Nuno Sepúlveda

Reappraisal of known malaria resistance loci in a large multicenter study

Many human genetic associations with resistance to malaria have been reported, but few have been reliably replicated. We collected data on 11,890 cases of severe malaria due to Plasmodium falciparum and 17,441 controls from 12 locations in Africa, Asia and Oceania. We tested 55 SNPs in 27 loci previously reported to associate with severe malaria. There was evidence of association at P < 1 × 10−4 with the HBB, ABO, ATP2B4, G6PD and CD40LG loci, but previously reported associations at 22 other loci did not replicate in the multicenter analysis. The large sample size made it possible to identify authentic genetic effects that are heterogeneous across populations or phenotypes, with a striking example being the main African form of G6PD deficiency, which reduced the risk of cerebral malaria but increased the risk of severe malarial anemia. The finding that G6PD deficiency has opposing effects on different fatal complications of P. falciparum infection indicates that the evolutionary origins of this common human genetic disorder are more complex than previously supposed.

A Central Role for Free Heme in the Pathogenesis of Severe Sepsis

Heme from red blood cells released in septic shock worsens organ dysfunction and increases the risk of death, but can be overcome by a scavenger of free heme. Casting Heme in a New Light Sepsis, or severe systemic infection, is a deadly disease that has always been difficult to treat. Despite modern-day antibiotics and intensive care management, patients with sepsis still have a high rate of major complications and death. These severe consequences are thought to be a result of simultaneous overwhelming infection and an overexuberant immune response, which together damage tissues and lead to organ dysfunction. One cell type that is injured during sepsis is the erythrocyte. As these red blood cells lyse, hemoglobin is released and oxidized, releasing free heme into the circulation. This heme is not an innocent bystander, however, as Larsen et al. now report. It increases inflammation and cell death, exacerbating the damage to the body and increasing the risk of death. The authors found that mice lacking heme oxygenase 1, the enzyme that breaks down heme into harmless by-products, have more free circulating heme, which makes them more susceptible to death from sepsis than are matching wild-type mice. In addition, giving extra heme to wild-type mice suffering from sepsis greatly increases their risk of organ dysfunction and death without affecting the number of bacteria in their blood. Moreover, hemopexin, a protein produced by the body to scavenge free heme, protects mice and human patients with sepsis from the deleterious effects of heme and decreases the risk of complications and death. Because these authors have shown that heme concentrations are associated with worse prognosis in sepsis patients, we may now have a new way to monitor patients’ health status and, eventually, to treat them. Measurements of heme and hemopexin in patients with sepsis may predict who needs more intensive interventions, potentially allowing for more timely treatment before organ failure ensues. In addition, high-risk patients could be given extra hemopexin or other heme-neutralizing substances to possibly save them from death caused by sepsis, even when all the current treatments fail. Low-grade polymicrobial infection induced by cecal ligation and puncture is lethal in heme oxygenase-1–deficient mice (Hmox1−/−), but not in wild-type (Hmox1+/+) mice. Here we demonstrate that the protective effect of this heme-catabolizing enzyme relies on its ability to prevent tissue damage caused by the circulating free heme released from hemoglobin during infection. Heme administration after low-grade infection in mice promoted tissue damage and severe sepsis. Free heme contributed to the pathogenesis of severe sepsis irrespective of pathogen load, revealing that it compromised host tolerance to infection. Development of lethal forms of severe sepsis after high-grade infection was associated with reduced serum concentrations of the heme sequestering protein hemopexin (HPX), whereas HPX administration after high-grade infection prevented tissue damage and lethality. Finally, the lethal outcome of septic shock in patients was also associated with reduced HPX serum concentrations. We propose that targeting free heme by HPX might be used therapeutically to treat severe sepsis.

When three is not a crowd : a Crossregulation Model of the dynamics and repertoire selection of regulatory CD4+ T cells

Summary:  Regulatory CD4+ T cells, enriched in the CD25 pool of healthy individuals, mediate natural tolerance and prevent autoimmune diseases. Despite their fundamental and potential clinical significance, regulatory T (TR) cells have not yet been incorporated in a coherent theory of the immune system. This article reviews experimental evidence and theoretical arguments supporting a model of TR cell dynamics, uncovering some of its most relevant biological implications. According to this model, the persistence and expansion of TR cell populations depend strictly on specific interactions they make with antigen‐presenting cells (APCs) and conventional effector T (TE) cells. This three‐partner crossregulation imposes that TR cells feed on the specific autoimmune activities they suppress, with implications ranging from their interactions with other cells to their repertoire selection in the periphery and in the thymus, and to the relationship between these cells and the innate immune system. These implications stem from the basic prediction that the peripheral dynamics sort the CD4+ T‐cell repertoire into two subsets: a less diverse set of small clones of autoreactive effector and regulatory cells that regulate each other’s growth, and a more diverse set of barely autoreactive TE cell clones, whose expansion is limited only by APC availability. It is argued that such partitioning of the repertoire sets the ground for self–non‐self discrimination.

Transforming Growth Factor Beta 2 and Heme Oxygenase 1 Genes Are Risk Factors for the Cerebral Malaria Syndrome in Angolan Children

Background Cerebral malaria (CM) represents a severe outcome of the Plasmodium falciparum infection. Recent genetic studies have correlated human genes with severe malaria susceptibility, but there is little data on genetic variants that increase the risk of developing specific malaria clinical complications. Nevertheless, susceptibility to experimental CM in the mouse has been linked to host genes including Transforming Growth Factor Beta 2 (TGFB2) and Heme oxygenase-1 (HMOX1). Here, we tested whether those genes were governing the risk of progressing to CM in patients with severe malaria syndromes. Methodology/Principal Findings We report that the clinical outcome of P. falciparum infection in a cohort of Angolan children (n = 430) correlated with nine TGFB2 SNPs that modify the risk of progression to CM as compared to other severe forms of malaria. This genetic effect was explained by two haplotypes harboring the CM-associated SNPs (Pcorrec. = 0.035 and 0.036). In addition, one HMOX1 haplotype composed of five CM-associated SNPs increased the risk of developing the CM syndrome (Pcorrec. = 0.002) and was under-transmitted to children with uncomplicated malaria (P = 0.036). Notably, the HMOX1-associated haplotype conferred increased HMOX1 mRNA expression in peripheral blood cells of CM patients (P = 0.012). Conclusions/Significance These results represent the first report on CM genetic risk factors in Angolan children and suggest the novel hypothesis that genetic variants of the TGFB2 and HMOX1 genes may contribute to confer a specific risk of developing the CM syndrome in patients with severe P. falciparum malaria. This work may provide motivation for future studies aiming to replicate our findings in larger populations and to confirm a role for these genes in determining the clinical course of malaria.

Candidate Polymorphisms and Severe Malaria in a Malian Population

Malaria is a major health burden in sub-Saharan African countries, including Mali. The disease is complex, with multiple genetic determinants influencing the observed variation in response to infection, progression, and severity. We assess the influence of sixty-four candidate loci, including the sickle cell polymorphism (HbS), on severe malaria in a case-control study consisting of over 900 individuals from Bamako, Mali. We confirm the known protective effects of the blood group O and the HbS AS genotype on life-threatening malaria. In addition, our analysis revealed a marginal susceptibility effect for the CD40 ligand (CD40L)+220C allele. The lack of statistical evidence for other candidates may demonstrate the need for large-scale genome-wide association studies in malaria to discover new polymorphisms. It also demonstrates the need for establishing the region-specific repertoire of functional variation in important genes, including the glucose-6-phosphatase deficiency gene, before embarking on focused genotyping.

African glucose-6-phosphate dehydrogenase alleles associated with protection from severe malaria in heterozygous females in Tanzania.

X-linked Glucose-6-phosphate dehydrogenase (G6PD) A- deficiency is prevalent in sub-Saharan Africa populations, and has been associated with protection from severe malaria. Whether females and/or males are protected by G6PD deficiency is uncertain, due in part to G6PD and malaria phenotypic complexity and misclassification. Almost all large association studies have genotyped a limited number of G6PD SNPs (e.g. G6PD202 / G6PD376), and this approach has been too blunt to capture the complete epidemiological picture. Here we have identified 68 G6PD polymorphisms and analysed 29 of these (i.e. those with a minor allele frequency greater than 1%) in 983 severe malaria cases and controls in Tanzania. We establish, across a number of SNPs including G6PD376, that only female heterozygotes are protected from severe malaria. Haplotype analysis reveals the G6PD locus to be under balancing selection, suggesting a mechanism of protection relying on alleles at modest frequency and avoiding fixation, where protection provided by G6PD deficiency against severe malaria is offset by increased risk of life-threatening complications. Our study also demonstrates that the much-needed large-scale studies of severe malaria and G6PD enzymatic function across African populations require the identification and analysis of the full repertoire of G6PD genetic markers.

Candidate Human Genetic Polymorphisms and Severe Malaria in a Tanzanian Population.

Human genetic background strongly influences susceptibility to malaria infection and progression to severe disease and death. Classical genetic studies identified haemoglobinopathies and erythrocyte-associated polymorphisms, as protective against severe disease. High throughput genotyping by mass spectrometry allows multiple single nucleotide polymorphisms (SNPs) to be examined simultaneously. We compared the prevalence of 65 human SNPs, previously associated with altered risk of malaria, between Tanzanian children with and without severe malaria. Five hundred children, aged 1–10 years, with severe malaria were recruited from those admitted to hospital in Muheza, Tanzania and compared with matched controls. Genotyping was performed by Sequenom MassArray, and conventional PCR was used to detect deletions in the alpha-thalassaemia gene. SNPs in two X-linked genes were associated with altered risk of severe malaria in females but not in males: heterozygosity for one or other of two SNPs in the G6PD gene was associated with protection from all forms of severe disease whilst two SNPs in the gene encoding CD40L were associated with respiratory distress. A SNP in the adenyl cyclase 9 (ADCY9) gene was associated with protection from acidosis whilst a polymorphism in the IL-1α gene (IL1A) was associated with an increased risk of acidosis. SNPs in the genes encoding IL-13 and reticulon-3 (RTN3) were associated with increased risk of cerebral malaria. This study confirms previously known genetic associations with protection from severe malaria (HbS, G6PD). It identifies two X-linked genes associated with altered risk of severe malaria in females, identifies mutations in ADCY9, IL1A and CD40L as being associated with altered risk of severe respiratory distress and acidosis, both of which are characterised by high serum lactate levels, and also identifies novel genetic associations with severe malaria (TRIM5) and cerebral malaria(IL-13 and RTN3). Further studies are required to test the generality of these associations and to understand their functional consequences.

Human candidate polymorphisms in sympatric ethnic groups differing in malaria susceptibility in mali.

Malaria still remains a major public health problem in Mali, although disease susceptibility varies between ethnic groups, particularly between the Fulani and Dogon. These two sympatric groups share similar socio-cultural factors and malaria transmission rates, but Fulani individuals tend to show significantly higher spleen enlargement scores, lower parasite prevalence, and seem less affected by the disease than their Dogon neighbours. We have used genetic polymorphisms from malaria-associated genes to investigate associations with various malaria metrics between the Fulanai and Dogon groups. Two cross sectional surveys (transmission season 2006, dry season 2007) were performed. Healthy volunteers from the both ethnic groups (n=939) were recruited in a rural setting. In each survey, clinical (spleen enlargement, axillary temperature, weight) and parasitological data (malaria parasite densities and species) were collected, as well as blood samples. One hundred and sixty six SNPs were genotyped and 5 immunoassays (AMA1, CSP, MSP1, MSP2, total IgE) were performed on the DNA and serum samples respectively. The data confirm the reduced malaria susceptibility in the Fulani, with a higher level of the protective O-blood group, and increased circulating antibody levels to several malaria antigens (p<10-15). We identified SNP allele frequency differences between the 2 ethnic groups in CD36, IL4, RTN3 and ADCY9. Moreover, polymorphisms in FCER1A, RAD50, TNF, SLC22A4, and IL13 genes were correlated with antibody production (p-value<0.003). Further work is required to understand the mechanisms underpinning these genetic factors.

Relevance of feline interferon omega for clinical improvement and reduction of concurrent viral excretion in retrovirus infected cats from a rescue shelter.

Abstract Feline Immnunodeficiency (FIV) and Feline Leukemia (FeLV) viruses are common infectious agents in stray cats and shelter environments. Recombinant feline interferon-ω (rFeIFNω) has shown an antiviral action not only against FIV and FeLV but also against herpesvirus (FHV-1) and calicivirus (FCV). Sixteen naturally infected FIV/FeLV cats were followed during rFeIFNω therapy in order to monitor clinical signs and to correlate with excretion of concomitant viruses (FCV, FHV-1, feline coronavirus (FCoV) and parvovirus (FPV)). Cats were submitted to clinical evaluations and concomitant virus excretion assessement. Comparing D0–D65, 10/16 cats improved clinical scores. Of the 10 cats positive for FHV-1 on D0, 4 were negative and 6 reduced viral loads. Of the 11 FCoV positive cats, 9 reduced viral loads. The 13 FCV positive cats and the FPV positive cat were negative on D65. In conclusion, rFeIFNω improves clinical signs and reduces concurrent viral excretion in naturally infected retroviral cats.

Differentiation of human thymic regulatory T cells at the double positive stage

Treg cells, best identified by the expression of the transcription factor FOXP3, play a crucial role in maintaining self‐tolerance. Natural Treg cells constitute an independent thymus‐derived T‐cell lineage whose developmental program in humans is still ill‐defined. Here, we provide evidence of a Treg‐cell differentiation pathway at the double positive (DP) stage, prior to commitment to the CD4+ or CD8+ lineage, in pediatric thymuses. FOXP3+ DP cells displayed a functional IL‐7 receptor and increased Bcl‐2 levels that may protect them from cell death/negative selection, and an activated/suppressive phenotype that was lost as CD4 single positive (SP) cells matured and acquired egress markers. A subpopulation of FOXP3+ DP thymocytes expressing CD103 likely represents the precursor of FOXP3+ CD8SP cells, which homogeneously expressed this mucosal‐homing molecule. Finally, co‐cultures of DP thymocytes with primary thymic epithelial cells and multiple linear regression analyses support that FOXP3+ SP cells are largely derived from FOXP3+ DP thymocytes. Overall, our data suggest that human Treg‐cell lineage commitment significantly occurs at the DP stage with possible implications for the diversity and autoreactivity of the natural Treg‐cell repertoire.