Sickle hemoglobin confers tolerance to Plasmodium infection.

Sickle human hemoglobin (Hb) confers a survival advantage to individuals living in endemic areas of malaria, the disease caused by Plasmodium infection. As demonstrated hereby, mice expressing sickle Hb do not succumb to experimental cerebral malaria (ECM). This protective effect is exerted irrespectively of parasite load, revealing that sickle Hb confers host tolerance to Plasmodium infection. Sickle Hb induces the expression of heme oxygenase-1 (HO-1) in hematopoietic cells, via a mechanism involving the transcription factor NF-E2-related factor 2 (Nrf2). Carbon monoxide (CO), a byproduct of heme catabolism by HO-1, prevents further accumulation of circulating free heme after Plasmodium infection, suppressing the pathogenesis of ECM. Moreover, sickle Hb inhibits activation and/or expansion of pathogenic CD8(+) T cells recognizing antigens expressed by Plasmodium, an immunoregulatory effect that does not involve Nrf2 and/or HO-1. Our findings provide insight into molecular mechanisms via which sickle Hb confers host tolerance to severe forms of malaria.

False positive testing for sickle hemoglobin in a blood donor with mild erythrocytosis and hemoglobin Geldrop St. Anna.

Solubility testing for sickle hemoglobin is commonly performed to identify blood suitable for patients with sickle cell disease. A 32-year-old Caucasian male blood donor's unit screened positive for sickle hemoglobin via solublity testing (Streck). As the donor was considered low risk for being positive for hemoglobin S (HbS), he self-referred to hematology for further evaluation. Testing with hemoglobin electrophoresis revealed the patient to be negative for HbS; however, 42 % fetal hemoglobin (HbF) was noted. Since this was higher than typically seen in hereditary persistence of HbF, deoxyribonucleic acid (DNA) sequencing of hemoglobin (Hb) was ordered through a referral laboratory. Hb gene sequencing revealed the patient to be heterozygous for Hb Geldrop St. Anna, a rare Hb variant. This variant has previously been shown to migrate in the HbF region with alkaline electrophoresis. The workup demonstrated that the oxygen dissociation curve was left-shifted consistent with slightly increased oxygen affinity of this variant. The patient's hematocrits (Hct) from his past donations were 53 % and 54 % about two years apart and his Hct at his hematology evaluation was 53 %. This report describes the first case of Hb Geldrop St. Anna in the United States and was associated with a false positive HbS screen. This Hb variant is considered to be benign and has an increased oxygen affinity that is associated with mild erythrocytosis. The donor was allowed to continue donating blood products.

Fetal hemoglobin in sickle cell anemia: a glass half full?

Fetal hemoglobin (HbF) modulates the phenotype of sickle cell anemia by inhibiting deoxy sickle hemoglobin (HbS) polymerization. The blood concentration of HbF, or the number of cells with detectable HbF (F-cells), does not measure the amount of HbF/F-cell. Even patients with high HbF can have severe disease because HbF is unevenly distributed among F-cells, and some cells might have insufficient concentrations to inhibit HbS polymerization. With mean HbF levels of 5%, 10%, 20%, and 30%, the distribution of HbF/F-cell can greatly vary, even if the mean is constant. For example, with 20% HbF, as few as 1% and as many as 24% of cells can have polymer-inhibiting, or protective, levels of HbF of ∼10 pg; with lower HbF, few or no protected cells can be present. Only when the total HbF concentration is near 30% is it possible for the number of protected cells to approach 70%. Rather than the total number of F-cells or the concentration of HbF in the hemolysate, HbF/F-cell and the proportion of F-cells that have enough HbF to thwart HbS polymerization is the most critical predictor of the likelihood of severe sickle cell disease.

Vasculopathy and pulmonary hypertension in sickle cell disease.

Sickle cell disease (SCD) is an autosomal recessive disorder in the gene encoding the ß-chain of hemoglobin. Deoxygenation causes the mutant hemoglobin S to polymerize, resulting in rigid, adherent red blood cells that are entrapped in the microcirculation and hemolyze. Cardinal features include severe painful crises and episodic acute lung injury, called acute chest syndrome. This population, with age, develops chronic organ injury, such as chronic kidney disease and pulmonary hypertension. A major risk factor for developing chronic organ injury is hemolytic anemia, which releases red blood cell contents into the circulation. Cell free plasma hemoglobin, heme, and arginase 1 disrupt endothelial function, drive oxidative and inflammatory stress, and have recently been referred to as erythrocyte damage-associated molecular pattern molecules (eDAMPs). Studies suggest that in addition to effects of cell free plasma hemoglobin on scavenging nitric oxide (NO) and generating reactive oxygen species (ROS), heme released from plasma hemoglobin can bind to the toll-like receptor 4 to activate the innate immune system. Persistent intravascular hemolysis over decades leads to chronic vasculopathy, with ∼10% of patients developing pulmonary hypertension. Progressive obstruction of small pulmonary arterioles, increase in pulmonary vascular resistance, decreased cardiac output, and eventual right heart failure causes death in many patients with this complication. This review provides an overview of the pathobiology of hemolysis-mediated endothelial dysfunction and eDAMPs and a summary of our present understanding of diagnosis and management of pulmonary hypertension in sickle cell disease, including a review of recent American Thoracic Society (ATS) consensus guidelines for risk stratification and management.

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.

Idiotypic cross-reactivity between antibodies of different specificities.

Cross-idiotypic specificity has been demonstrated between antibody populations of different specificities using antibodies directed toward human sickle cell hemoglobin (HbS). A site-specific antibody directed toward the beta6-position of HbS, anti-Val, was used to elicit an anti-idiotypic response in rabbits. Using this anti-idiotypic serum, idiotypic cross-reactivity was demonstrated between antibody populations that bind to human adult hemoglobin (HbA). It was demonstrated that in the case of the goat antibodies, these idiotypically cross-reacting antibodies are directed towards the beta6-position of the hemoglobin molecule. However, they differ in their specificity, binding to this site on HbA, whereas anti-Val binds only to HbS. The sheep antibody populations directed toward HbS differ qualitatively from those of the goat. Using rabbit anti-idiotypic serum specific for sheep anti-Val, cross-reactivity could be demonstrated with antibodies directed toward the alpha-chain of the hemoglobin molecule, as well as the beta-chain. There was also a low level of cross-reactivity with antibodies from a goat immunized with HbA.

Evolution of an idiotypic determinant: anti-Val.

An antibody population which reacts only with human sickle cell hemoglobin (HbS) and not with normal human hemoglobin, has been isolated from goat, sheep, and guinea pig antisera. These antibody populations termed anti-Val (Val), isolated from an individual goat (no. 6) and sheep (no. 26) have been used to elicit anti-idiotypic responses in rabbits. These anti-idiotypic sera were used to study the idiotypic cross-reactions between the goat and sheep anti-Val. Strong cross-reactions were present using either Ra anti-goat anti-Val or Ra anti-sheep anti-Val. Guinea pig anti-Val did not cross-react with these anti-idiotypic sera. Binding of HbS to the anti-Val of the goat and sheep could be blocked by the anti-idiotypic sera, but the binding of HbS to the guinea pig anti-Val could not. These data demonstrate idiotypic cross-reactivity between two closely related species.

The indirect health effects of malaria estimated from health advantages of the sickle cell trait.

Most estimates of the burden of malaria are based on its direct impacts; however, its true burden is likely to be greater because of its wider effects on overall health. Here we estimate the indirect impact of malaria on children's health in a case-control study, using the sickle cell trait (HbAS), a condition associated with a high degree of specific malaria resistance, as a proxy indicator for an effective intervention. We estimate the odds ratios for HbAS among cases (all children admitted to Kilifi County Hospital during 2000-2004) versus community controls. As expected, HbAS protects strongly against malaria admissions (aOR 0.26; 95%CI 0.22-0.31), but it also protects against other syndromes, including neonatal conditions (aOR 0.79; 0.67-0.93), bacteraemia (aOR 0.69; 0.54-0.88) and severe malnutrition (aOR 0.67; 0.55-0.83). The wider health impacts of malaria should be considered when estimating the potential added benefits of effective malaria interventions.

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.

Towards a point-of-care strip test to diagnose sickle cell anemia.

A rapid test to identify patients with sickle cell disease could have important benefits in low-resource settings. Sickle cell anemia (SCA) affects about 300,000 newborns each year, the majority of whom are born in sub-Saharan Africa. Low-cost therapies are available to treat SCA, but most countries in sub-Saharan Africa lack robust neonatal screening programs needed to identify patients in need of treatment. To address this need, we developed and evaluated a competitive lateral flow assay that identifies patients with SCA (genotype HbSS) in 15 minutes using undiluted whole blood. A small volume of blood (0.5 µL- 3 µL) is mixed with antibody-coated blue latex beads in a tube and applied to the strip. Strips are then placed in a well of running buffer and allowed to run for 10 minutes. Laboratory evaluation with samples containing different proportions of hemoglobin A (HbA) and hemoglobin S (HbS) indicated that the test should enable identification of SCA patients but not persons with sickle cell trait (SCT). We evaluated the test using 41 samples from individuals with SCA, SCT, and normal blood. With visual inspection or quantitative analysis, we found a 98% accuracy when differentiating SCA from normal and SCT samples as a group (90% sensitivity and 100% specificity for identifying SCA). This work demonstrates important steps towards making a lateral flow test for hemoglobinopathies more appropriate for point-of-care use; further work is needed before the test is appropriate for clinical use.

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.

Blood viscosity is lower in trained than in sedentary sickle cell trait carriers.

The aim of this study was to compare blood and plasma viscosities, as well as the hematocrit/blood viscosity ratio (HVR), between trained and sedentary SCT carriers. Thirty African male SCT carriers from the city of Dakar (Senegal) participated in the study: one group composed of 15 trained SCT carriers (TSCTc) and one group composed of 15 sedentary individuals (SSCTc). Blood was sampled in resting condition and blood and plasma viscosities were measured using a cone-plate viscometer. After the determination of hematocrit by microcentrifugation, HVR was determined for each subject. Blood and plasma viscosities, as well as hematocrit, were significantly reduced in TSCTc compared to SSCTc. As a consequence, HRV was greater in TSCTc. These findings provide evidence that SCT carriers should be encouraged to practice regular physical activity to limit the cardiovascular strain usually caused by their blood hyperviscosity.

Malaria: a vaccine concept based on sickle haemoglobin's augmentation of an innate autoimmune process to band 3.

The protection from malaria afforded by sickle haemoglobin (and certain other haemoglobinopathies) suggests that it may be possible to utilise a common property that their erythrocytes share with both malaria-infected erythrocytes and senescent erythrocytes to develop a vaccine. All three conditions cause clustering of a specific protein molecule, band 3, on their erythrocyte's surface and this protein, when present on senescent erythrocytes at least, results in the immune recognition and removal of these by naturally occurring antibodies. It is hypothesised that if an up-regulated immune response to this protein on sickle cells is responsible for the benefit afforded to malaria patients then a vaccine using antigenic band 3 peptides may provide similar protection.

Serum immunoglobulin (G, A and M) classes and IgG subclasses in sickle cell anaemia.

Serum concentrations of immunoglobulins G, A, M and IgG subclasses were determined by single radial immunodiffusion assay in a population of sickle cell anaemia patients resident in the tropics. Fifty apparently healthy subjects of haemoglobin genotype AA, of comparable age, sex and socioeconomic status (SES), and in the same environment as the patients, were included as controls. Three indices of morbidity in SCA, namely frequency of crisis, degree of anaemia and the number of organ complications, were used to derive a severity score for each patient; and thus categorize the subjects into severity groups. Immunoglobulin levels were then correlated with the indices of morbidity as well as the derived severity score. IgG, IgA, IgM, IgG1 and IgG3 levels were significantly raised in the SCA subjects when they were compared as a group with the controls. When separated into disease severity groups, the mildly affected patients were found to have virtually normal levels of immunoglobulins. Total IgG concentration and level of the IgG3 subclass showed significant positive correlation with frequency of crisis and derived severity score. Markedly raised levels of IgG and IgG3 may be predictive of severity in sickle cell anaemia.

Carbon-fiber microelectrode amperometry reveals sickle-cell-induced inflammation and chronic morphine effects on single mast cells.

Sickle cell disease, caused by a mutation of hemoglobin, is characterized by a complex pathophysiology including an important inflammatory component. Mast cells are tissue-resident leukocytes known to influence a range of immune functions in a variety of different ways, largely through the secretion of biologically active mediators from preformed granules. However, it is not understood how mast cells influence the inflammatory environment in sickle cell disease. A notable consequence of sickle cell disease is severe pain. Therefore, morphine is often used to treat this disease. Because mast cells express opioid receptors, it is pertinent to understand how chronic morphine exposure influences mast cell function and inflammation in sickle cell disease. Herein, carbon-fiber microelectrode amperometry (CFMA) was used to monitor the secretion of immunoactive mediators from single mast cells. CFMA enabled the detection and quantification of discrete exocytotic events from single mast cells. Mast cells from two transgenic mouse models expressing human sickle hemoglobin (hBERK1 and BERK) and a control mouse expressing normal human hemoglobin (HbA-BERK) were monitored using CFMA to explore the impact of sickle-cell-induced inflammation and chronic morphine exposure on mast cell function. This work, utilizing the unique mechanistic perspective provided by CFMA, describes how mast cell function is significantly altered in hBERK1 and BERK mice, including decreased serotonin released compared to HbA-BERK controls. Furthermore, morphine was shown to significantly increase the serotonin released from HbA-BERK mast cells and demonstrated the capacity to reverse the observed sickle-cell-induced changes in mast cell function.