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1.  Iron Status and Helicobacter pylori Infection in Symptomatic Children: An International Multi-Centered Study 
PLoS ONE  2013;8(7):e68833.
Iron deficiency (ID) and iron deficiency anaemia (IDA) are global major public health problems, particularly in developing countries. Whilst an association between H. pylori infection and ID/IDA has been proposed in the literature, currently there is no consensus. We studied the effects of H. pylori infection on ID/IDA in a cohort of children undergoing upper gastrointestinal endoscopy for upper abdominal pain in two developing and one developed country.
In total 311 children (mean age 10.7±3.2 years) from Latin America - Belo Horizonte/Brazil (n = 125), Santiago/Chile (n = 105) - and London/UK (n = 81), were studied. Gastric and duodenal biopsies were obtained for evaluation of histology and H. pylori status and blood samples for parameters of ID/IDA.
The prevalence of H. pylori infection was 27.7% being significantly higher (p<0.001) in Latin America (35%) than in UK (7%). Multiple linear regression models revealed H. pylori infection as a significant predictor of low ferritin and haemoglobin concentrations in children from Latin-America. A negative correlation was observed between MCV (r = −0.26; p = 0.01) and MCH (r = −0.27; p = 0.01) values and the degree of antral chronic inflammation, and between MCH and the degree of corpus chronic (r = −0.29, p = 0.008) and active (r = −0.27, p = 0.002) inflammation.
This study demonstrates that H. pylori infection in children influences the serum ferritin and haemoglobin concentrations, markers of early depletion of iron stores and anaemia respectively.
PMCID: PMC3701645  PMID: 23861946
2.  Characterising Cytokine Gene Expression Signatures in Patients with Severe Sepsis 
Mediators of Inflammation  2013;2013:164246.
Introduction. Severe sepsis in humans may be related to an underlying profound immune suppressive state. We investigated the link between gene expression of immune regulatory cytokines and the range of illness severity in patients with infection and severe sepsis. Methods. A prospective observational study included 54 ICU patients with severe sepsis, 53 patients with infection without organ failure, and 20 healthy controls. Gene expression in peripheral blood mononuclear cells (PBMC) was measured using real-time polymerase chain reaction. Results. Infection differed from health by decreased expression of the IL2, and IL23 and greater expression of IL10 and IL27. Severe sepsis differed from infection by having decreased IL7, IL23, IFNγ, and TNFα gene expression. An algorithm utilising mRNA copy number for TNFα, IFNγ, IL7, IL10, and IL23 accurately distinguished sepsis from severe sepsis with a receiver operator characteristic value of 0.88. Gene expression was similar with gram-positive and gram-negative infection and was similar following medical and surgical severe sepsis. Severity of organ failure was associated with serum IL6 protein levels but not with any index of cytokine gene expression in PBMCs. Conclusions. Immune regulatory cytokine gene expression in PBMC provides a robust method of modelling patients' response to infection.
PMCID: PMC3713593  PMID: 23935244
3.  Citrullination of proteins: a common post-translational modification pathway induced by different nanoparticles in vitro and in vivo 
Nanomedicine (London, England)  2012;7(8):1181-1195.
Rapidly expanding manufacture and use of nanomaterials emphasize the requirements for thorough assessment of health outcomes associated with novel applications. Post-translational protein modifications catalyzed by Ca2+-dependent peptidylargininedeiminases have been shown to trigger immune responses including autoantibody generation, a hallmark of immune complexes deposition in rheumatoid arthritis. Therefore, the aim of the study was to assess if nanoparticles are able to promote protein citrullination.
Materials & methods
Human A549 and THP-1 cells were exposed to silicon dioxide, carbon black or single-walled carbon nanotubes. C57BL/6 mice were exposed to respirable single-walled carbon nanotubes. Protein citrullination, peptidylargininedeiminases activity and target proteins were evaluated.
The studied nanoparticles induced protein citrullination both in cultured human cells and mouse lung tissues. Citrullination occurred via the peptidylargininedeiminase-dependent mechanism. Cytokeratines 7, 8, 18 and plectins were identified as intracellular citrullination targets.
Nanoparticle exposure facilitated post-translational citrullination of proteins.
PMCID: PMC3465773  PMID: 22625207
autoimmunity; high content analysis; immune system; inflammation; nanomaterial; nanoparticle; peptidylargininedeiminase; post-translational modification; protein citrullination; rheumatoid arthritis
4.  Dendrimers branch out to support mucosal integrity 
EMBO Molecular Medicine  2012;4(9):860-862.
PMCID: PMC3491819  PMID: 22911657
dendrimer; diarrhoea; interleukin-6; nanobiotechnology; shigellosis
5.  Effect of immune pressure on Hepatitis C virus evolution: insights from a single source outbreak 
Hepatology (Baltimore, Md.)  2011;53(2):396-405.
The host's immune response to the Hepatitis C virus (HCV) can result in the selection of characteristic mutations (adaptations) that enable the virus to escape this response. The ability of the virus to mutate at these sites is dependent on the incoming virus, fitness cost incurred by the mutation and the benefit to the virus in escaping the response. Studies examining viral adaptation in chronic HCV infection have shown that these characteristic immune escape mutations can be observed at the population level as human leucocyte antigen (HLA)-specific viral polymorphisms. We examined 63 individuals with chronic HCV infection who were infected from a single HCV genotype 1b source. Our aim was to determine the extent to which the host's immune pressure affects HCV diversity and how the sequence of the incoming virus, including pre-existing escape mutations, can influence subsequent mutations in recipients and infection outcome.
HCV sequences from these individuals revealed 29 significant associations between specific HLA types within the new hosts and variations within their viruses which likely represent new viral adaptations. These associations do not overlap with previously reported adaptations for genotype 1a and 3a, possibly reflecting a combination of constraint due to the incoming virus and genetic distance between the strains. However, these sites only accounted for a portion of sites where viral diversity was observed in the new hosts. Furthermore, pre-existing viral adaptations in the incoming (source) virus were likely to have influenced outcome in the new hosts.
PMCID: PMC3044208  PMID: 21246583
HCV; viral adaptation; CD8+ T-cells; viral diversity; single source outbreak
6.  Multiple common variants for celiac disease influencing immune gene expression 
Nature genetics  2010;42(4):295-302.
We performed a second-generation genome wide association study of 4,533 celiac disease cases and 10,750 controls. We genotyped 113 selected SNPs with PGWAS<10−4, and 18 SNPs from 14 known loci, in a further 4,918 cases and 5,684 controls. Variants from 13 new regions reached genome wide significance (Pcombined<5×10−8), most contain immune function genes (BACH2, CCR4, CD80, CIITA/SOCS1/CLEC16A, ICOSLG, ZMIZ1) with ETS1, RUNX3, THEMIS and TNFRSF14 playing key roles in thymic T cell selection. A further 13 regions had suggestive association evidence. In an expression quantitative trait meta-analysis of 1,469 whole blood samples, 20 of 38 (52.6%) tested loci had celiac risk variants correlated (P<0.0028, FDR 5%) with cis gene expression.
PMCID: PMC2847618  PMID: 20190752
7.  Evaluation of 6 candidate genes on chromosome 11q23 for coeliac disease susceptibility: a case control study 
BMC Medical Genetics  2010;11:76.
Recent whole genome analysis and follow-up studies have identified many new risk variants for coeliac disease (CD, gluten intolerance). The majority of newly associated regions encode candidate genes with a clear functional role in T-cell regulation. Furthermore, the newly discovered risk loci, together with the well established HLA locus, account for less than 50% of the heritability of CD, suggesting that numerous additional loci remain undiscovered. Linkage studies have identified some well-replicated risk regions, most notably chromosome 5q31 and 11q23.
We have evaluated six candidate genes in one of these regions (11q23), namely CD3E, CD3D, CD3G, IL10RA, THY1 and IL18, as risk factors for CD using a 2-phase candidate gene approach directed at chromosome 11q. 377 CD cases and 349 ethnically matched controls were used in the initial screening, followed by an extended sample of 171 additional coeliac cases and 536 additional controls.
Promotor SNPs (-607, -137) in the IL18 gene, which has shown association with several autoimmune diseases, initially suggested association with CD (P < 0.05). Follow-up analyses of an extended sample supported the same, moderate effect (P < 0.05) for one of these. Haplotype analysis of IL18-137/-607 also supported this effect, primarily due to one relatively rare haplotype IL18-607C/-137C (P < 0.0001), which was independently associated in two case-control comparisons. This same haplotype has been noted in rheumatoid arthritis.
Haplotypes of the IL18 promotor region may contribute to CD risk, consistent with this cytokine's role in maintaining inflammation in active CD.
PMCID: PMC2880976  PMID: 20478055
8.  Statistical Methods for Analysis of High-Throughput RNA Interference Screens 
Nature methods  2009;6(8):569-575.
RNA interference (RNAi) has become a powerful technique for reverse genetics and drug discovery and, in both of these areas, large-scale high-throughput RNAi screens are commonly performed. The statistical techniques used to analyze these screens are frequently borrowed directly from small-molecule screening; however small-molecule and RNAi data characteristics differ in meaningful ways. We examine the similarities and differences between RNAi and small-molecule screens, highlighting particular characteristics of RNAi screen data that must be addressed during analysis. Additionally, we provide guidance on selection of analysis techniques in the context of a sample workflow.
PMCID: PMC2789971  PMID: 19644458
9.  Deoxycholate induces COX-2 expression via Erk1/2-, p38-MAPK and AP-1-dependent mechanisms in esophageal cancer cells 
BMC Cancer  2009;9:190.
The progression from Barrett's metaplasia to adenocarcinoma is associated with the acquirement of an apoptosis-resistant phenotype. The bile acid deoxycholate (DCA) has been proposed to play an important role in the development of esophageal adenocarcinoma, but the precise molecular mechanisms remain undefined. The aim of this study was to investigate DCA-stimulated COX-2 signaling pathways and their possible contribution to deregulated cell survival and apoptosis in esophageal adenocarcinoma cells.
Following exposure of SKGT-4 cells to DCA, protein levels of COX-2, MAPK and PARP were examined by immunoblotting. AP-1 activity was assessed by mobility shift assay. DCA-induced toxicity was assessed by DNA fragmentation and MTT assay.
DCA induced persistent activation of the AP-1 transcription factor with Fra-1 and JunB identified as the predominant components of the DCA-induced AP-1 complex. DCA activated Fra-1 via the Erk1/2- and p38 MAPK while Erk1/2 is upstream of JunB. Moreover, DCA stimulation mediated inhibition of proliferation with concomitant low levels of caspase-3-dependent PARP cleavage and DNA fragmentation. Induction of the anti-apoptotic protein COX-2 by DCA, via MAPK/AP-1 pathway appeared to balance the DCA mediated activation of pro-apoptotic markers such as PARP cleavage and DNA fragmentation. Both of these markers were increased upon COX-2 suppression by aspirin pretreatment prior to DCA exposure.
DCA regulates both apoptosis and COX-2-regulated cell survival in esophageal cells suggesting that the balance between these two opposing signals may determine the transformation potential of DCA as a component of the refluxate.
PMCID: PMC2704223  PMID: 19534809
10.  Novel celiac disease genetic determinants related to the immune response 
Nature genetics  2008;40(4):395-402.
Our celiac disease genome-wide association study identified IL2/IL21 region risk variants. We genotyped 1,020 of the most associated non-HLA markers in a further 1,643 cases and 3,406 controls. Joint analysis including the genome-wide association study data (767 cases, 1422 controls) identified seven new risk regions (P <5×10-7). Six regions harbor genes controlling immune responses, including: CCR3, IL12A, IL18RAP, RGS1, SH2B3 (nsSNP rs3184504), TAGAP. Whole blood IL18RAP mRNA expression correlated with IL18RAP genotype. Type 1 diabetes and celiac disease share HLA-DQ, IL2/IL21, CCR3 and SH2B3 risk regions. Extensive genome-wide association study follow-up has identified additional celiac disease risk variants in relevant biological pathways.
PMCID: PMC2673512  PMID: 18311140
11.  Loss of viral fitness and cross-recognition by CD8+ T cells limit HCV escape from a protective HLA-B27–restricted human immune response  
There is an association between expression of the MHC class I molecule HLA-B27 and protection following human infection with either HIV or HCV. In both cases, protection has been linked to HLA-B27 presentation of a single immunodominant viral peptide epitope to CD8+ T cells. If HIV mutates the HLA-B27–binding anchor of this epitope to escape the protective immune response, the result is a less-fit virus that requires additional compensatory clustered mutations. Here, we sought to determine whether the immunodominant HLA-B27–restricted HCV epitope was similarly constrained by analyzing the replication competence and immunogenicity of different escape mutants. Interestingly, in most HLA-B27–positive patients chronically infected with HCV, the escape mutations spared the HLA-B27–binding anchor. Instead, the escape mutations were clustered at other sites within the epitope and had only a modest impact on replication competence. Further analysis revealed that the cluster of mutations is required for efficient escape because a combination of mutations is needed to impair T cell recognition of the epitope. Artificially introduced mutations at the HLA-B27–binding anchors were found to be either completely cross-reactive or to lead to substantial loss of fitness. These results suggest that protection by HLA-B27 in HCV infection can be explained by the requirement to accumulate a cluster of mutations within the immunodominant epitope to escape T cell recognition.
PMCID: PMC2631298  PMID: 19139562
12.  Natural selection and the molecular basis of electrophoretic variation at the coagulation F13B locus 
Electrophoretic analysis of protein variation at the coagulation F13B locus has previously revealed three alleles, with alleles 1, 2, and 3 each being at high frequency in European, African, and Asian populations, respectively. To determine if this unusual pattern of interpopulation differentiation reflects local natural selection or neutral genetic drift, we re-sequenced 4.6 kb of the gene, encompassing all exons, splice junctions, and 1.4 kb of the promoter, in African, European, and Asian samples. These analyses revealed three major lineages, which correspond to the common protein alleles and differ from each other at a non-synonymous substitution in exon 3 and a novel splice acceptor in intron K. There is previous evidence that these lineages are not functionally equivalent; we therefore carried out case–control analyses and confirmed that variability at F13B modulates susceptibility and/or survivorship in coronary artery disease (P<0.05) and type II diabetes within the coronary artery disease cohort (P<0.01). Tajima's D and Fu and Li's tests did not indicate significant departures from neutral expectations. However, publicly available data from SeattleSNPs and HapMap do indicate highly unusual levels of population differentiation (P=0.003) and an excess of allele-specific, extended haplotype homozygosity within the African population (P=0.0125). Possible causes of this putative signal of selection include hematophagous organisms, infection by pathogens that cause disseminated intravascular coagulation, and metabolic or dietary factors.
PMCID: PMC2986062  PMID: 18716611
coagulation factors; population genetics; classical marker genetics; natural selection; alternative splicing
13.  A genome-wide association study for celiac disease identifies risk variants in the region harboring IL2 and IL21 
Nature genetics  2007;39(7):827-829.
We tested 310,605 single-nucleotide polymorphisms for association in 778 celiac disease cases and 1422 controls. Outside the HLA, the most significant finding (rs13119723, P=2.0 × 10−7, empirical genome-wide significance P=0.045) was in the KIAA1109/Tenr/IL2/IL21 linkage disequilibrium block. Association was independently confirmed in two further collections (strongest at rs6822844, 24kB 5' of IL21, meta-analysis P=1.3 × 10−14, OR 0.63), suggesting genetic variation in this region predisposes to celiac disease.
PMCID: PMC2274985  PMID: 17558408
14.  Septic shock is correlated with asymmetrical dimethyl arginine levels, which may be influenced by a polymorphism in the dimethylarginine dimethylaminohydrolase II gene: a prospective observational study 
Critical Care  2006;10(5):R139.
Asymmetrical dimethyl arginine (ADMA) is an endogenous non-selective inhibitor of nitric oxide synthase that may influence the severity of organ failure and the occurrence of shock secondary to an infectious insult. Levels may be genetically determined by a promoter polymorphism in a regulatory gene encoding dimethylarginine dimethylaminohydrolase II (DDAH II), which functions by metabolising ADMA to citrulline. The aim of this study was to examine the association between ADMA levels and the severity of organ failure and shock in severe sepsis and also to assess the influence of a promoter polymorphism in DDAH II on ADMA levels.
A prospective observational study was designed, and 47 intensive care unit (ICU) patients with severe sepsis and 10 healthy controls were enrolled. Serum ADMA and IL-6 were assayed on admission to the ICU and seven days later. Allelic variation for a polymorphism at position -449 in the DDAH II gene was assessed in each patient. Clinical and demographic details were also collected.
On day 1 more ADMA was detectable in the ICU group than in the control group (p = 0.005). Levels subsequently increased during the first week in ICU (p = 0.001). ADMA levels were associated with vasopressor requirements on day one (p = 0.001). ADMA levels and Sequential Organ Failure Assessment scores were directly associated on day one (p = 0.0001) and day seven (p = 0.002). The degree of acidaemia and lactaemia was directly correlated with ADMA levels at both time points (p < 0.01). On day seven, IL-6 was directly correlated with ADMA levels (p = 0.006). The variant allele with G at position -449 in the DDAH II gene was associated with increased ADMA concentrations at both time points (p < 0.05).
Severity of organ failure, inflammation and presence of early shock in severe sepsis are associated with increased ADMA levels. ADMA concentrations may be influenced by a polymorphism in the DDAH II gene.
PMCID: PMC1751072  PMID: 17002794
15.  Detection of a Tyrosine Phosphatase LAR on Intestinal Epithelial Cells and Intraepithelial Lymphocytes in the Human Duodenum 
Mediators of Inflammation  2005;2005(1):23-30.
Studies of tyrosine phosphorylation in the human duodenum have indicated that proliferating cells in the middle portion of the duodenal crypt were devoid of this feature, suggesting that tyrosine kinase activation is not a dominant factor in crypt cell proliferation, and that consequently tyrosine phosphatase activity may be a more critical factor in crypt cell development. We investigated the expression of the leukocyte common antigen-related receptor (LAR) family of tyrosine phosphatases. A flow cytometry system was used to examine cells from the surface, mid-portion, and lower part of the crypt. Individual cell populations were immunostained with anti-LAR antibodies using phycoerythrin-conjugated anti-CD3 to discriminate between epithelial cells (CD3−) and intraepithelial lymphocytes (CD3+). Epithelial cells expressed LAR throughout the crypt. Expression of LAR was maximal in the mid-portion of the crypt with lower expression at the top of the villi. Intraepithelial lymphocytes expressed low levels of LAR at the tips of the villi with stronger expression extending towards the base of the crypt. These findings were confirmed by immunohistochemistry on paraffin-fixed sections. Of note, peripheral blood lymphocytes expressed less LAR than IEL. These observations suggest the possibility that tyrosine phosphatase LAR may be of importance in the regulation of crypt cell proliferation. Moreover, as the extracellular domain of LAR has homology with adhesion molecules, the finding of this molecule on IEL could suggest a possible functional role in homing of this unique lymphocyte.
PMCID: PMC1513056  PMID: 15770063
16.  The Neutrophil-Activating Protein (Hp-Nap) of Helicobacter pylori Is a Protective Antigen and a Major Virulence Factor 
The Journal of Experimental Medicine  2000;191(9):1467-1476.
Helicobacter pylori infection induces the appearance of inflammatory infiltrates, consisting mainly of neutrophils and monocytes, in the human gastric mucosa. A bacterial protein with neutrophil activating activity (HP-NAP) has been previously identified, but its role in infection and immune response is still largely unknown. Here, we show that vaccination of mice with HP-NAP induces protection against H. pylori challenge, and that the majority of infected patients produce antibodies specific for HP-NAP, suggesting an important role of this factor in immunity. We also show that HP-NAP is chemotactic for human leukocytes and that it activates their NADPH oxidase to produce reactive oxygen intermediates, as demonstrated by the translocation of its cytosolic subunits to the plasma membrane, and by the lack of activity on chronic granulomatous disease leukocytes. This stimulating effect is strongly potentiated by tumor necrosis factor α and interferon γ and is mediated by a rapid increase of the cytosolic calcium concentration. The activation of leukocytes induced by HP-NAP is completely inhibited by pertussis toxin, wortmannin, and PP1. On the basis of these results, we conclude that HP-NAP is a virulence factor important for the H. pylori pathogenic effects at the site of infection and a candidate antigen for vaccine development.
PMCID: PMC2213429  PMID: 10790422
neutrophils; monocytes; NADPH oxidase; chemotaxis; Helicobacter

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