
(at protein levels #1 mg/ml) (Figure 1). Also, addition of pooled
Hp up to 4 mg/ml to plasma samples did not affect results.
; 96-well plates were coated with rabbit anti-human Hp (H-
8636, Sigma) as capture antibody. Samples were diluted 1 in
10,000 in 3% BSA/PBS and Hp standards (100 ng/ml, 80 ng/ml,
60 ng/ml, 40 ng/ml, 20 ng/ml and 0 ng/ml) prepared using
pooled Hp (Sigma) in 3% BSA/PBS. Detection was by mono-
clonal anti-Hp (Sigma; H-6395) followed by sheep anti-mouse IgG
alkaline phosphatase conjugate (Sigma; A3563). Substrate (p-
nitrophenyl phosphate) was added and OD read at 405 nm. A
standard curve was drawn to determine levels of Hp .
Hypohaptoglobinemia is defined as levels ,0.18 mg/ml .
Levels of CRP were determined using
a commercial ELISA kit (American Laboratory Products Com-
pany, Windham, NH, USA). The cut off value to define an APR is
$10 mg/ml .
Albumin concentrations were determined by mix-
ing 280 ml bromocresol green reagent (Abbott Laboratories Ltd,
UK) and 2.4 ml sample in duplicate and reading optical density at
630 nm. Blanks (water), standards (25, 57 g/l) and a quality
control (29 g/l) were also prepared. The mean blank value was
subtracted from all mean values. A graph was drawn using the two
calibration points (straight line up to 60 g/l) and sample values
read off. Hypoalbuminemia is defined as levels ,35 mg/ml .
Plasma Hp levels were determined by ELISA
Differences between categorical variables were assessed using
chi-square tests. Differences between categorical variables and
continuous variables were assessed using Mann Whitney U or
Kruskal-Wallis for non-normal data and t-tests for normal data.
Non-parametric analyses between continuous variables were
assessed by Spearman correlations.
A general linear model was used to examine the effect of
variables of interest on Hpr levels in Gabon. Since Hpr, Hp and
CRP levels showed heteroscedasticity, the levels were log
transformed before analysis. Variables of interest included CRP
Figure 1. Specificity of Haptoglobin-related protein (Hpr)
ELISA. To investigate whether the Hpr ELISA was specific for Hpr
rather than Hp, it was performed using various dilutions pooled Hp
(Sigma) (stock concentration=100 mg/ml). To determine whether the
ELISA detected Hpr in HDL, it was performed using various dilutions of
a purified HDL fraction (stock concentration=22.5 mg/ml total protein
of which 300 mg/ml was found to be Hpr as determined using the Hpr
standard). To show that the ELISA could detect Hpr in plasma, it was
performed with dilutions of pooled sera (Courtesy of John Radcliffe
blood bank). All results are means of duplicate readings and are minus
readings from blank wells.
Levels of HPR in Gabon
PLOS ONE | www.plosone.org3 November 2012 | Volume 7 | Issue 11 | e49816
concentration (continuous and 2 categories), Hp (continuous and 2
categories), albumin (continuous and 2 categories), Plasmodium
(log10(Plasmodium+1) and 0/1), Hp genotype (3 and 6 categories),
age (continuous) and sex. Variables significantly associated with
Hpr in the univariate analysis were added to a multivariate model
and variables with P.0.05 removed in a stepwise fashion. 539
children contributed to the final model (missing values, n=14).
SPSS for Windows version 12.0 (SPSS, Inc., Chicago, IL) was used
for data analysis.
Hpr Levels in Gabonese and Caucasian Children
Levels of Hpr and Hp were determined by ELISA in children
from Gabon (aged 1–12 years, 53% male) and Caucasian children
(aged 3–4, 47% male) from the United Kingdom. The median
concentration of Hpr in Gabon was 0.28 mg/ml which was
significantly higher than healthy Caucasian controls (0.049 mg/
ml), overall and with age-specific comparisons (Table 1,
P,0.0001). In Gabon, Hpr levels decreased with increasing age
group and Hpr levels were higher in females compared to males
(Table 1, P,0.001). No differences in Hpr levels with respect to
gender were observed in Caucasians (Table 1, P=0.61). Hp levels
were higher in Caucasian (median [inter-quartile range] 0.3 mg/
ml [0.19–0.48]) compared to Gabonese children (0.13 mg/ml
[0.03–0.44]), but this was not statistically significant (P=0.4). In
both Caucasian and Gabonese children Hpr was positively
correlated with Hp (rs=0.51, P,0.001 and rs=0.29, P,0.001,
respectively) (Figure 2).
Association between Hpr, Acute Phase Proteins,
Parasitaemia and Haptoglobin Polymorphisms in
Children from Gabon
Table 2 summarises Hpr, acute phase proteins and parasito-
logical findings of the study children as well as frequencies of
haptoglobin polymorphisms. In addition to high Hpr levels, CRP
levels were also high in Gabonese children, greater than 80% of
whom were defined as having an APR (CRP.10 mg/ml) related
to malaria parasitemia. Of note, Hpr levels increased five-fold in
Gabonese children with an APR compared with UK children or
Gabonese children without an APR. Hp levels were low in this
population where 53.5% of children were defined as having
hypohaptoglobinemia (Hp,0.18 mg/mL), presumably due to
malaria-induced hemolysis .
We wished to identify variables that were associated with Hpr
levels at the population level. Variables chosen for investigation
were APR proteins (Hp, CRP and albumin), Plasmodium spp
(prevalence and density), demographics (age and sex) as well as Hp
genotype and subtype. Univariate analyses of variables of interest
were performed on log (Hpr) levels. Hpr was significantly
positively associated with Hp and albumin (P#0.004) but not
CRP (P=0.96) (Table 3). Hpr levels decreased with age (P=0.001)
and parasitemia (P=0.003). Hpr was also associated with sex and
Hp genotype (in the order1–1.2–1.2–2) and Hp subtype (overall
P#0.002) (Table 3).
A multivariate general linear model was then constructed to
examine the effect of multiple variables on Hpr levels. The
resulting fitted model predicts the median Hpr level for the
population controlling for relevant variables. Variables chosen for
the final model included age, sex, Hp genotype and Hp
concentration. The median Hpr level in the population, after
controlling for relevant variables, was 0.27 mg/ml (95% CI 0.25,
0.29). Hpr levels decreased with age (b=20.04, 95% CI 0.06,
20.02, P,0.0001) and females had higher Hpr levels (0.29 mg/ml
95% CI 0.27, 0.32) compared to males (0.25 mg/ml 95% CI 0.23,
0.27, P=0.004). Hp levels were positively associated with Hpr
levels (b=0.1, 95% CI 0.07, 0.14, P,0.0001). Hpr levels also
varied according to Hp1Fand Hp1Salleles (overall P=0.0002).
Children with the Hp1-1 genotype, 1S-1S had lower Hpr levels
(0.2 mg/ml, 95% CI 0.15, 0.26) than children of 1S-1F (0.28 mg/
ml, 95% CI 0.24, 0.33, P=0.09) and 1F-1F genotype (0.29 mg/
ml, 95% CI 0.24, 0.36, P=0.018). There was no significant
difference in Hpr levels between 2-1F (0.26 mg/ml, 95% CI 0.23,
0.3) and 2-1S genotypes (0.24 mg/ml, 95% CI 0.21, 0.29,
P=0.55). Hpr levels in Hp2-2 were 0.21 mg/ml (95% CI 0.17,
0.24) which were significantly lower than 1F-1F, 1F-1S, and 2-1F
Figure 2. Haptoglobin related protein levels are correlated
with haptoglobin levels in A) Gabonese and B) Caucasian
children. Scatterplot of raw Hpr values (mg/ml) plotted against raw Hp
values (mg/ml). Associations were assessed by Spearmans rho. Hpr was
positively correlated with Hp in A) Gabonese children (rs=0.29,
P,0.001), and B) Caucasian children (rs=0.51, P,0.001).
Levels of HPR in Gabon
PLOS ONE | www.plosone.org4November 2012 | Volume 7 | Issue 11 | e49816
Data shown represent the first large-scale population study of
Hpr levels in humans. Previous laboratory investigations, using
a small number of samples, reported lower Hpr levels in the range
of 0.02–0.05 mg/ml [15,27]. This was similar to median levels
found in our Caucasian controls (0.049 mg/mL) but significantly
lower than those of the Gabonese children (0.28 mg/ml) described
in this study. Several explanations can be put forward for these
higher levels in Gabonese children. These include host genetics
and prevalence of an APR. It is possible that an increase in gene
copy number may contribute to higher plasma levels of Hpr in
Gabonese compared to Caucasian children. Up to 6 copies of the
Hpr gene have been found in the chromosomes of African-
Americans, whereas only one copy was found in Caucasian-
Americans . The low frequency of increased Hpr gene copy
number in African-Americans genotyped (28%) suggests that it is
unlikely to account for the relatively high proportion of children
with levels of Hpr.0.1 mg/ml (91%) found in this Gabonese
study population. The high prevalence of an APR due to malaria
infection seems more likely to be the cause of elevated median
concentrations, although considerable individual variation in levels
was observed, as discussed below.
Hpr levels were found to decrease with age in the Gabonese
children. This may be dependent upon intrinsic factors related to
the age of the host. It is interesting to note that some acute phase
proteins show age-related profiles, e.g. levels of Hp decrease with
age, whereas others are unrelated to age, e.g. CRP [33,34]. Levels
of Hpr were higher in females than in males. This finding may be
related to metabolism of HDL. It has been shown in a cohort of
10–15 year old males that testosterone decreased levels of HDL-
cholesterol and apolipoprotein A-I, whereas estradiol increased
levels of HDL-cholesterol . Hpr is found circulating in HDL
and thus changes in HDL levels will result in changes in Hpr
levels. Furthermore, there is a marked decrease in plasma levels of
HDL during infection and inflammation .
Univariate analysis showed that Hpr was correlated with the
acute phase protein Hp, but not CRP or albumin in Gabonese
children. Hp levels are determined by several factors, including IL-
6-dependent APR, Hp genotype, parasite density and hemolysis
[7,21]. This correlation between Hpr and Hp would suggest that
the production and/or clearance of both these molecules may be
under the influence of the same mechanisms. The positive
correlations in Hpr and Hp levels in Gabonese children, the
majority of whom are undergoing an APR, leads us to suggest that
Hpr may also be an APP. Whilst longitudinal analyses within
individuals will be required to prove that this is the case, it is
plausible that during an APR both Hp and Hpr may be
upregulated simultaneously as IL-6 responsive elements (IL-6RE)
have been identified in both the Hp and Hpr promoters .
Indeed if Hpr is an acute phase protein, it is a moderate one, i.e.
a protein showing a 1- to 10-fold increase during an APR as there
was a positive correlation with Hp but no direct correlation
between Hpr and CRP levels . This is not unexpected;
although the CRP gene also contains IL-6RE ,the relative
kinetics of acute phase proteins differ during an APR, IL-6 and IL-
1b acting synergistically to induce promotion of CRP production
. Thus CRP levels rapidly increase several hundred-fold and
fall rapidly whereas Hp levels rise 2-4-fold and persist .
Albumin is a negative acute phase protein, i.e. production is
reduced in an APR .
Hpr levels were inversely correlated with parasitemia. Hp levels
also decrease with increasing parasite density due to intravascular
hemolysis and subsequent clearance of Hb-Hp complexes .
Thus, levels of Hp and Hpr may be correlated due to similar
clearance patterns. In support of this possible conclusion, we have
also shown that Hpr correlates with Hp in a malaria endemic
region of Papua New Guinea, in the absence of an APR (Imrie H,
unpublished observations). Whilst Hpr is able to bind Hb, in
contrast to Hp-Hb, Hpr-Hb does not promote any high-affinity
binding to the scavenger receptor CD163, which clears Hp-Hb
Table 1. Hpr levels in Gabonese and Caucasian children.
nHpr mg/mlnHpr mg/ml
Overall563 0.28 [0.17–0.46]360.049 [0.041–
1–4173 0.36 [0.21–0.5]360.049 [0.041–
5–9318 0.26 [0.17–0.44]
10–1461 0.22 [0.15–0.34]
Female259 0.32 [0.18–0.5]190.054 [0.041–0.08] ,0.001
Male294 0.25 [0.16–0.4]170.047 [0.041–
Values represent median [interquartile range]. P-values represent comparisons
between Caucasians and Gabonese children.
Table 2. Acute phase protein levels, parasitaemia and
frequency of haptoglobin polymorphisms in Gabonese
C-reactive protein (mg/mL)
C-reactive protein (.10 mg/mL)1
Albumin (mg/mL) 40.61(11.5)
Plasmodium prevalence296 (53.6)
Continuous data are shown as median value [inter-quartile range] or mean
(standard deviation) and categorical data are n (%). Note for C-reactive protein,
albumin and plasmodium prevalence n=550.
1C-reactive protein .10 mg/mL was the cut-off used to define an acute phase
2The cut off value for hypohaptoglobinaemia is ,0.18 mg/mL .
3The cut off value for low albumin is ,35 mg/mL .
4The frequency of species in this population has been published previously .
Levels of HPR in Gabon
PLOS ONE | www.plosone.org5November 2012 | Volume 7 | Issue 11 | e49816
complexes from the circulation . Nevertheless, the mechanism
of clearance of Hpr-Hb may be related to the clearance of Hp-Hb
complexes. It is usual for some plasma Hp (0.9%) to be associated
with HDL particles  and thus we speculate that when HDL
associated Hp binds to Hb, some HDL containing Hpr may be
cleared concurrently. Alternatively other scavenger receptors may
also exist which bind to both Hp-Hb and Hpr-Hb.
Hpr levels were also associated with Hp genotype; Hp2-2
individuals had significantly lower levels of Hpr compared to
Hp1-1 and Hp 2-1 individuals. This may be related to the
differential affinity of Hp genotypes for binding Hb and subsequent
patterns of clearance. Whilst the affinity of the higher molecular
weight polymers for Hb is generally lower than that of Hp1-1, their
plasma levels are lower, reflecting the clearance of a greater
molecular weight per molecule. Thus levels are in the phenotype
order: Hp1-1.Hp2-1.Hp2-2 [9,41]. Hpr concentration was also
related to Hp subtype, with Hp1S-1S having lower levels of Hpr
compared to 1F-1F. Functional differences between the Hp sub-
phenotypes are unknown and we have reported that subtype (1S or
1F) does not affect plasma concentration of Hp in these children
Our results show that levels of Hpr vary between and within
populations from the United Kingdom and Gabon. Since all
humans are refractory to infection with T. b. brucei, there can be no
trypanolytic advantage in having higher levels in endemic areas.
The closely-related Hp is a multifunctional protein (e.g. antibody-
like properties, immunomodulation, iron metabolism) .We
speculate that Hpr likewise has more than one role, possibly in
innate resistance to other pathogens. Hp levels modulate TLF
activity: low Hp levels (due to acute intravascular hemolysis) result
in increased TLF activity in vitro (10–40 fold), whereas, high levels
of Hp suppress both the lytic capacity of TLF in vitro and the
ability to clear trypanosomes in vivo in a mouse model . Thus
low Hp levels, as occur in malaria, might also increase other
activities of Hpr. We speculate that Hpr may have a role in
resistance to malaria. It has been suggested that the binding of Hp-
Hb to CD163 elicits IL-6 and IL-10 secretion. It may be that Hpr-
Hb complexes also have immunomodulatory effects.
Our population-based observations indicate that a more
dynamic view of the relative roles of Hpr and Hpr-Hb complexes
needs to be considered in understanding innate immunity to
African trypanosomes and possibly other pathogens that cause
ahaptoglobinemia, such as the newly discovered Plasmodium spp of
humans and primates .
In vivo population data presented here show individual variation
in Hpr and Hp levels caused by complex dynamics of Hpr and Hp
levels in children living under the burden of malaria infection in
African settings with consequences for the ability to control
Caucasian samples were kindly provided by AJ Pollard and D Kelly,
Oxford Vaccine Group and the Oxford Biomedical Research Centre,
Department of Paediatrics, University of Oxford.
Conceived and designed the experiments: HI KD. Performed the
experiments: HI FM-N. Analyzed the data: FF HI. Contributed
reagents/materials/analysis tools: PD AL SH KD. Wrote the paper: HI
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