RESUMEN
Molecular analyses of lung aspirates from Gambian children with severe pneumonia detected pathogens more frequently than did culture and showed a predominance of bacteria, principally Streptococcus pneumoniae, >75% being of serotypes covered by current pneumococcal conjugate vaccines. Multiple pathogens were detected frequently, notably Haemophilus influenzae (mostly nontypeable) together with S. pneumoniae.
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Haemophilus influenzae/aislamiento & purificación , Pulmón/microbiología , Neumonía Bacteriana/diagnóstico , Neumonía Bacteriana/microbiología , Streptococcus pneumoniae/aislamiento & purificación , África Occidental , Proteínas Bacterianas/genética , Proteínas Portadoras/genética , Preescolar , Coinfección , Gambia , Infecciones por Haemophilus/diagnóstico , Infecciones por Haemophilus/microbiología , Haemophilus influenzae/genética , Humanos , Inmunoglobulina D/genética , Lactante , Lipoproteínas/genética , Tipificación de Secuencias Multilocus , Vacunas Neumococicas , Neumonía Bacteriana/diagnóstico por imagen , Neumonía Neumocócica/diagnóstico , Neumonía Neumocócica/microbiología , Neumonía Viral/diagnóstico , Neumonía Viral/virología , Reacción en Cadena de la Polimerasa , Radiografía , Serogrupo , Streptococcus pneumoniae/genética , Virus/aislamiento & purificaciónRESUMEN
We previously described the NOD.c3c4 mouse, which is protected from type 1 diabetes (T1D) because of protective alleles at multiple insulin-dependent diabetes (Idd) genes, but develops autoimmune biliary disease (ABD) resembling primary biliary cirrhosis (PBC). In this paper, we characterize the NOD.ABD strain, which is genetically related to the NOD.c3c4 strain but develops both ABD and T1D. Histologically, NOD.ABD biliary disease is indistinguishable from that in NOD.c3c4 mice. The frequency of effector memory (CD44(+)CD62L(-)) and central memory (CD44(+)CD62L(+)) CD8 T cells is significantly increased in the intrahepatic lymphocyte fraction of NOD.ABD mice, and NOD.ABD CD8 T cells produce more IFN-γ and TNF-α, compared with controls. NOD.ABD splenocytes can transfer ABD and T1D to NOD.c3c4 scid mice, but only T1D to NOD scid mice, suggesting that the genetic origin of the target organ and/or its innate immune cells is critical to disease pathogenesis. The disease transfer model, importantly, shows that biliary duct damage (characteristic of PBC) and inflammation precede biliary epithelial cell proliferation. Unlike T1D where both CD4 and CD8 T cells are required for disease transfer, purified NOD.ABD CD8 T cells can transfer liver inflammation into NOD.c3c4 scid recipients, and disease transfer is ameliorated by cotransferring T regulatory cells. Unlike NOD.c3c4 mice, NOD.ABD mice do not develop anti-nuclear or anti-Smith autoantibodies; however, NOD.ABD mice do develop the antipyruvate dehydrogenase Abs typical of human PBC. The NOD.ABD strain is a model of immune dysregulation affecting two organ systems, most likely by mechanisms that do not completely coincide.
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Conductos Biliares/inmunología , Conductos Biliares/patología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/patología , Cirrosis Hepática Biliar/inmunología , Cirrosis Hepática Biliar/patología , Traslado Adoptivo , Animales , Cruzamientos Genéticos , Diabetes Mellitus Tipo 1/genética , Modelos Animales de Enfermedad , Femenino , Humanos , Células K562 , Cirrosis Hepática Biliar/genética , Masculino , Ratones , Ratones Congénicos , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones SCID , Especificidad de Órganos/genética , Especificidad de Órganos/inmunologíaRESUMEN
Dizygotic (DZ) twinning has a genetic component and is common among sub-Saharan Africans; in The Gambia its frequency is up to 3% of live births. Variation in PTX3, encoding Pentraxin 3, a soluble pattern recognition receptor that plays an important role both in innate immunity and in female fertility, has been associated with resistance to Mycobacterium tuberculosis pulmonary disease and to Pseudomonas aeruginosa infection in cystic fibrosis patients. We tested whether PTX3 variants in Gambian women associate with DZ twinning, by genotyping five PTX3 single nucleotide polymorphisms (SNPs) in 130 sister pairs (96 full sibs and 34 half sibs) who had DZ twins. Two, three and five SNP haplotypes differed in frequency between twinning mothers and those without a history of twinning (from P = 0.006 to 3.03e-06 for two SNP and three SNP haplotypes, respectively). Twinning mothers and West African tuberculosis-controls from a previous study shared several frequent haplotypes. Most importantly, our data are consistent with an independently reported association of PTX3 and female fertility in a sample from Ghana. Taken together, these results indicate that selective pressure on PTX3 variants that affect the innate immune response to infectious agents, could also produce the observed high incidence of DZ twinning in Gambians.
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Proteína C-Reactiva/genética , Inmunidad Innata/genética , Polimorfismo de Nucleótido Simple , Componente Amiloide P Sérico/genética , Gemelos Dicigóticos/genética , Alelos , Estudios de Casos y Controles , Cromosomas Humanos Par 3 , Femenino , Gambia , Frecuencia de los Genes , Orden Génico , Haplotipos , Humanos , Desequilibrio de LigamientoRESUMEN
Systemic bacteraemia has been reported in children with severe Plasmodium falciparum malaria in Sub Saharan Africa, making the identification or exclusion of concurrent infections a prerequisite for adequate treatment and studies of the immune responses to particular infections. Given the overlap in clinical signs in humans between malaria and, for example, pneumonia, the true cause of severe illness is sometimes difficult to establish. Traditional microbiological culture methods employed to detect systemic bacteraemia are often time consuming and have modest sensitivity. Therefore, molecular methods have become increasingly used in the diagnosis of septicaemia. Here, we evaluated the usefulness of both broad-range 16S rRNA PCR, in conjunction with DNA sequencing and species-specific PCR targeting of Streptococcus pneumoniae and non-typhoidal Salmonella, to screen for bacterial co-infections in blood samples from children enrolled in a malaria pathogenesis study. PCR revealed no test-positive results for these pathogens and DNA sequencing of 16S rRNA amplicons identified the presence of bacterial genomic DNA (most probably from environmental bacterial sources) in a large proportion of samples. We demonstrate that the issue of potential mixed bacteraemic infection and/or background bacterial genomic DNA, which may relate to co-migration of PCR amplicons on agarose gels, can be overcome by using denaturing gradient gel electrophoresis (DGGE). PCR for Plasmodium spp. was also performed on genomic DNA from bloods from Gambian children with pneumonia, in order to estimate the prevalence of Plasmodium/pneumonia co-infections in the study population. While 12.2% of samples were test-positive, parasite density was very low and did not vary significantly between cases and controls.
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Infecciones Bacterianas/diagnóstico , Malaria Falciparum/diagnóstico , Adolescente , Infecciones Bacterianas/epidemiología , Infecciones Bacterianas/metabolismo , Estudios de Casos y Controles , Niño , Preescolar , Coinfección , ADN Bacteriano/análisis , Femenino , Gambia/epidemiología , Genoma Bacteriano , Humanos , Lactante , Malaria Falciparum/metabolismo , Masculino , Reacción en Cadena de la Polimerasa , Prevalencia , ARN Ribosómico 16S/análisis , Análisis de Secuencia de ADNRESUMEN
BACKGROUND: The aetiology of the autoimmune disease type 1 diabetes (T1D) involves many genetic and environmental factors. Evidence suggests that innate immune responses, including the action of interferons, may also play a role in the initiation and/or pathogenic process of autoimmunity. In the present report, we have adopted a linkage disequilibrium (LD) mapping approach to test for an association between T1D and three regions encompassing 13 interferon alpha (IFNA) genes, interferon omega-1 (IFNW1), interferon beta-1 (IFNB1), interferon gamma (IFNG) and the interferon consensus-sequence binding protein 1 (ICSBP1). RESULTS: We identified 238 variants, most, single nucleotide polymorphisms (SNPs), by sequencing IFNA, IFNB1, IFNW1 and ICSBP1, 98 of which where novel when compared to dbSNP build 124. We used polymorphisms identified in the SeattleSNP database for INFG. A set of tag SNPs was selected for each of the interferon and interferon-related genes to test for an association between T1D and this complex gene family. A total of 45 tag SNPs were selected and genotyped in a collection of 472 multiplex families. CONCLUSION: We have developed informative sets of SNPs for the interferon and interferon related genes. No statistical evidence of a major association between T1D and any of the interferon and interferon related genes tested was found.
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Diabetes Mellitus Tipo 1/genética , Predisposición Genética a la Enfermedad , Interferones/genética , Polimorfismo Genético , Enfermedades Autoinmunes/genética , Bases de Datos Genéticas , Exones , Salud de la Familia , Femenino , Ligamiento Genético , Humanos , Interferón Tipo I/genética , Interferón-alfa/genética , Interferón beta/genética , Interferón gamma/genética , Desequilibrio de Ligamiento , Masculino , Modelos Estadísticos , Familia de Multigenes , Polimorfismo de Nucleótido Simple , Análisis de Secuencia de ADNRESUMEN
The monocyte chemotactic protein-1 (MCP-1) is a chemokine that plays an important role in the recruitment of monocytes to M. tuberculosis infection sites, and previous studies have reported that genetic variants in MCP1 are associated with differential susceptibility to pulmonary tuberculosis (PTB). We examined eight MCP1 single nucleotide polymorphisms (SNPs) in a multi-ethnic, case-control design that included: 321 cases and 346 controls from Guinea-Bissau, 258 cases and 271 controls from The Gambia, 295 cases and 179 controls from the U.S. (African-Americans), and an additional set of 237 cases and 144 controls of European ancestry from the U.S. and Argentina. Two locus interactions were also examined for polymorphisms in MCP1 and interleukin 12B (IL12B), another gene implicated in PTB risk. Examination of previously associated MCP1 SNPs rs1024611 (-2581A/G), rs2857656 (-362G/C) and rs4586 (+900C/T) did not show evidence for association. One interaction between rs2857656 and IL12B SNP rs2288831 was observed among Africans but the effect was in the opposite direction in Guineans (OR = 1.90, p = 0.001) and Gambians (OR = 0.64, p = 0.024). Our data indicate that the effect of genetic variation within MCP1 is not clear cut and additional studies will be needed to elucidate its role in TB susceptibility.
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Quimiocina CCL2/genética , Epistasis Genética , Subunidad p40 de la Interleucina-12/genética , Polimorfismo Genético , Tuberculosis Pulmonar/diagnóstico , Tuberculosis Pulmonar/genética , Adolescente , Adulto , Negro o Afroamericano , Anciano , Argentina , Población Negra , Estudios de Casos y Controles , Quimiocina CCL2/biosíntesis , Etnicidad , Femenino , Gambia , Predisposición Genética a la Enfermedad , Variación Genética , Guinea Bissau , Humanos , Masculino , Persona de Mediana Edad , Estados Unidos , Población BlancaRESUMEN
We examined whether polymorphisms in interleukin-12B (IL12B) associate with susceptibility to pulmonary tuberculosis (PTB) in two West African populations (from The Gambia and Guinea-Bissau) and in two independent populations from North and South America. Nine polymorphisms (seven SNPs, one insertion/deletion, one microsatellite) were analyzed in 321 PTB cases and 346 controls from Guinea-Bissau and 280 PTB cases and 286 controls from The Gambia. For replication we studied 281 case and 179 control African-American samples and 221 cases and 144 controls of European ancestry from the US and Argentina. First-stage single locus analyses revealed signals of association at IL12B 3' UTR SNP rs3212227 (unadjusted allelic pâ=â0.04; additive genotypic pâ=â0.05, ORâ=â0.78, 95% CI [0.61-0.99]) in Guinea-Bissau and rs11574790 (unadjusted allelic pâ=â0.05; additive genotypic pâ=â0.05, ORâ=â0.76, 95% CI [0.58-1.00]) in The Gambia. Association of rs3212227 was then replicated in African-Americans (rs3212227 allelic pâ=â0.002; additive genotypic pâ=â0.05, ORâ=â0.78, 95% CI [0.61-1.00]); most importantly, in the African-American cohort, multiple significant signals of association (seven of the nine polymorphisms tested) were detected throughout the gene. These data suggest that genetic variation in IL12B, a highly relevant candidate gene, is a risk factor for PTB in populations of African ancestry, although further studies will be required to confirm this association and identify the precise mechanism underlying it.
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Variación Genética , Subunidad p40 de la Interleucina-12/genética , Tuberculosis Pulmonar/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Argentina/epidemiología , Población Negra/genética , Estudios de Casos y Controles , Estudios de Cohortes , Femenino , Gambia/epidemiología , Frecuencia de los Genes , Estudios de Asociación Genética , Variación Genética/fisiología , Genética de Población , Guinea Bissau/epidemiología , Humanos , Desequilibrio de Ligamiento , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple/fisiología , Tuberculosis Pulmonar/epidemiología , Tuberculosis Pulmonar/etnología , Estados Unidos/epidemiología , Adulto JovenRESUMEN
BACKGROUND: Pneumonia remains the leading cause of death in young children globally and improved diagnostics are needed to better identify cases and reduce case fatality. Metabolomics, a rapidly evolving field aimed at characterizing metabolites in biofluids, has the potential to improve diagnostics in a range of diseases. The objective of this pilot study is to apply metabolomic analysis to childhood pneumonia to explore its potential to improve pneumonia diagnosis in a high-burden setting. METHODOLOGY/PRINCIPAL FINDINGS: Eleven children with World Health Organization (WHO)-defined severe pneumonia of non-homogeneous aetiology were selected in The Gambia, West Africa, along with community controls. Metabolomic analysis of matched plasma and urine samples was undertaken using Ultra Performance Liquid Chromatography (UPLC) coupled to Time-of-Flight Mass Spectrometry (TOFMS). Biomarker extraction was done using SIMCA-P+ and Random Forests (RF). 'Unsupervised' (blinded) data were analyzed by Principal Component Analysis (PCA), while 'supervised' (unblinded) analysis was by Partial Least Squares-Discriminant Analysis (PLS-DA) and Orthogonal Projection to Latent Structures (OPLS). Potential markers were extracted from S-plots constructed following analysis with OPLS, and markers were chosen based on their contribution to the variation and correlation within the data set. The dataset was additionally analyzed with the machine-learning algorithm RF in order to address issues of model overfitting and markers were selected based on their variable importance ranking. Unsupervised PCA analysis revealed good separation of pneumonia and control groups, with even clearer separation of the groups with PLS-DA and OPLS analysis. Statistically significant differences (p<0.05) between groups were seen with the following metabolites: uric acid, hypoxanthine and glutamic acid were higher in plasma from cases, while L-tryptophan and adenosine-5'-diphosphate (ADP) were lower; uric acid and L-histidine were lower in urine from cases. The key limitation of this study is its small size. CONCLUSIONS/SIGNIFICANCE: Metabolomic analysis clearly distinguished severe pneumonia patients from community controls. The metabolites identified are important for the host response to infection through antioxidant, inflammatory and antimicrobial pathways, and energy metabolism. Larger studies are needed to determine whether these findings are pneumonia-specific and to distinguish organism-specific responses. Metabolomics has considerable potential to improve diagnostics for childhood pneumonia.
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Metabolómica , Neumonía/sangre , Neumonía/orina , Adolescente , Biomarcadores/análisis , Biomarcadores/sangre , Biomarcadores/orina , Niño , Preescolar , Cromatografía Líquida de Alta Presión , Gambia , Haemophilus influenzae/genética , Haemophilus influenzae/aislamiento & purificación , Humanos , Masculino , Proyectos Piloto , Neumonía/diagnóstico , Neumonía/microbiología , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/aislamiento & purificaciónRESUMEN
The introduction of molecular diagnostic methods is crucial for improved understanding of the aetiology and epidemiology of bacterial infections in communities in resource poor settings. A blood sample from a 7 month old patient diagnosed with malaria in 2001 in a Gambian outpatient clinic was reported as culture negative after it was subjected to traditional bacterial culture protocols. We re-addressed the analysis of the blood sample from this case more recently (after 6.5 years in archival storage) in pilot work establishing 16S rRNA PCR in our molecular laboratory. Initial 16S rRNA PCR results confirmed the presence of bacterial DNA in the sample. 16S rRNA sequence analysis identified the organism as Campylobacter spp. In light of the molecular evidence we successfully grew the organism using appropriate culture conditions and subsequently biochemically confirmed that the isolate was Campylobacter jejuni. PCR and DNA sequencing of a set of seven C. jejuni housekeeping genes and in silico Multilocus Sequence Typing (MLST) analysis revealed that the isolate exhibits a novel sequence type (ST) of C. jejuni (ST 2928) and belongs to ST-443 clonal complex. This study demonstrates the potential for molecular tools to enhance the diagnosis of bacterial infections, which remain a major killer globally, not least in children in the developing world. Improvements in diagnostics are needed, and will be important not only for sick individuals but also for populations, where better measures of disease burden will contribute significantly to the improvement of public health policy.
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Campylobacter jejuni/aislamiento & purificación , Genes Bacterianos , Secuencia de Bases , Campylobacter jejuni/genética , Cartilla de ADN , Electroforesis en Gel de Agar , Gambia , Humanos , Reacción en Cadena de la Polimerasa , ARN Ribosómico 16S/genéticaRESUMEN
Candidate imprinted transcriptional units in the mouse genome were identified systematically from 27,663 FANTOM2 full-length mouse cDNA clones by expression profiling. Large-scale cDNA microarrays were used to detect differential expression dependent upon chromosomal parent of origin by comparing the mRNA levels in the total tissue of 9.5 dpc parthenogenote and androgenote mouse embryos. Of the FANTOM2 transcripts, 2114 were identified as candidates on the basis of the array data. Of these, 39 mapped to known imprinted regions of the mouse genome, 56 were considered as nonprotein-coding RNAs, and 159 were natural antisense transcripts. The imprinted expression of two transcripts located in the mouse chromosomal region syntenic to the human Prader-Willi syndrome region was confirmed experimentally. We further mapped all candidate imprinted transcripts to the mouse and human genome and were shown in correlation with the imprinting disease loci. These data provide a major resource for understanding the role of imprinting in mammalian inherited traits.