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BACKGROUND: While many placental herpesvirus genomes have been fully sequenced, the complete genome of a marsupial herpesvirus has not been described. Here we present the first genome sequence of a metatherian herpesvirus, Macropodid herpesvirus 1 (MaHV-1). RESULTS: The MaHV-1 viral genome was sequenced using an Illumina MiSeq sequencer, de novo assembly was performed and the genome was annotated. The MaHV-1 genome was 140 kbp in length and clustered phylogenetically with the primate simplexviruses, sharing 67% nucleotide sequence identity with Human herpesviruses 1 and 2. The MaHV-1 genome contained 66 predicted open reading frames (ORFs) homologous to those in other herpesvirus genomes, but lacked homologues of UL3, UL4, UL56 and glycoprotein J. This is the first alphaherpesvirus genome that has been found to lack the UL3 and UL4 homologues. We identified six novel ORFs and confirmed their transcription by RT-PCR. CONCLUSIONS: This is the first genome sequence of a herpesvirus that infects metatherians, a taxonomically unique mammalian clade. Members of the Simplexvirus genus are remarkably conserved, so the absence of ORFs otherwise retained in eutherian and avian alphaherpesviruses contributes to our understanding of the Alphaherpesvirinae. Further study of metatherian herpesvirus genetics and pathogenesis provides a unique approach to understanding herpesvirus-mammalian interactions.
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Herpesviridae/genética , Animais , Genoma Viral/genética , Herpesviridae/classificação , Fases de Leitura Aberta/genética , Proteínas Virais/genéticaRESUMO
BACKGROUND: Meleagrid herpesvirus 1 (MeHV-1) infectious bacterial artificial chromosomes (iBACs) are ideal vectors for the development of recombinant vaccines for the poultry industry. However, the full potential of iBACS as vectors can only be realised after thorough genetic characterisation, including identification of those genetic locations that are non-essential for virus replication. Generally, transposition has proven to be a highly effective strategy for rapid and efficient mutagenesis of iBAC clones. The current study describes the characterisation of 34 MeHV-1 mutants containing transposon insertions within the pMeHV1-C18 iBAC genome. METHODS: Tn5 and MuA transposition methods were used to generate a library of 76 MeHV-1 insertion mutants. The capacity of each mutant to facilitate the recovery of infectious MeHV-1 was determined by the transfection of clone DNA into chicken embryo fibroblasts. RESULTS: Attempts to recover infectious virus from the modified clones identified 14 genetic locations that were essential for MeHV-1 replication in cell culture. Infectious MeHV-1 was recovered from the remaining 14 intragenic insertion mutants and six intergenic insertion mutants, suggesting that the respective insertion locations are non-essential for MeHV-1 replication in cell culture. CONCLUSIONS: The essential and non-essential designations for those MeHV-1 genes characterised in this study were generally in agreement with previous reports for other herpesviruses homologues. However, the requirement for the mardivirus-specific genes LORF4A and LORF5 are reported for the first time. These findings will help direct future work on the development of recombinant poultry vaccines using MeHV-1 as a vector by identifying potential transgene insertion sites within the viral genome.
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Loci Gênicos , Genoma Viral , Herpesvirus Meleagrídeo 1/genética , Animais , Galinhas , Cromossomos Artificiais Bacterianos , Elementos de DNA Transponíveis , Fibroblastos/virologia , Mutagênese Insercional , TransfecçãoRESUMO
Meleagrid herpesvirus 1 (MeHV-1 or turkey herpesvirus) has been widely used as a vaccine in commercial poultry. Initially, these vaccine applications were for the prevention of Marek's disease resulting from Gallid herpesvirus 2 infections, while more recently MeHV-1 has been used as recombinant vector for other poultry infections. The construction of herpesvirus infectious clones that permit propagation and manipulation of the viral genome in bacterial hosts has advanced the studies of herpesviral genetics. The current study reports the construction of five MeHV-1 infectious clones. The in vitro properties of viruses recovered from these clones were indistinguishable from the parental MeHV-1. In contrast, the rescued MeHV-1 viruses were significantly attenuated when used in vivo. Complete sequencing of the infectious clones identified the absence of two regions of the MeHV-1 genome compared to the MeHV-1 reference sequence. These analyses determined the rescued viruses have seven genes, UL43, UL44, UL45, UL56, HVT071, sorf3 and US2 either partially or completely deleted. In addition, single nucleotide polymorphisms were identified in all clones compared with the MeHV-1 reference sequence. As a consequence of one of the polymorphisms identified in the UL13 gene, four of the rescued viruses were predicted to encode a serine/threonine protein kinase lacking two of three domains required for activity. Thus four of the recovered viruses have a total of eight missing or defective genes. The implications of these findings in the context of herpesvirus biology and infectious clone construction are discussed.
Assuntos
Genes Virais , Herpesvirus Meleagrídeo 1/genética , Herpesvirus Meleagrídeo 1/fisiologia , Mutação , Deleção de Sequência , Replicação Viral , Animais , Células Cultivadas , Galinhas , DNA Viral/química , DNA Viral/genética , Fibroblastos/virologia , Dados de Sequência Molecular , Genética Reversa , Análise de Sequência de DNARESUMO
BACKGROUND AND AIM: The etiology of Crohn's disease (CD) implicates both genetic and environmental factors. Smoking behavior is one environmental risk factor to play a role in the development of CD. The study aimed to assess the contribution of the interleukin 23 receptor (IL23R) in determining disease susceptibility in two independent cohorts of CD, and to investigate the interactions between IL23R variants, smoking behavior, and CD-associated genes, NOD2 and ATG16L1. METHODS: Ten IL23R single-nucleotide polymorphisms (SNPs) were genotyped in 675 CD cases, and 1255 controls from Brisbane, Australia (dataset 1). Six of these SNPs were genotyped in 318 CD cases and 533 controls from Canterbury, New Zealand (dataset 2). Case-control analysis of genotype and allele frequencies, and haplotype analysis for all SNPs was conducted. RESULTS: We demonstrate a strong increased CD risk for smokers in both datasets (odds ratio 3.77, 95% confidence interval 2.88-4.94), and an additive interaction between IL23Râ SNPs and cigarette smoking. Ileal involvement was a consistent marker of strong SNP-CD association (P ≤ 0.001), while the lowest minor allele frequencies for location were found in those with colonic CD (L2). Three haplotype blocks were identified across the 10 IL23Râ SNPs conferring different risk of CD. Haplotypes conferred no further risk of CD when compared with single SNP analyses. CONCLUSION: IL23R gene variants determine CD susceptibility in the Australian and New Zealand population, particularly ileal CD. A strong additive interaction exists between IL23Râ SNPs and smoking behavior resulting in a dramatic increase in disease risk depending upon specific genetic background.
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Doença de Crohn/etiologia , Doença de Crohn/genética , Receptores de Interleucina/genética , Fumar/efeitos adversos , Adolescente , Adulto , Austrália , Estudos de Casos e Controles , Estudos de Coortes , Feminino , Predisposição Genética para Doença/genética , Genótipo , Humanos , Masculino , Nova Zelândia , Polimorfismo de Nucleotídeo Único/genética , Fatores de Risco , Adulto JovemRESUMO
Insects collected in dry traps can degrade rapidly, especially in warm, humid environments where many biodiversity and biosecurity surveillance activities are undertaken. Degradation can severely impact diagnostics, as trap catches can become difficult to identify to species level using morphological characters or, of increasing importance, molecular approaches. This is especially problematic for biosecurity surveillance of exotic tephritid fruit flies, where diagnostics are heavily reliant on morphological characters. We tested the effects of differing temperature and humidity conditions on mock samples of tephritid fruit flies in a controlled environment and compared our results to field trap catches. DNA degradation was quantified using real-time PCR assays, including one assay newly developed and tested here. We observed a correlation between increasing DNA degradation and increasing temperature and humidity. The greatest DNA degradation occurred under combined high humidity (90% relative humidity) and constant high temperature (35 °C). Unexpectedly, fluctuating temperature did not have a significant impact on DNA. Other factors, such as trap design, time in the field, and rainfall, did not significantly correlate with DNA quality across the field samples tested. When plotted against mock samples, field samples clustered together, with no clear pattern or predictability regarding the quantity of DNA preserved, indicating other untested environmental variables may be at play. Predictably, increased exposure time was found to have a detrimental effect on DNA quality for all treatments. These findings will improve the delivery of surveillance activities through the implementation of shorter trap clearance timeframes and improved trap designs and procedures.
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The propagation of herpesvirus genomes as infectious bacterial artificial chromosomes (iBAC) has enabled the application of highly efficient strategies to investigate gene function across the genome. One of these strategies, transposition, has been used successfully on a number of herpesvirus iBACs to generate libraries of gene disruption mutants. Gene deletion studies aimed at determining the dispensable gene repertoire of the Meleagrid herpesvirus 1 (MeHV-1) genome to enhance the utility of this virus as a vaccine vector have been conducted in this report. A MeHV-1 iBAC was used in combination with the Tn5 and MuA transposition systems in an attempt to generate MeHV-1 gene interruption libraries. However, these studies demonstrated that Tn5 transposition events into the MeHV-1 genome occurred at unexpectedly low frequencies. Furthermore, characterization of genomic locations of the rare Tn5 transposon insertion events indicated a nonrandom distribution within the viral genome, with seven of the 24 insertions occurring within the gene encoding infected cell protein 4. Although insertion events with the MuA system occurred at higher frequency compared with the Tn5 system, fewer insertion events were generated than has previously been reported with this system. The characterization and distribution of these MeHV-1 iBAC transposed mutants is discussed at both the nucleotide and genomic level, and the properties of the MeHV-1 genome that could influence transposition frequency are discussed.
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Genoma Viral , Herpesvirus Meleagrídeo 1/genética , Mutagênese Insercional , Transposases/genética , Cromossomos Artificiais Bacterianos/genética , Elementos de DNA Transponíveis , Deleção de Genes , Biblioteca Gênica , Herpesvirus Meleagrídeo 1/metabolismo , Análise de Sequência de DNA/veterinária , Transposases/metabolismoRESUMO
Insect identification and preservation of voucher specimens is integral to pest diagnostic and surveillance activities; yet bulk-trapped insects are a diagnostic challenge due to high catch numbers and the susceptibility of samples to environmental damage. Many insect trap catches rely on examination of morphological characters for species identifications, which is a time consuming and highly skilled task, hence there is a need for more efficient molecular approaches. Many bulk DNA extraction methods require destructive sampling of specimens, resulting in damaged, or fully destroyed, voucher specimens. We developed an inexpensive, rapid, bulk DNA isolation method that preserves specimens as pinned vouchers to a standard that allows for post-extraction morphological examination and inclusion in insect reference collections. Our protocol was validated using a group of insects that are time-consuming to identify when trapped in large numbers-the dacine fruit flies (Diptera: Tephritidae: Dacinae). In developing our method, we evaluated existing protocols against the following criteria: effect on morphology; suitability for large trap catches; cost; ease of handling; and application to downstream molecular diagnostic analyses such as real-time PCR and metabarcoding. We found that the optimum method for rapid isolation of DNA extraction was immersing flies in a NaOH:TE buffer at 75°C for 10 minutes, without the need for proteinase K or detergents. This HotSOAK method produced sufficient high-quality DNA whilst preserving morphological characters suitable for species-level identification with up to 20,000 flies in a sample. The lysates performed well in down-stream analyses such as loop-mediated isothermal amplification (LAMP) and real-time PCR applications, while for metabarcoding PCR the lysate required an additional column purification step. Development of this method is a key step required for upscaling our capacity to accurately detect insects captured in bulk traps, whether for biodiversity, biosecurity, or pest management objectives.
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Insetos , Tephritidae , Animais , Análise Custo-Benefício , Insetos/genética , Tephritidae/genética , Drosophila/genética , DNA/genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Bactrocera tryoni and Bactrocera neohumeralis are morphologically similar sibling pest fruit fly species that possess different biological attributes, geographic distributions, and host ranges. The need to differentiate between the two species is critical for accurate pest status assessment, management, biosecurity, and maintenance of reference colonies. While morphologically similar, adults may be separated based on subtle characters; however, some characters exhibit intraspecific variability, creating overlap between the two species. Additionally, there is currently no single molecular marker or rapid diagnostic assay that can reliably distinguish between B. neohumeralis and B. tryoni; therefore, ambiguous samples remain undiagnosed. Here we report the first molecular marker that can consistently distinguish between B. tryoni and B. neohumeralis. Our diagnostic region consists of two adjacent single nucleotide polymorphisms (SNPs) within the pangolin (pan) gene region. We confirmed the genotypes of each species are consistent across their distributional range, then developed a tetra-primer amplification refractory mutation system (ARMS) PCR assay for rapid diagnosis of the species. The assay utilizes four primers in multiplex, with two outer universal primers, and two internal primers: one designed to target two adjacent SNPs (AA) present in B. tryoni and the other targeting adjacent SNPs present in B. neohumeralis (GG). The assay accurately discriminates between the two species, but their SNP genotypes are shared with other nontarget tephritid fruit fly species. Therefore, this assay is most suited to adult diagnostics where species confirmation is necessary in determining ambiguous surveillance trap catches; maintaining pure colony lines; and in Sterile Insect Technique management responses.
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The cue-lure-responding New Guinea fruit fly, Bactrocera trivialis, poses a biosecurity risk to neighbouring countries, e.g., Australia. In trapping programs, lure caught flies are usually morphologically discriminated from non-target species; however, DNA barcoding can be used to confirm similar species where morphology is inconclusive, e.g., Bactrocera breviaculeus and B. rufofuscula. This can take days-and a laboratory-to resolve. A quicker, simpler, molecular diagnostic assay would facilitate a more rapid detection and potential incursion response. We developed LAMP assays targeting cytochrome c oxidase subunit I (COI) and Eukaryotic Translation Initiation Factor 3 Subunit L (EIF3L); both assays detected B. trivialis within 25 min. The BtrivCOI and BtrivEIF3L assay anneal derivatives were 82.7 ± 0.8 °C and 83.3 ± 1.3 °C, respectively, detecting down to 1 × 101 copies/µL and 1 × 103 copies/µL, respectively. Each assay amplified some non-targets from our test panel; however notably, BtrivCOI eliminated all morphologically similar non-targets, and combined, the assays eliminated all non-targets. Double-stranded DNA gBlocks were developed as positive controls; anneal derivatives for the COI and EIF3L gBlocks were 84.1 ± 0.7 °C and 85.8 ± 0.2 °C, respectively. We recommend the BtrivCOI assay for confirmation of suspect cue-lure-trapped B. trivialis, with BtrivEIF3L used for secondary confirmation when required.
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Tephritidae , Animais , Austrália , Drosophila , Técnicas de Diagnóstico Molecular , Nova Guiné , Técnicas de Amplificação de Ácido Nucleico , Tephritidae/genéticaRESUMO
OBJECTIVES: Crohn's disease (CD; MIM 266600) is one of the most common forms of inflammatory bowel disease (IBD), and represents a significant burden to health care in developed countries. Our aim was to determine whether a gene in the IBD linkage region on chromosome 19q13, with a role in Paneth cell secretion and T-cell activation, conferred genetic susceptibility to the development of CD. METHODS: In total, 792 CD cases and 1,244 controls of Australian origin (Caucasian) were genotyped for seven single-nucleotide polymorphisms (SNPs) in the gene encoding the intermediate conductance calcium-activated potassium channel protein (KCNN4) at 19q13.2. CD cases were phenotyped using the Montreal classification. The replication set comprised an additional 326 CD cases and 951 population-based Caucasian controls. Analysis of the KCNN4 mRNA transcript was carried out using quantitative reverse transcriptase-PCR. RESULTS: KCNN4 SNP rs2306801 was associated with CD (primary P=0.0008, odds ratio (OR) (95% confidence interval (CI)): 0.76 (0.65-0.89); replication P=0.01, OR (95% CI): 0.77 (0.61-0.97). Stratification by disease location identified the association between SNP rs2306801 and ileal CD (P=0.01). Non-inflamed ileal mucosa from CD patients carrying any of the common disease-predisposing NOD2 variants (R702W, G908R, 1007fs) had significantly reduced levels of KCNN4 mRNA expression (P=0.001). KCNN4 protein expression was detected in Paneth cells, and in T cells in inflamed lamina propria. CONCLUSIONS: Our data implicate the role of KCNN4 in ileal CD. The dual roles of KCNN4 in Paneth cell secretion and T-cell activation and also its nature as a potassium channel make it an important and practical therapeutic target.
Assuntos
Doença de Crohn/genética , Íleo/patologia , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Adulto , Alelos , Austrália , Distribuição de Qui-Quadrado , Colo/patologia , Doença de Crohn/patologia , Feminino , Frequência do Gene , Estudos de Associação Genética , Ligação Genética , Predisposição Genética para Doença , Genótipo , Haplótipos , Humanos , Imuno-Histoquímica , Inflamação/genética , Inflamação/patologia , Masculino , Pessoa de Meia-Idade , Nova Zelândia , Polimorfismo de Nucleotídeo Único , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estatísticas não ParamétricasRESUMO
OBJECTIVES: Crohn's disease (CD) and ulcerative colitis (UC) are the two most common forms of inflammatory bowel disease (IBD), representing a significant health-care burden. A variant in the autophagy gene ATG16L1 (T300A) has been newly identified as a CD susceptibility locus by genome-wide association. Our aim was to assess the contribution of T300A in determining disease susceptibility and phenotype in two independent Australian IBD cohorts and explore the relationship between T300A and known CD risk factors (NOD2[nucleotide-binding oligomerization domain containing 2] status and smoking). METHODS: In total, 669 CD and 543 UC cases, and 1,244 controls (study 1), 154 CD cases and 420 controls (study 2), and 702 unaffected parents from both groups were genotyped. We conducted case-control and family association analyses, and investigated relationships between T300A and disease subgroups and between NOD2 status and cigarette smoking (CD only). RESULTS: The strong association between CD and T300A was confirmed (P < 0.001), with a two-fold increase in disease risk associated with the GG genotype (odds ratio [OR] 1.96, 95% confidence interval [CI] 1.49-2.58), while ileal CD risk was almost three-fold (OR 2.73, CI 1.87-4.0). ATG16L1 and NOD2 were found to contribute independently to CD risk. A greater than seven-fold increased CD risk was observed for current smokers with a GG genotype (vs nonsmoking AA genotype; P < 0.001, OR 7.65, CI 4.21-13.91). A significant inverse association was found between T300A and UC (P= 0.002). This was strongest for patients with extensive, severe disease. CONCLUSIONS: We confirm the strong association between T300A and CD, specifically ileal subphenotype, and also report the first strong association of this variant with UC.
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Proteínas de Transporte/genética , DNA/genética , Doenças Inflamatórias Intestinais/genética , Polimorfismo Genético , Vigilância da População , Proteínas Relacionadas à Autofagia , Intervalos de Confiança , Genótipo , Humanos , Doenças Inflamatórias Intestinais/epidemiologia , Razão de Chances , Prevalência , Prognóstico , Estudos Prospectivos , Queensland/epidemiologia , Fatores de Risco , Fumar/efeitos adversos , Fumar/epidemiologiaRESUMO
BACKGROUND: The first major Crohn's disease (CD) susceptibility gene, NOD2, implicates the innate intestinal immune system and other pattern recognition receptors in the pathogenesis of this chronic, debilitating disorder. These include the Toll-like receptors, specifically TLR4 and TLR5. A variant in the TLR4 gene (A299G) has demonstrated variable association with CD. We aimed to investigate the relationship between TLR4 A299G and TLR5 N392ST, and an Australian inflammatory bowel disease cohort, and to explore the strength of association between TLR4 A299G and CD using global meta-analysis. METHODS: Cases (CD = 619, ulcerative colitis = 300) and controls (n = 360) were genotyped for TLR4 A299G, TLR5 N392ST, and the 4 major NOD2 mutations. Data were interrogated for case-control analysis prior to and after stratification by NOD2 genotype. Genotype-phenotype relationships were also sought. Meta-analysis was conducted via RevMan. RESULTS: The TLR4 A299G variant allele showed a significant association with CD compared to controls (P = 0.04) and a novel NOD2 haplotype was identified which strengthened this (P = 0.003). Furthermore, we identified that TLR4 A299G was associated with CD limited to the colon (P = 0.02). In the presence of the novel NOD2 haplotype, TLR4 A299G was more strongly associated with colonic disease (P < 0.001) and nonstricturing disease (P = 0.009). A meta-analysis of 11 CD cohorts identified a 1.5-fold increase in risk for the variant TLR4 A299G allele (P < 0.00001). CONCLUSIONS: TLR 4 A299G appears to be a significant risk factor for CD, in particular colonic, nonstricturing disease. Furthermore, we identified a novel NOD2 haplotype that strengthens the relationship between TLR4 A299G and these phenotypes.
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Doença de Crohn/genética , DNA/genética , Mutação , Proteína Adaptadora de Sinalização NOD2/genética , Receptor 4 Toll-Like/genética , Adolescente , Adulto , Doença de Crohn/epidemiologia , Feminino , Frequência do Gene , Haplótipos , Humanos , Incidência , Masculino , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Estudos Prospectivos , Queensland/epidemiologiaRESUMO
Bovine viral diarrhoea virus 1 (BVDV-1) is strongly associated with several important diseases of cattle, such as bovine respiratory disease, diarrhoea and haemoragic lesions. To date many subgenotypes have been reported for BVDV-1, currently ranging from subgenotype 1a to subgenotype 1u. While BVDV-1 has a world-wide distribution, the subgenotypes have a more restricted geographical distribution. As an example, BVDV-1 subgenotypes 1a and 1b are frequently detected in North America and Europe, while the subgenotype 1c is rarely detected. In contrast, BVDV-1 subgenotype 1c is by far the most commonly reported in Australia. Despite this, uneven distribution of the biological importance of the subgenotypes remains unclear. The aim of this study was to characterise the in vivo properties of five strains of BVDV-1 subgenotype 1c in cattle infection studies. No overt respiratory signs were reported in any of the infected cattle regardless of strain. Consistent with other subgenotypes, transient pyrexia and leukopenia were commonly identified, while thrombocytopenia was not. The quantity of virus detected in the nasal secretions of transiently infected animals suggested the likelihood of horizontal transmission was very low. Further studies are required to fully understand the variability and importance of the BVDV-1 subgenotype 1c.
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Antigenic variation in Plasmodium falciparum erythrocyte membrane protein 1, caused by a switch in transcription of the encoding var gene, is an important feature of malaria. In this study, we quantified the relative abundance of var gene transcripts present in P. falciparum parasite clones using real-time reverse transcription-polymerase chain reaction (RT-PCR) and conventional RT-PCR combined with cloning and sequencing, with the aim of directly comparing the results obtained. When there was sufficient abundance of RNA for the real-time RT-PCR assay to be operating within the region of good reproducibility, RT-PCR and real-time RT-PCR tended to identify the same dominant transcript, although some transcript-specific issues were identified. When there were differences in the estimated relative amounts of minor transcripts, the RT-PCR assay tended to produce higher estimates than real-time RT-PCR. These results provide valuable information comparing RT-PCR and real-time RT-PCR analysis of samples with small quantities of RNA as might be expected in the analysis of field or clinical samples.
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Variação Antigênica/genética , Perfilação da Expressão Gênica/métodos , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Animais , Células Cultivadas , Primers do DNA/química , DNA de Protozoário/isolamento & purificação , Humanos , RNA de Protozoário/isolamento & purificação , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Sensibilidade e EspecificidadeAssuntos
Colite Ulcerativa/genética , Antígenos de Histocompatibilidade Classe I/genética , Polimorfismo Genético , Povo Asiático/genética , Colite Ulcerativa/sangue , Colite Ulcerativa/etnologia , Frequência do Gene , Predisposição Genética para Doença , Antígenos de Histocompatibilidade Classe I/sangue , Humanos , Fenótipo , Medição de Risco , Fatores de Risco , Índice de Gravidade de DoençaRESUMO
BACKGROUND AND AIMS: Angiotensin II (AII) is a powerful splanchnic vasoconstrictor with pro-inflammatory and pro-fibrotic properties. Angiotensin converting enzyme (ACE) inhibitors and AII Receptor Antagonists (ARBs) are therapeutic in animal models of colitis. The aim of this case-control study is to determine the expression of angiotensinogen and related genes in human ileal Crohn's disease. METHODS: Using quantitative real-time polymerase chain reaction (RT-PCR), we measured mRNA expression levels of angiotensinogen (AGT), hypoxia inducible factor (HIF)1α and melanoma cell adhesion molecule (MCAM; CD146) in 101 human samples (69 biopsy, 12 resection) from affected ileum (inflamed CD cases, n=36) and unaffected ileum (non-inflamed CD cases, n=45 and controls, n=20). Immunohistochemistry for angiotensinogen was also performed. The study was of case-control design in a tertiary care setting. RESULTS: Ileal expression of AGT was lower in CD cases compared to controls (p<0.0001), in inflamed CD samples (p=0.017) and current smokers (p=0.02). HIF1α expression was lower in non-inflamed CD biopsy samples than controls (p=0.006). The presence of disease-associated NOD2 variants was associated with increased expression of markers of angiogenesis (HIF1α p=0.009; MCAM p=0.007) in inflamed CD samples. Angiotensinogen immunohistochemical staining supported the quantitative RT-PCR expression findings. CONCLUSIONS: Angiotensinogen expression is down regulated in human ileal CD, particularly in the presence of inflammation and current cigarette smoking, implicating the mesenteric vasculature and mucosal hypoxia as co-factors in ileal CD pathogenesis. A novel reduction in HIF1α expression in non-inflamed ileal mucosa in CD patients was also demonstrated.
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Proteínas Angiogênicas/análise , Angiotensinogênio/análise , Doença de Crohn/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/análise , Íleo/química , Neovascularização Patológica , Adulto , Idoso , Proteínas Angiogênicas/genética , Angiotensinogênio/genética , Antígeno CD146/genética , Estudos de Casos e Controles , Doença de Crohn/genética , Doença de Crohn/patologia , Feminino , Regulação da Expressão Gênica , Genótipo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Íleo/patologia , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Neovascularização Patológica/genética , Proteína Adaptadora de Sinalização NOD2/genética , Fenótipo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Fumar/efeitos adversos , Adulto JovemRESUMO
Plasmodium falciparum undergoes antigenic variation by switching the expressed erythrocyte membrane protein (PfEMP)1. This family of proteins plays an important role in the development of chronic, recrudescent P. falciparum malaria, acquired immunity and severe malaria. However, little is known about the switching mechanism or switching rates in the human host. Here, we estimate the switch rate of var genes, using recently published data describing the var gene transcripts detected in blood taken from human volunteers during acute P. falciparum infections and a mathematical model of the in-host dynamics. The overall switch rate of PfEMP1 predicted during the initial stage of infection ( approximately 18% switching parasites per generation) is much higher than previously reported. The implications of the predicted switching rates are discussed.
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Variação Antigênica/genética , Genes de Troca/fisiologia , Malária Falciparum/imunologia , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Animais , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Expressão Gênica/fisiologia , Humanos , Malária Falciparum/genética , Malária Falciparum/parasitologia , Modelos Genéticos , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Fatores de TempoRESUMO
In Plasmodium falciparum a highly polymorphic multi-copy gene family, var, encodes the variant surface antigen P. falciparum erythrocyte membrane protein 1 (PfEMP1), which has an important role in cytoadherence and immune evasion. Using previously described universal PCR primers for the first Duffy binding-like domain (DBLalpha) of var we analysed the DBLalpha repertoires of Dd2 (originally from Thailand) and eight isolates from the Solomon Islands (n=4), Philippines (n=2), Papua New Guinea (n=1) and Africa (n=1). We found 15-32 unique DBLalpha sequence types among these isolates and estimated detectable DBLalpha repertoire sizes ranging from 33-38 to 52-57 copies per genome. Our data suggest that var gene repertoires generally consist of 40-50 copies per genome. Eighteen DBLalpha sequences appeared in more than one Asia-Pacific isolate with the number of sequences shared between any two isolates ranging from 0 to 6 (mean=2.0 +/-1.6). At the amino acid level DBLalpha sequence similarity within isolates ranged from 45.2 +/- 7.1 to 50.2 +/- 6.9%, and was not significantly different from the DBLalpha amino acid sequence similarity among isolates (P>0.1). Comparisons with published sequences also revealed little overlap among DBLalpha sequences from different regions. High DBLalpha sequence diversity and minimal overlap among these isolates suggest that the global var gene repertoire is immense, and may potentially be selected for by the host's protective immune response to the var gene products, PfEMP1.
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Variação Genética , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Animais , Ásia , Asiático , Sistema do Grupo Sanguíneo Duffy/metabolismo , Dados de Sequência Molecular , Oceano Pacífico , Plasmodium falciparum/isolamento & purificação , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
BACKGROUND AND AIMS: Peroxisome proliferator-activated receptor (PPAR) γ is a transcription factor, highly expressed in colonic epithelial cells, adipose tissue and macrophages, with an important role in the regulation of inflammatory pathways. The common PPARγ variants C161T and Pro12Ala have recently been associated with Ulcerative Colitis (UC) and an extensive UC phenotype respectively, in a Chinese population. PPARγ Pro12Ala variant homozygotes appear to be protected from the development of Crohn's disease (CD) in European Caucasians. METHODS: A case-control study was performed for both variants (CD n=575, UC n=306, Controls n=360) using a polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis in an Australian IBD cohort. A transmission disequilibrium test was also performed using CD trios for the PPARγ C161T variant. Genotype-phenotype analyses were also undertaken. RESULTS: There was no significant difference in genotype distribution data or allele frequency between CD and UC patients and controls. There was no difference in allele transmission for the C161T variant. No significant relationship between the variants and disease location was observed. CONCLUSIONS: We were unable to replicate in a Caucasian cohort the recent association between PPARγ C161T and UC or between PPARγ Pro12Ala and an extensive UC phenotype in a Chinese population. There are significant ethnic differences in genetic susceptibility to IBD and its phenotypic expression.
Assuntos
Doenças Inflamatórias Intestinais/genética , PPAR gama/genética , Polimorfismo de Nucleotídeo Único , Estudos de Casos e Controles , Estudos de Coortes , Colite Ulcerativa/genética , Doença de Crohn/genética , Frequência do Gene , Estudos de Associação Genética , Predisposição Genética para Doença , Genótipo , Projeto HapMap , Humanos , FenótipoRESUMO
Plasmodium falciparum erythrocyte membrane protein 1, which is encoded by the var multigene family, is expressed on the surface of P. falciparum-infected erythrocytes and has been implicated in many of the complications associated with falciparum malaria. Transcriptional switching of var is commonly investigated using in vitro cultured parasites, because parasite material from patients is limited. We investigated the affect of short-term in vitro cultivation on var gene transcription in patient samples. A significant reduction in the overall abundance of var transcripts was observed during the first approximately 10 days of culture. The rate of down-regulation was not constant among all var genes; genes with an upsA, -D, and -E 5' flanking region had a significantly faster rate than genes with an upsB or -C flanking region. These results have significant implications for the investigation of associations between var transcription and clinical manifestations using parasites that have been enriched by in vitro culture.