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1.  Association of a NOD2 Gene Polymorphism and T-Helper 17 Cells With Presumed Ocular Toxoplasmosis 
The Journal of Infectious Diseases  2012;207(1):152-163.
Retinochoroiditis manifests in patients infected with Toxoplasma gondii. Here, we assessed 30 sibships and 89 parent/case trios of presumed ocular toxoplasmosis (POT) to evaluate associations with polymorphisms in the NOD2 gene. Three haplotype-tagging single-nucleotide polymorphisms (tag-SNPs) within the NOD2 gene were genotyped. The family-based association test showed that the tag-SNP rs3135499 is associated with retinochoroiditis (P = .039). We then characterized the cellular immune response of 59 cases of POT and 4 cases of active ocular toxoplasmosis (AOT). We found no differences in levels of interferon γ (IFN-γ) and interleukin 2 produced by T-helper 1 cells when comparing patients with AOT or POT to asymptomatic individuals. Unexpectedly, we found an increased interleukin 17A (IL-17A) production in patients with POT or OAT. In patients with POT or AOT, the main cellular source of IL-17A was CD4+CD45RO+T-bet−IFN-γ− T-helper 17 cells. Altogether, our results suggest that NOD2 influences the production of IL-17A by CD4+ T lymphocytes and might contribute to the development of ocular toxoplasmosis.
PMCID: PMC3523795  PMID: 23100559
NOD2; IL-17; Th17; T lymphocytes; ocular toxoplasmosis; Toxoplasma gondii
2.  Cytokine Responses to Novel Antigens in a Peri-Urban Population in Brazil Exposed to Leishmania infantum chagasi 
Visceral leishmaniasis (VL) is fatal if untreated, and there are no vaccines for this disease. High levels of CD4-derived interferon-γ (IFN-γ) in the presence of low levels of interleukin-10 (IL-10) predicts vaccine success. Tumor necrosis factor-α (TNF-α) is also important in this process. We characterized human immune responses in three groups exposed to Leishmania infantum chagasi in Brazil: 1) drug-cured VL patients (recovered VL); 2) asymptomatic persons with positive Leishmania-specific delayed-type hypersensitivity skin reactions (DTH+); and 3) DTH-negative household contacts. Magnitude of DTH correlated with crude Leishmania antigen–driven IFN-γ, TNF-α, and IL-5, but not IL-10. DTH+ persons showed equivalent levels of IFN-γ, but higher levels of IL-10, to tryparedoxin peroxidase and Leishmania homolog of receptor for activated C kinase compared with recovered VL patients. The IFN-γ:IL-10 and TNF-α:IL-10 ratios were higher in recovered VL patients than in DTH+ persons. Seven of 11 novel candidates (R71, L37, N52, L302.06, M18, J41, and M22) elicited cytokine responses (36–71% of responders) in recovered VL patients and DTH+ persons. This result confirmed their putative status as cross-species vaccine/immunotherapeutic candidates.
PMCID: PMC3516316  PMID: 22826477
3.  Genome-wide association study identifies new psoriasis susceptibility loci and an interaction between HLA-C and ERAP1 
Strange, Amy | Capon, Francesca | Spencer, Chris CA | Knight, Jo | Weale, Michael E | Allen, Michael H | Barton, Anne | Band, Gavin | Bellenguez, Céline | Bergboer, Judith GM | Blackwell, Jenefer M | Bramon, Elvira | Bumpstead, Suzannah J | Casas, Juan P | Cork, Michael J | Corvin, Aiden | Deloukas, Panos | Dilthey, Alexander | Duncanson, Audrey | Edkins, Sarah | Estivill, Xavier | Fitzgerald, Oliver | Freeman, Colin | Giardina, Emiliano | Gray, Emma | Hofer, Angelika | Hüffmeier, Ulrike | Hunt, Sarah E | Irvine, Alan D | Jankowski, Janusz | Kirby, Brian | Langford, Cordelia | Lascorz, Jesús | Leman, Joyce | Leslie, Stephen | Mallbris, Lotus | Markus, Hugh S | Mathew, Christopher G | McLean, WH Irwin | McManus, Ross | Mössner, Rotraut | Moutsianas, Loukas | Naluai, Åsa T | Nestle, Frank O | Novelli, Giuseppe | Onoufriadis, Alexandros | Palmer, Colin NA | Perricone, Carlo | Pirinen, Matti | Plomin, Robert | Potter, Simon C | Pujol, Ramon M | Rautanen, Anna | Riveira-Munoz, Eva | Ryan, Anthony W | Salmhofer, Wolfgang | Samuelsson, Lena | Sawcer, Stephen J | Schalkwijk, Joost | Smith, Catherine H | Ståhle, Mona | Su, Zhan | Tazi-Ahnini, Rachid | Traupe, Heiko | Viswanathan, Ananth C | Warren, Richard B | Weger, Wolfgang | Wolk, Katarina | Wood, Nicholas | Worthington, Jane | Young, Helen S | Zeeuwen, Patrick LJM | Hayday, Adrian | Burden, A David | Griffiths, Christopher EM | Kere, Juha | Reis, André | McVean, Gilean | Evans, David M | Brown, Matthew A | Barker, Jonathan N | Peltonen, Leena | Donnelly, Peter | Trembath, Richard C
Nature genetics  2010;42(11):985-990.
PMCID: PMC3749730  PMID: 20953190
4.  Cytokine Responses to Novel Antigens in an Indian Population Living in an Area Endemic for Visceral Leishmaniasis 
There are no effective vaccines for visceral leishmaniasis (VL), a neglected parasitic disease second only to malaria in global mortality. We previously identified 14 protective candidates in a screen of 100 Leishmania antigens as DNA vaccines in mice. Here we employ whole blood assays to evaluate human cytokine responses to 11 of these antigens, in comparison to known defined and crude antigen preparations.
Whole blood assays were employed to measure IFN-γ, TNF-α and IL-10 responses to peptide pools of the novel antigens R71, Q51, L37, N52, L302.06, J89, M18, J41, M22, M63, M57, as well as to recombinant proteins of tryparedoxin peroxidase (TRYP), Leishmania homolog of the receptor for activated C kinase (LACK) and to crude soluble Leishmania antigen (SLA), in Indian patients with active (n = 8) or cured (n = 16) VL, and in modified Quantiferon positive (EHC+ve, n = 20) or modified Quantiferon negative (EHC−ve, n = 9) endemic healthy controls (EHC).
Active VL, cured VL and EHC+ve groups showed elevated SLA-specific IFN-γ, but only active VL patients produced IL-10 and EHC+ve did not make TNF-α. IFN-γ to IL-10 and TNF-α to IL-10 ratios in response to TRYP and LACK antigens were higher in cured VL and EHC+ve exposed individuals compared to active VL. Five of the eleven novel candidates (R71, L37, N52, J41, and M22) elicited IFN-γ and TNF-α, but not IL-10, responses in cured VL (55–87.5% responders) and EHC+ve (40–65% responders) subjects.
Our results are consistent with an important balance between pro-inflammatory IFNγ and TNFγ cytokine responses and anti-inflammatory IL-10 in determining outcome of VL in India, as highlighted by response to both crude and defined protein antigens. Importantly, cured VL patients and endemic Quantiferon positive individuals recognise 5 novel vaccine candidate antigens, confirming our recent data for L. chagasi in Brazil, and their potential as cross-species vaccine candidates.
Author Summary
Visceral leishmaniasis is a parasitic infection that results in death in susceptible people unless they are treated. Current drugs are expensive and toxic, and there are no vaccines in use in humans. We know that it is possible to become immune to infection with this parasite because people who have been cured using drug treatment are resistant to further infection. In addition, a large percentage of people infected with the parasite remain asymptomatic and develop a specific immune response that can be measured using crude leishmanial antigens. We hypothesized that these resistant people might hold the key to understanding the kind of immune response required for protection. In this paper we compared the immune response to a series of novel vaccine candidates in people with active disease, in those drug-cured from the disease, and in the naturally resistant individuals. We show that immune individuals make strong cytokine responses to five of eleven novel vaccine candidates that were tested, making them ideal candidates to take forward in the development of a defined vaccine against leishmaniasis.
PMCID: PMC3493615  PMID: 23150744
5.  Common variants near ATM are associated with glycemic response to metformin in type 2 diabetes 
Nature genetics  2010;43(2):117-120.
Metformin is the most commonly used pharmacological therapy for type 2 diabetes. We carried out a GWA study on glycaemic response to metformin in 1024 Scottish patients with type 2 diabetes. Replication was in two cohorts consisting of 1783 Scottish patients and 1113 patients from the UK Prospective Diabetes Study. In a meta-analysis (n=3920) we observed an association (P=2.9 *10−9) for a SNP rs11212617 at a locus containing the ataxia telangiectasia mutated (ATM) gene with an odds ratio of 1.35 (95% CI 1.22 to 1.49) for treatment success. In a rat hepatoma cell line, inhibition of ATM with KU-55933 attenuated the phosphorylation and activation of AMPK in response to metformin. We conclude that ATM, a gene known to be involved in DNA repair and cell cycle control, plays a role in the effect of metformin upstream of AMPK, and variation in this gene alters glycaemic response to metformin.
PMCID: PMC3030919  PMID: 21186350
6.  The -2518 bp promoter polymorphism at CCL2/MCP1 influences susceptibility to mucosal but not localized cutaneous leishmaniasis in Brazil 
Mucosal leishmaniasis (ML) follows localized cutaneous leishmaniasis (CL) caused by Leishmania braziliensis. Proinflammatory responses mediate CL self-healing but are exaggerated in ML. Proinflammatory monocyte chemoattractant protein 1 (MCP-1; encoded by CCL2) is associated with CL. We explore its role in CL/ML through analysis of the regulatory CCL2 -2518bp promoter polymorphism in CL/ML population samples and families from Brazil. Genotype frequencies were compared among ML/CL cases and control groups using logistic regression and the family-based association test (FBAT). MCP-1 was measured in plasma and macrophages. The GG recessive genotype at CCL2 -2518bp was more common in patients with ML (N=67) than in neighborhood control (NC; N=60) subjects (OR 1.78; 95% CI 1.01–3.14; P=0.045), than in NC combined with leishmanin skin-test positive (N=60) controls (OR 4.40; 95% CI 1.42–13.65; P=0.010), and than in controls combined with CL (N=60) patients (OR 2.78; 95% CI 1.13–6.85; P=0.045). No associations were observed for CL compared to any groups. FBAT (91 ML and 223 CL cases in families) confirmed recessive association of ML with allele G (Z=2.679; P=0.007). Higher levels of MCP-1 occurred in plasma (P=0.03) and macrophages (P<0.0001) from GG compared to AA individuals. These results suggest that high MCP-1 increases risk of ML.
PMCID: PMC2878927  PMID: 20430117
Mucosal leishmaniasis; genetic association; MCP-1; CCL2
7.  P2X7 receptor-mediated killing of an intracellular parasite, Toxoplasma gondii, by human and murine macrophages1 
The P2X7 receptor (P2X7R)4 is highly expressed on the macrophage cell surface and activation of infected cells by extracellular ATP has been shown to kill intracellular bacteria and parasites. Furthermore, single nucleotide polymorphisms (SNPs) that decrease receptor function reduce the ability of human macrophages to kill Mycobacterium tuberculosis and are associated with extrapulmonary tuberculosis. In this paper we show that macrophages from people with the 1513C (rs3751143) loss-of-function P2X7R SNP are less effective in killing intracellular Toxoplasma gondii after exposure to ATP compared with macrophages from people with the 1513A wild-type allele. Supporting a P2X7R-specific effect on T. gondii, macrophages from P2X7R knock-out mice (P2X7R−/−) are unable to kill T. gondii as effectively as macrophages from wild-type mice. We show that P2X7R-mediated T. gondii killing occurs in parallel with host cell apoptosis and is independent of NO production.
PMCID: PMC2931343  PMID: 20488797
8.  TLR1/2 Activation during Heterologous Prime-Boost Vaccination (DNA-MVA) Enhances CD8+ T Cell Responses Providing Protection against Leishmania (Viannia) 
Leishmania (Viannia) parasites present particular challenges, as human and murine immune responses to infection are distinct from other Leishmania species, indicating a unique interaction with the host. Further, vaccination studies utilizing small animal models indicate that modalities and antigens that prevent infection by other Leishmania species are generally not protective.
Using a newly developed mouse model of chronic L. (Viannia) panamensis infection and the heterologous DNA prime – modified vaccinia virus Ankara (MVA) boost vaccination modality, we examined whether the conserved vaccine candidate antigen tryparedoxin peroxidase (TRYP) could provide protection against infection/disease.
Heterologous prime – boost (DNA/MVA) vaccination utilizing TRYP antigen can provide protection against disease caused by L. (V.) panamensis. However, protection is dependent on modulating the innate immune response using the TLR1/2 agonist Pam3CSK4 during DNA priming. Prime-boost vaccination using DNA alone fails to protect. Prior to infection protectively vaccinated mice exhibit augmented CD4 and CD8 IFNγ and memory responses as well as decreased IL-10 and IL-13 responses. IL-13 and IL-10 have been shown to be independently critical for disease in this model. CD8 T cells have an essential role in mediating host defense, as CD8 depletion reversed protection in the vaccinated mice; vaccinated mice depleted of CD4 T cells remained protected. Hence, vaccine-induced protection is dependent upon TLR1/2 activation instructing the generation of antigen specific CD8 cells and restricting IL-13 and IL-10 responses.
Given the general effectiveness of prime-boost vaccination, the recalcitrance of Leishmania (Viannia) to vaccine approaches effective against other species of Leishmania is again evident. However, prime-boost vaccination modality can with modulation induce protective responses, indicating that the delivery system is critical. Moreover, these results suggest that CD8 T cells should be targeted for the development of a vaccine against infection caused by Leishmania (Viannia) parasites. Further, TLR1/2 modulation may be useful in vaccines where CD8 T cell responses are critical.
Author Summary
Leishmania (Viannia) are the predominant agents of leishmaniasis in Latin America. Given the fact that leishmaniasis is a zoonosis, eradication is unlikely; a vaccine could provide effective prevention of disease. However, these parasites present a challenge and we do not fully understand what elements of the host immune defense prevent disease. We examined the ability of vaccination to protect against L. (Viannia) infection using the highly immunogenic heterologous prime-boost (DNA-modified vaccinia virus) modality and a single Leishmania antigen (TRYP). Although this mode of vaccination can induce protection against other leishmaniases (cutaneous, visceral), no protection was observed against L. (V.) panamensis. However, we found that if the vaccination was modified and the innate immune response was activated through Toll-like receptor1/2(TLR1/2) during the DNA priming, vaccinated mice were protected. Protection was dependent on CD8 T cells. Vaccinated mice had higher CD8 T cell responses and decreased levels of cytokines known to promote infection. Given the long-term persistence of CD8 T cell memory, these findings are encouraging for vaccine development. Further, these results suggest that modulation of TLR1/2 signaling could improve the efficacy of DNA-based vaccines, especially where CD8 T cell activation is critical, thereby contributing to effective and affordable anti parasitic vaccines.
PMCID: PMC3114751  PMID: 21695103
9.  Geographic Information Systems and Applied Spatial Statistics Are Efficient Tools to Study Hansen's Disease (Leprosy) and to Determine Areas of Greater Risk of Disease 
Applied Spatial Statistics used in conjunction with geographic information systems (GIS) provide an efficient tool for the surveillance of diseases. Here, using these tools we analyzed the spatial distribution of Hansen's disease in an endemic area in Brazil. A sample of 808 selected from a universe of 1,293 cases was geocoded in Mossoró, Rio Grande do Norte, Brazil. Hansen's disease cases were not distributed randomly within the neighborhoods, with higher detection rates found in more populated districts. Cluster analysis identified two areas of high risk, one with a relative risk of 5.9 (P = 0.001) and the other 6.5 (P = 0.001). A significant relationship between the geographic distribution of disease and the social economic variables indicative of poverty was observed. Our study shows that the combination of GIS and spatial analysis can identify clustering of transmissible disease, such as Hansen's disease, pointing to areas where intervention efforts can be targeted to control disease.
PMCID: PMC2813173  PMID: 20134009
11.  HLA and Infectious Diseases †  
Clinical Microbiology Reviews  2009;22(2):370-385.
Summary: Following their discovery in the early 1970s, classical human leukocyte antigen (HLA) loci have been the prototypical candidates for genetic susceptibility to infectious disease. Indeed, the original hypothesis for the extreme variability observed at HLA loci (H-2 in mice) was the major selective pressure from infectious diseases. Now that both the human genome and the molecular basis of innate and acquired immunity are understood in greater detail, do the classical HLA loci still stand out as major genes that determine susceptibility to infectious disease? This review looks afresh at the evidence supporting a role for classical HLA loci in susceptibility to infectious disease, examines the limitations of data reported to date, and discusses current advances in methodology and technology that will potentially lead to greater understanding of their role in infectious diseases in the future.
PMCID: PMC2668228  PMID: 19366919
12.  Post-Genomic Research on Leishmaniasis 
Trends in parasitology  2008;24(9):401-405.
Scientific conferences, a major feature of academic life, rarely provide the opportunity for self appraisal of a research field. During a recent meeting on leishmaniasis research in the post-genomic era, ~60 researchers participated in group discussions that aimed to provide a critical self-appraisal of the state of the field and to highlight major roadblocks likely to prevent translation of new research into tools for leishmaniasis control. These discussions demonstrated a surprising concordance of views and highlighted a number of critical areas for future development.
PMCID: PMC2817896  PMID: 18684668
13.  Host genetic and epigenetic factors in toxoplasmosis 
Memorias do Instituto Oswaldo Cruz  2009;104(2):162-169.
Analysing human genetic variation provides a powerful tool in understanding risk factors for disease. Toxoplasma gondii acquired by the mother can be transmitted to the fetus. Infants with the most severe clinical signs in brain and eye are those infected early in pregnancy when fetal immunity is least well developed. Genetic analysis could provide unique insight into events in utero that are otherwise difficult to determine. We tested the hypothesis that propensity for T. gondii to cause eye disease is associated with genes previously implicated in congenital or juvenile onset ocular disease. Using mother-child pairs from Europe (EMSCOT) and child/parent trios from North America (NCCCTS), we demonstrated that ocular and brain disease in congenital toxoplasmosis associate with polymorphisms in ABCA4 encoding ATP-binding cassette transporter, subfamily A, member 4 previously associated with juvenile onset retinal dystrophies including Stargardt's disease. Polymorphisms at COL2A1 encoding type II collagen, previously associated with Stickler syndrome, associated only with ocular disease in congenital toxoplasmosis. Experimental studies showed that both ABCA4 and COL2A1 show isoform-specific epigenetic modifications consistent with imprinting, which provided an explanation for the patterns of inheritance observed. These genetic and epigenetic risk factors provide unique insight into molecular pathways in the pathogenesis of disease.
PMCID: PMC2735098  PMID: 19430638
congenital infection; toxoplasmosis; genetics; epigenetics
14.  A prime/boost DNA/Modified vaccinia virus Ankara vaccine expressing recombinant Leishmania DNA encoding TRYP is safe and immunogenic in outbred dogs, the reservoir of zoonotic visceral leishmaniasis 
Vaccine  2009;27(7):1080-1086.
Previous studies demonstrated safety, immunogenicity and efficacy of DNA/modified vaccinia virus Ankara (MVA) prime/boost vaccines expressing tryparedoxin peroxidase (TRYP) and Leishmania homologue of the mammalian receptor for activated C kinase (LACK) against Leishmania major challenge in mice, which was consistent with results from TRYP protein/adjuvant combinations in non-human primates. This study aimed to conduct safety and immunogenicity trials of these DNA/MVA vaccines in dogs, the natural reservoir host of Leishmania infantum, followed-up for 4 months post-vaccination.
In a cohort of 22 uninfected outbred dogs, blinded randomised administration of 1000 μg (high dose) or 100 μg (low dose) DNA prime (day 0) and 1 × 108 pfu MVA boost (day 28) was shown to be safe and showed no clinical side effects. High dose DNA/MVA vaccinated TRYP dogs produced statistically higher mean levels of the type-1 pro-inflammatory cytokine IFN-γ than controls in whole blood assays (WBA) stimulated with the recombinant vaccine antigen TRYP, up to the final sampling at day 126, and in the absence of challenge with Leishmania. TRYP vaccinated dogs also demonstrated significantly higher TRYP-specific total IgG and IgG2 subtype titres than in controls, and positive in vivo intradermal reactions at day 156 in the absence of natural infection, observed in 6/8 TRYP vaccinated dogs. No significant increases in IFN-γ in LACK-stimulated WBA, or in LACK-specific IgG levels, were detected in LACK vaccinated dogs compared to controls, and only 2/9 LACK vaccinated dogs demonstrated DTH responses at day 156. In all groups, IgG1 subclass responses and antigen-specific stimulation of IL-10 were similar to controls demonstrating an absence of Th2/Treg response, as expected in the absence of in vivo restimulation or natural/experimental challenge with Leishmania.
These collective results indicate significant antigen-specific type-1 responses and in vivo memory phase cellular immune responses, consistent with superior potential for protective vaccine immunogenicity of DNA/MVA TRYP over LACK.
PMCID: PMC2663027  PMID: 19095029
Leishmania infantum; Tryparedoxin peroxidase; Prime/boost DNA/MVA vaccination
15.  Genes at Human Chromosome 5q31.1 Regulate Delayed Type Hypersensitivity Responses Associated with Leishmania chagasi Infection 
Genes and immunity  2007;8(7):539-551.
Visceral leishmaniasis (VL) caused by Leishmania chagasi is endemic to northeast Brazil. A positive delayed-type hypersensitivity skin test response (DTH+) is a marker for acquired resistance to disease, clusters in families, and may be genetically controlled. Twenty-three single nucleotide polymorphisms (SNPs) were genotyped in the cytokine 5q23.3-q31.1 region IRF1-IL5-IL13-IL4-IL9-LECT2-TGFBI in 102 families (323 DTH+; 190 DTH−; 123 VL individuals) from a VL endemic region in northeast Brazil. Data from 20 SNPs were analysed for association with DTH+/− status and VL using family-based, stepwise conditional logistic regression analysis. Independent associations were observed between the DTH+ phenotype and markers in separate linkage disequilibrium blocks in LECT2 (OR 2.25; P=0.005; 95% CI=1.28-3.97) and TGFBI (OR 1.94; P=0.003; 95% CI=1.24-3.03). VL child/parent trios gave no evidence of linkage and association, but the DTH− phenotype was associated with SNP rs2070874 at IL4 (OR 3.14; P=0.006; 95% CI=1.38-7.14), and SNP rs30740 between LECT2 and TGFBI (OR 3.00; P=0.042; 95% CI=1.04-8.65). These results indicate several genes in the immune response gene cluster at 5q23.3-q31.1 influence outcomes of L. chagasi infection in this region of Brazil.
PMCID: PMC2435172  PMID: 17713557
16.  Typhoid Fever and Genetic Polymorphisms at the Natural Resistance-Associated Macrophage Protein 1 (NRAMP1) 
The Journal of infectious diseases  2001;183(7):1156-1160.
Control of Salmonella enterica serovar Typhimurium (S. typhimurium) infection in the mouse model of typhoid fever is critically dependent on the natural resistance-associated macrophage protein 1, Nramp1. Here we examined the role of genetic polymorphisms in the human homologue, NRAMP1, in resistance to typhoid fever in southern Vietnam. Patients with blood-culture confirmed typhoid fever and healthy controls were genotyped for six polymorphic markers within and near NRAMP1 on chromosome 2q35. Four single base pair polymorphisms (274 C/T, 469+14 G/C, 1465-85 G/A, D543N), a (GT)n repeat in the promoter region of NRAMP1 and D2S1471, a microsatellite marker approximately 130 kb downstream of NRAMP1, were examined. The allelic and genotypic frequencies for each polymorphism were compared in cases and controls. No allelic association was identified between the NRAMP1 alleles and typhoid fever susceptibility. In addition, neither homozygotes or heterozygotes for any of the NRAMP1 variants were at increased risk of typhoid fever.
PMCID: PMC2413323  PMID: 11237848
17.  IFNG and IFNGR1 gene polymorphisms and susceptibility to post kala-azar dermal leishmaniasis in Sudan 
Genes and immunity  2006;8(1):75-78.
Post kala-azar dermal leishmanaisis (PKDL) in Sudan is associated with elevated interferon-γ. To study interferon-γ pathways in PKDL, we genotyped 80 trios from the Masalit ethnic group for polymorphisms at −470 ins/delTT, -270T/C, -56T/C and +95T/C in IFNGR1, and at −179G/A and +874T/A in IFNG. No associations occurred at IFNG. Global association with haplotypes comprising all 4 markers at IFNGR1 (χ210df = 21.97, P=0.015) was observed, associated with a significant (χ21df=4.54, P=0.033) bias in transmission of the haplotype insTT T T T, and less (χ21df=5.59, P=0.018) than expected transmission of insTT C C C. When compared with data on malaria associations from The Gambia, the results suggest a complex pattern of haplotypic variation at the IFNGR1 promoter locus associated with different infectious disease in African populations that reflect the complex roles of IFN-γ in parasite killing versus inflammation and pathogenesis.
PMCID: PMC2330095  PMID: 17136124
PKDL; leishmaniasis; association; IFNGR1
18.  Genetic Predisposition to Self-Curing Infection with the Protozoan Leishmania chagasi: A Genome Wide Scan 
The Journal of infectious diseases  2007;196(8):1261-1269.
The protozoan Leishmania chagasi can cause disseminated, fatal visceral leishmaniasis (VL) or asymptomatic human infection. We hypothesized that genetic factors contribute to this variable response to infection. A family study was performed in endemic neighborhoods near Natal, northeast Brazil. Subjects were assessed for VL or asymptomatic infection, defined as a positive delayed type hypersensitivity (DTH) skin test response to Leishmania antigen without disease symptoms. A genome scan of 405 microsatellite markers in 1254 subjects was analyzed for regions of linkage. The results indicated loci of potential linkage to DTH response on chromosomes 2, 13, 15 and 19, and a novel region of potential interest for VL on chromosome 9. An understanding of the genetic factors determining whether an individual will develop symptomatic or asymptomatic infection with L. chagasi may illuminate proteins essential for immune protection against this parasitic disease; findings could reveal strategies for immunotherapy or prevention.
PMCID: PMC2330094  PMID: 17955446
Visceral leishmaniasis; Delayed Type Hypersensitivity; Linkage Analysis; genetic susceptibility
19.  Genetic susceptibility to infectious diseases: big is beautiful (and will bigger be even better?) 
The Lancet infectious diseases  2006;6(10):653-663.
Genetic epidemiology, including twin studies, provides robust evidence that genetic variation in human populations contributes to susceptibility to infectious disease. One of the major limitations of studies to date that attempt to identify the genes and mechanisms underlying this susceptibility has been lack of power due to small sample size. With the development of novel technologies, burgeoning information on the human genome, the HapMap project, and human genetic diversity, we are at the beginning of a new era in the study of genetics of complex diseases. This review looks afresh at the epidemiological evidence supporting a role for genetics in susceptibility to infectious disease, examines the somewhat limited achievements to date, and discusses current advances in methodology and technology that will potentially lead to translational data in the future.
PMCID: PMC2330096  PMID: 17008174
20.  Slc11a1, Formerly Nramp1, Is Expressed in Dendritic Cells and Influences Major Histocompatibility Complex Class II Expression and Antigen-Presenting Cell Function▿  
Infection and Immunity  2007;75(10):5059-5067.
Solute carrier family 11 member a1 (Slc11a1; formerly Nramp1) encodes a late endosomal/lysosomal protein/divalent cation transporter that regulates iron homeostasis in macrophages. During macrophage activation, Slc11a1 has multiple pleiotropic effects on gene regulation and function, including gamma interferon-induced class II expression and antigen-presenting cell function. The wild-type allele at Slc11a1 has been associated with a bias in Th1 cell function in vivo, which is beneficial in resistance to infection against intracellular macrophage pathogens but detrimental in contributing to development of type 1 diabetes. The extent to which this depends on macrophage versus dendritic cell (DC) function is not known. Here we show that Slc11a1 is expressed in late endosomes and/or lysosomes of CD11c+ DCs. DCs from mutant and congenic wild-type mice upregulate interleukin-12 (IL-12) and IL-10 mRNA in response to lipopolysaccharide (LPS) stimulation, but the ratio of IL-10 to IL-12 is higher in unstimulated DCs and DCs stimulated for 15 h with LPS from mutant mice than from wild-type mice. DCs from wild-type mice upregulate major histocompatibility complex class II in response to LPS more efficiently than DCs from mutant mice. Unstimulated DCs from wild-type and mutant mice present ovalbumin (OVA) peptide with an efficiency equivalent to that of an OVA-specific CD4 T-cell line, but DCs from wild-type mice are more efficient at processing and presenting OVA or Leishmania activator of cell kinase (LACK) protein to OVA- and LACK-specific T cells. These data indicate that wild-type Slc11a1 expressed in DCs may play a role both in determining resistance to infectious disease and in susceptibility to autoimmune disease such as type 1 diabetes.
PMCID: PMC2044529  PMID: 17620357
21.  Heterologous Priming-Boosting with DNA and Modified Vaccinia Virus Ankara Expressing Tryparedoxin Peroxidase Promotes Long-Term Memory against Leishmania major in Susceptible BALB/c Mice▿  
Infection and Immunity  2006;75(2):852-860.
Leishmaniasis affects 12 million people, but there are no vaccines in routine clinical use. Th1 polarizing vaccines that elicit long-term protection are required to prevent disease in susceptible populations. We recently showed that heterologous priming-boosting with tryparedoxin peroxidase (TRYP) DNA followed by TRYP-modified vaccinia virus Ankara (TRYP MVA) protected susceptible BALB/c mice from Leishmania major. Here we compared treatment with TRYP DNA with treatment with TRYP DNA/TRYP MVA. We found that equivalent levels of protection during the postvaccination effector phase correlated with equivalent levels of serum immunoglobulin G2a and gamma interferon (IFN-γ) in draining lymph nodes. In contrast, challenge infection during the memory phase revealed that there was enhanced clinical efficacy with TRYP DNA/TRYP MVA. This correlated with higher levels of effector phase splenic IFN-γ, sustained prechallenge levels of memory phase IFN-γ, and a more polarized post-L. major challenge Th1 response compared to the Th2/Treg response. Thus, TRYP DNA/TRYP MVA, but not TRYP DNA alone, provides long-term protection against murine leishmaniasis.
PMCID: PMC1828487  PMID: 17101647
22.  Genetic susceptibility to infectious diseases: big is beautiful, but will bigger be even better? 
The Lancet Infectious Diseases  2006;6(10):653-663.
Genetic epidemiology, including twin studies, provides robust evidence that genetic variation in human populations contributes to susceptibility to infectious disease. One of the major limitations of studies that attempt to identify the genes and mechanisms that underlie this susceptibility has been lack of power caused by small sample size. With the development of novel technologies, burgeoning information on the human genome, the HapMap project, and human genetic diversity, we are at the beginning of a new era in the study of the genetics of complex diseases. This review looks afresh at the epidemiological evidence that supports a role for genetics in susceptibility to infectious disease, examines the somewhat limited achievements to date, and discusses current advances in methodology and technology that will potentially lead to translational data in the future.
PMCID: PMC2330096  PMID: 17008174
23.  Interleukin-4 (IL-4) and IL-10 Collude in Vaccine Failure for Novel Exacerbatory Antigens in Murine Leishmania major Infection  
Infection and Immunity  2005;73(11):7620-7628.
Leishmaniasis affects 12 million people but there are no vaccines in routine use. Recently, we used DNA vaccination in a susceptible BALB/c high-dose model of infection to screen 100 novel Leishmania major genes as vaccine candidates. In addition to finding novel protective antigens, we identified several antigens that reproducibly exacerbated disease. Here we examined the immune response to two of these antigens, lmd29 and 584C, that were originally identified in an expressed sequence tag cDNA sequencing project. We show that, in addition to exacerbating disease in susceptible BALB/c mice, these antigens retain a propensity to exacerbate disease in resistant C57BL/6 mice. This ability to exacerbate disease was lost when susceptible BALB/c mice were rendered resistant by disruption of the genes encoding interleukin-4 (IL-4) alone, IL-4/IL-13, or IL-4, IL-5, IL-9, and IL-13. Failure to exacerbate disease was associated with reduced IL-5 and IL-10 production in IL-4 knockout mice. Treatment of lmd29-vaccinated mice with anti-IL-10 receptor antibody prior to challenge infection converted exacerbation in wild-type BALB/c mice into highly significant antigen-specific protection. These studies demonstrate that some highly immunogenic antigens of L. major, while having an intrinsic capacity to exacerbate disease in the context of otherwise T helper 1-promoting DNA vaccine delivery, can be rendered protective by the removal of functional IL-10.
PMCID: PMC1273906  PMID: 16239566
24.  DNA-Salmonella enterica Serovar Typhimurium Primer-Booster Vaccination Biases towards T Helper 1 Responses and Enhances Protection against Leishmania major Infection in Mice  
Infection and Immunity  2004;72(8):4924-4928.
Successful resolution of infections by intracellular pathogens requires gamma interferon (IFN-γ). DNA vaccines promote T helper 1 (Th1) responses by triggering interleukin-12 (IL-12) release by dendritic cells (DC) through Toll-like receptor 9 (TLR9). In humans TLR9 is restricted to plasmacytoid DC. Here we show that DNA-Salmonella enterica serovar Typhimurium primer-booster vaccination, which provides alternative ligands to bind TLR4 on myeloid DC, strongly biases towards Th1 responses compared to vaccination with DNA alone. This results in higher immunoglobulin G2a (IgG2a) responses compared to IgG1 responses, higher IFN-γ responses compared to IL-10 CD4+-T-cell responses, and enhanced protection against Leishmania major infection in susceptible BALB/c mice.
PMCID: PMC470645  PMID: 15271962
25.  From genomes to vaccines: Leishmania as a model. 
The 35 Mb genome of Leishmania should be sequenced by late 2002. It contains approximately 8500 genes that will probably translate into more than 10 000 proteins. In the laboratory we have been piloting strategies to try to harness the power of the genome-proteome for rapid screening of new vaccine candidate. To this end, microarray analysis of 1094 unique genes identified using an EST analysis of 2091 cDNA clones from spliced leader libraries prepared from different developmental stages of Leishmania has been employed. The plan was to identify amastigote-expressed genes that could be used in high-throughput DNA-vaccine screens to identify potential new vaccine candidates. Despite the lack of transcriptional regulation that polycistronic transcription in Leishmania dictates, the data provide evidence for a high level of post-transcriptional regulation of RNA abundance during the developmental cycle of promastigotes in culture and in lesion-derived amastigotes of Leishmania major. This has provided 147 candidates from the 1094 unique genes that are specifically upregulated in amastigotes and are being used in vaccine studies. Using DNA vaccination, it was demonstrated that pooling strategies can work to identify protective vaccines, but it was found that some potentially protective antigens are masked by other disease-exacerbatory antigens in the pool. A total of 100 new vaccine candidates are currently being tested separately and in pools to extend this analysis, and to facilitate retrospective bioinformatic analysis to develop predictive algorithms for sequences that constitute potentially protective antigens. We are also working with other members of the Leishmania Genome Network to determine whether RNA expression determined by microarray analyses parallels expression at the protein level. We believe we are making good progress in developing strategies that will allow rapid translation of the sequence of Leishmania into potential interventions for disease control in humans.
PMCID: PMC1692919  PMID: 11839176

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