Antibody responses to P. falciparum Apical Membrane Antigen 1(AMA-1) in relation to haemoglobin S (HbS), HbC, G6PD and ABO blood groups among Fulani and Masaleit living in Western Sudan.

Fulani and Masaleit are two sympatric ethnic groups in western Sudan who are characterised by marked differences in susceptibility to Plasmodium falciparum malaria. It has been demonstrated that Glucose-6-phosphate dehydrogenase (G6PD) deficiency and Sickle cell trait HbAS carriers are protected from the most severe forms of malaria. This study aimed to investigate a set of specific IgG subclasses against P. falciparum Apical Membrane Antigen 1 (AMA-1 3D7), haemoglobin variants and (G6PD) in association with malaria susceptibility among Fulani ethnic group compared to sympatric ethnic group living in Western Sudan. A total of 124 children aged 5-9 years from each tribe living in an area of hyper-endemic P. falciparum unstable malaria transmission were recruited and genotyped for the haemoglobin (Hb) genes, (G6PD) and (ABO) blood groups. Furthermore, the level of plasma IgG antibody subclasses against P. falciparum antigen (AMA-1) were measured using enzyme linked immunosorbent assays (ELISA). Higher levels of anti-malarial IgG1, IgG2 and IgG3 but not IgG4 antibody were found in Fulani when compared to Masaleit. Individuals carrying the HbCC phenotype were significantly associated with higher levels of IgG1 and IgG2. Furthermore, individuals having the HbAS phenotype were associated with higher levels of specific IgG2 and IgG4 antibodies. In addition, patients with G6PD A/A genotype were associated with higher levels of specific IgG2 antibody compared with those carrying the A/G and G/G genotypes. The results indicate that the Fulani ethnic group show lower frequency of HbAS, HbSS and HbAC compared to the Masaleit ethnic group. The inter-ethnic analysis shows no statistically significant difference in G6PD genotypes (P value = 0.791). However, the intra-ethnic analysis indicates that both ethnic groups have less A/A genotypes and (A) allele frequency of G6PD compared to G/G genotypes, while the HbSA genotype was associated with higher levels of IgG2 (AMA-1) and IgG4 antibodies. In addition, patients carrying the G6PD A/A genotype were associated with higher levels of specific IgG2 antibody compared with those carrying the A/G and G/G genotypes. The present results revealed that the Fulani ethnic group has statistically significantly lower frequency of abnormal haemoglobin resistant to malaria infection compared to the Masaleit ethnic group.

Hemoglobin S and C heterozygosity enhances neither the magnitude nor breadth of antibody responses to a diverse array of Plasmodium falciparum antigens.

BACKGROUND: Heterozygous states of hemoglobin (Hb) A and HbS (HbAS, sickle-cell trait) or HbC (HbAC) protect against Plasmodium falciparum malaria by unclear mechanisms. Several studies suggest that HbAS and HbAC accelerate the acquisition of immunity to malaria, possibly by enhancing P. falciparum-specific antibody responses. METHODS: We used a protein microarray representing 491 P. falciparum proteins expressed during exoerythrocytic and erythrocytic stages of the life cycle to test the hypothesis that HbAS and HbAC enhance the P. falciparum-specific IgG response compared with normal HbAA. Plasma samples were collected from Malian children aged 2-10 years before and after a 6-month malaria season and were probed against the microarray. Immunoglobulin G (IgG) profiles of children with HbAA (n = 106), HbAS (n = 15), and HbAC (n = 20) were compared. RESULTS: Although the magnitude and breadth of P. falciparum-specific IgG responses increased with age and from before to after the malaria season in each antigen category, Hb type did not independently predict significant differences in P. falciparum-specific IgG profiles. CONCLUSIONS: These data do not support the hypothesis that HbAS and HbAC protect against malaria by enhancing P. falciparum-specific antibody responses. It remains possible that HbAS and HbAC protect against malaria by enhancing antibody responses to antigens not studied here or through other immune mechanisms.

Non-opsonising aggregates of IgG and complement in haemoglobin C erythrocytes.

Haemoglobin C (HbC) differs from normal HbA by a lysine for glutamate substitution at position 6 of beta-globin. Heterozygous AC and homozygous CC phenotypes are associated with shortened erythrocyte life spans and mild anaemia. AC and CC erythrocytes contain elevated amounts of membrane-associated haemichromes, band 3 clusters, and immunoglobulin G (IgG) in vivo. These findings led us to investigate whether AC and CC erythrocytes might expose elevated levels of IgG and complement, two opsonins that have been implicated in the phagocytic clearance of senescent and sickle erythrocytes. Surprisingly, we found IgG, complement, and other plasma proteins co-localised in aggregates beneath the membrane of circulating AC and CC erythrocytes. These observations, and our finding of similar aggregates in erythrocytes heterozygous or homozygous for haemoglobin S (sickle-cell haemoglobin), suggest that the vast majority of membrane-associated IgG and complement detected in these abnormal erythrocytes is intracellular and does not contribute to the eventual opsonic clearance of these cells. Phagocytosis studies with macrophages provide evidence in support of this suggestion. Studies of erythrocyte clearance that involve the detection of membrane-associated IgG and complement as putative opsonins should investigate the possibility that these plasma proteins reside in the erythrocyte interior, and not on the cell surface.

Screening for sickle cell disease on dried blood: a new approach evaluated on 27,000 Belgian newborns.

SETTING: Early diagnosis of sickle cell disease decreases morbidity. However, cost-effective screening programmes are not yet available. METHODS: We explored the feasibility of systematic screening performed on dried blood harvested from five-day-old newborns. RESULTS: A total of 27,010 samples were collected in Belgian maternity units between June 2003 and February 2005, and the presence of haemoglobin (Hb) C or S in the eluted blood was examined by an enzyme-linked immunosorbent assay (ELISA) test performed with a monoclonal antibody detecting both mutated forms. As this antibody slightly cross-reacts with Hb A, better specificity is achieved if the test is performed not later than day 5. Among the 27,010 samples, 132 (0.49%) were positive. Molecular biology tests performed on dried blood from positive samples showed that 106 of these babies were heterozygotes for the Hb S mutation and three were heterozygotes for the Hb C mutation, while three newborns were SS homozygotes (0.011%). Seventeen samples (0.063%) were false-positives as we could not detect any mutation. CONCLUSIONS: We have developed a new immunological approach in the field of haemoglobinopathy neonatal screening. This ELISA test is cheap (0.2 euro/test or 1800 euro/detected SS homozygote) and could be centralized. Its cost-effectiveness in the whole Belgian population is comparable with that of screening for phenylketonuria or congenital adrenal hyperplasia. Further improvements should obviously be achieved in order to better discriminate heterozygotes and homozygotes, but the accessibility and the low cost of the test are relevant arguments for the screening extension in a wide range of countries, especially in Central Africa.

Red blood cells that do and red blood cells that don't: how to resist a persistent parasite.

Sixty years ago, Haldane proposed that certain abnormalities in red blood cells could be selected as malaria-resistance genes. Population studies have confirmed that many human polymorphisms confer resistance to severe malaria, although the mechanisms of protection remain unknown. A recent article proposes a new mechanism for explaining the protective effects of hemoglobin C (HbC). HbC-containing red blood cells have modified displays of malaria surface proteins that reduce parasite adhesiveness and could reduce the risk of severe disease.

HBsAg as the antigen component of circulating immune complexes in HIV-infected patients.

Seeing the same transmission pattern of HIV and HBV coinfection by these two agents is not an uncommon feature. Immunity impairment due to HIV infection can be the cause of a higher rate of HBV replication with less intensive liver damage and less effective immune response to HBV, while the pathological course in both infections involves elevated levels of circulating immune complexes (CIC). These were the reasons for us to examine the frequency of HBsAg involvement as the antigen component of circulating immune complexes formed in sera of HIV-infected patients in different stages of HIV disease. We tested 67 sera of HIV-positive patients in different stages of HIV disease for the presence of HBsAg and HIV antigen p24 (with and without acid dissociation of immune complexes), for the presence of anti-Hbc antibodies and circulating immune complexes. HBsAg was positive in 13.8% sera prior to and 33.8% after acid pretreatment. Anti-HBc antibodies were present in 76.9% serum samples tested. Fifty percent of sera were positive for both HBsAg and p24 antigen after dissociation of immune complexes. The level of CIC was elevated in 65.9% of sera. Our results suggest that HBsAg is commonly associated in immune complexes formed in the sera of HIV-infected patients and that they may simultaneously contain HIV and HBsAg in patients coinfected with both agents. This may contribute to their mutual interaction and influence the diagnosis and follow-up of patients.

Utilization of monoclonal-antibody-based assay (HemoCard in screening for and differentiating between genotypes of sickle cell disease and other hemoglobinopathies.

Sickle cell disease covers a group of conditions in which pathology may be attributed to the presence of sickle hemoglobin (HbS). The identification of HbS and other variants including those in combination with HbS is commonly achieved by cellulose acetate electrophoresis at alkaline pH. Because many hemoglobin variants with similar charges have similar electrophoretic migration patterns, they are difficult to differentiate by electrophoresis. The HemoCard assays address this concern through the use of monoclonal antibodies capable of specifically recognizing the unique amino acid substitution in the variant hemoglobin. The panel of HemoCard monoclonal antibodies confirms the absence and presence of HbA, HbC, HbE, HbS, and other sickling hemoglobin variants. The combination of alkaline cellulose acetate electrophoresis and HemoCard assays allows the technologist to reach a final conformation of both common and much less common sickle cell disease genotypes, combinations of HbS with other hemoglobins that ordinarily do not produce sickle cell disease, and other clinically important hemoglobinopathies including HbE/beta-thalassemia and hemoglobin C disease.

Simple and rapid enzyme-linked immunosorbent assay for the detection of hemoglobin C[alpha 2 beta 2 6(A3)Glu----Lys] in cord blood using a monoclonal antibody.

We have generated a murine hybridoma that secretes a monoclonal antibody (mAb) that is highly specific for hemoglobin C (HbC) [alpha 2 beta 2 6(A3)Glu----Lys] and shows no cross reactivity with HbA, HbA2, HbF, HbS, HbE, or Hb O-Arab. Using this antibody, we developed a simple and rapid enzyme linked immunosorbent assay (ELISA) technique for the detection of HbC in both adult and cord blood. The assay can be carried out using either whole blood samples or hemolysates. With as little as 10 microliters/well of whole blood or 5 micrograms Hb/well of hemolysates, and, with dilutions of the antibody up to 10(-5), we were able to detect HbC unequivocally in cord blood samples. The ELISA procedure could detect HbC in proportions as low as 0.01%. This simple diagnostic test represents a technological advance in Hb identification and can easily be used for mass screening (96 samples in less than 45 min) to detect HbC. Furthermore, this assay, when employed in conjunction with an mAb specific for beta 6GLU of HbA, allows the discrimination between HbC homozygotes, heterozygotes, and normals.