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1.  An NKG2D-mediated human lymphoid stress-surveillance response with high inter-individual variation* 
Science translational medicine  2011;3(113):113ra124.
Microbes and viruses provoke immune responses because certain of their molecular determinants engage and activate dendritic cells (DC). However, evidence is growing for lymphocyte activation by tissue dysregulation. Thus, murine γδ T cells and NK cells can respond rapidly in vivo to Major Histocompatibility Complex (MHC) class I–related “stress-antigens” displayed by cells experiencing DNA damage and/or other physico-chemical stress. Such “lymphoid stress-surveillance” (LSS) can limit tumor formation, but may also promote immunopathology. MICA is a highly polymorphic human stress-antigen implicated in tumor-surveillance, inflammation, and transplant rejection. However, neither the generality of LSS in humans, nor a functional context for MICA polymorphism has been established. Here we show that MICA coding-sequence polymorphisms substantially affect RNA and protein expression. All donors tested showed LSS responses of γδ T and NK cells, but unexpectedly each was individually “tuned”. Hence, some responded optimally to high MICA expression, while others responded better to poorly-expressed MICA alleles, challenging the orthodoxy that higher stress-antigen levels promote greater responsiveness. The routine clinical monitoring of individual tuning should provide practical insight into individual variation in tumor immune-surveillance, transplant rejection and inflammation, and introduce new perspectives on immuno-evasion and immune-suppression in these scenarios.
doi:10.1126/scitranslmed.3002922
PMCID: PMC3966512  PMID: 22133594
2.  Can a combined screening/treatment programme prevent premature failure of renal transplants due to chronic rejection in patients with HLA antibodies: study protocol for the multicentre randomised controlled OuTSMART trial 
Trials  2014;15:30.
Background
Renal transplantation is the best treatment for kidney failure, in terms of length and quality of life and cost-effectiveness. However, most transplants fail after 10 to 12 years, consigning patients back onto dialysis. Damage by the immune system accounts for approximately 50% of failing transplants and it is possible to identify patients at risk by screening for the presence of antibodies against human leukocyte antigens. However, it is not clear how best to treat patients with antibodies. This trial will test a combined screening and treatment protocol in renal transplant recipients.
Methods/Design
Recipients >1 year post-transplantation, aged 18 to 70 with an estimated glomerular filtration rate >30 mL/min will be randomly allocated to blinded or unblinded screening arms, before being screened for the presence of antibodies. In the unblinded arm, test results will be revealed. Those with antibodies will have biomarker-led care, consisting of a change in their anti-rejection drugs to prednisone, tacrolimus and mycophenolate mofetil. In the blinded arm, screening results will be double blinded and all recruits will remain on current therapy (standard care). In both arms, those without antibodies will be retested every 8 months for 3 years. The primary outcome is the 3-year kidney failure rate for the antibody-positive recruits, as measured by initiation of long-term dialysis or re-transplantation, predicted to be approximately 20% in the standard care group but <10% in biomarker-led care. The secondary outcomes include the rate of transplant dysfunction, incidence of infection, cancer and diabetes mellitus, an analysis of adherence with medication and a health economic analysis of the combined screening and treatment protocol. Blood samples will be collected and stored every 4 months and will form the basis of separately funded studies to identify new biomarkers associated with the outcomes.
Discussion
We have evidence that the biomarker-led care regime will be effective at preventing graft dysfunction and expect this to feed through to graft survival. This trial will confirm the benefit of routine screening and lead to a greater understanding of how to keep kidney transplants working longer.
Trial registration
Current Controlled Trials ISRCTN46157828.
doi:10.1186/1745-6215-15-30
PMCID: PMC3906093  PMID: 24447519
Human leukocyte antigen antibodies; renal transplantation; premature allograft failure; chronic rejection; screening; tacrolimus; mycophenolate mofetil; clinical trial; randomised controlled trial; randomized controlled trial; KCTU; CTU; trials unit; diabetes; kidney; graft failure; biomarker; biomarker-led care; DSA; non-DSA; multicentre; immunosuppression; usual care; treatment as usual; standard care; blinded; unblinded; EME funded; NIHR; optimised; optimized; intention-to-treat; per protocol
3.  RNA Binding by the NS3 Protease of the Hepatitis C Virus 
Virus research  2012;169(1):80-90.
The hepatitis C virus (HCV) nonstructural protein 3 (NS3) is essential for the processing of the HCV polyprotein, the replication of HCV RNA, and to short circuit innate immunity signaling. NS3 contains an N-terminal domain with protease activity and a C-terminal domain with helicase activity. The two domains communicate with each other along with other HCV and cellular proteins. Herein we show that RNAs can bind directly to the active site cleft of the NS3 protease domain (NS3P) and inhibit proteolysis of peptide substrates. RNAs that are less apt to form intramolecular structures have a stronger inhibitory activity than RNAs with more stable base paired regions. Two mutations in the protease domain that resulted in decreased affinity to ssRNA were also defective in RNA-induced ATPase activity from the helicase domain of NS3. The coordinated effects on inhibition of protease activity and stimulation of ATPase activity raise the possibility that RNA serves as a regulatory switch for the two processes.
doi:10.1016/j.virusres.2012.07.007
PMCID: PMC3467334  PMID: 22814430
Hepatitis C Virus; positive-strand RNA virus; NS3 protease; nucleic acid inhibitors; RNA binding; ATPase activation
4.  Strategy for the creation of clinical grade hESC line banks that HLA-match a target population 
EMBO Molecular Medicine  2012;5(1):10-17.
Here, we describe a pre-derivation embryo haplotyping strategy that we developed in order to maximize the efficiency and minimize the costs of establishing banks of clinical grade hESC lines in which human leukocyte antigen (HLA) haplotypes match a significant proportion of the population. Using whole genome amplification followed by medium resolution HLA typing using PCR amplification with sequence-specific primers (PCR-SSP), we have typed the parents, embryos and hESC lines from three families as well as our eight clinical grade hESC lines and shown that this technical approach is rapid, reliable and accurate. By employing this pre-derivation strategy where, based on HLA match, embryos are selected for a GMP route on day 3–4 of development, we would have drastically reduced our cGMP laboratory running costs.
doi:10.1002/emmm.201201973
PMCID: PMC3569650  PMID: 23161805
clinical grade stem cells; clinical grade human embryonic stem cell validation and banking; HLA typing; human embryonic stem cells; single cell analyses
5.  Minimum information about a marker gene sequence (MIMARKS) and minimum information about any (x) sequence (MIxS) specifications 
Yilmaz, Pelin | Kottmann, Renzo | Field, Dawn | Knight, Rob | Cole, James R | Amaral-Zettler, Linda | Gilbert, Jack A | Karsch-Mizrachi, Ilene | Johnston, Anjanette | Cochrane, Guy | Vaughan, Robert | Hunter, Christopher | Park, Joonhong | Morrison, Norman | Rocca-Serra, Philippe | Sterk, Peter | Arumugam, Manimozhiyan | Bailey, Mark | Baumgartner, Laura | Birren, Bruce W | Blaser, Martin J | Bonazzi, Vivien | Booth, Tim | Bork, Peer | Bushman, Frederic D | Buttigieg, Pier Luigi | Chain, Patrick S G | Charlson, Emily | Costello, Elizabeth K | Huot-Creasy, Heather | Dawyndt, Peter | DeSantis, Todd | Fierer, Noah | Fuhrman, Jed A | Gallery, Rachel E | Gevers, Dirk | Gibbs, Richard A | Gil, Inigo San | Gonzalez, Antonio | Gordon, Jeffrey I | Guralnick, Robert | Hankeln, Wolfgang | Highlander, Sarah | Hugenholtz, Philip | Jansson, Janet | Kau, Andrew L | Kelley, Scott T | Kennedy, Jerry | Knights, Dan | Koren, Omry | Kuczynski, Justin | Kyrpides, Nikos | Larsen, Robert | Lauber, Christian L | Legg, Teresa | Ley, Ruth E | Lozupone, Catherine A | Ludwig, Wolfgang | Lyons, Donna | Maguire, Eamonn | Methé, Barbara A | Meyer, Folker | Muegge, Brian | Nakielny, Sara | Nelson, Karen E | Nemergut, Diana | Neufeld, Josh D | Newbold, Lindsay K | Oliver, Anna E | Pace, Norman R | Palanisamy, Giriprakash | Peplies, Jörg | Petrosino, Joseph | Proctor, Lita | Pruesse, Elmar | Quast, Christian | Raes, Jeroen | Ratnasingham, Sujeevan | Ravel, Jacques | Relman, David A | Assunta-Sansone, Susanna | Schloss, Patrick D | Schriml, Lynn | Sinha, Rohini | Smith, Michelle I | Sodergren, Erica | Spor, Aymé | Stombaugh, Jesse | Tiedje, James M | Ward, Doyle V | Weinstock, George M | Wendel, Doug | White, Owen | Whiteley, Andrew | Wilke, Andreas | Wortman, Jennifer R | Yatsunenko, Tanya | Glöckner, Frank Oliver
Nature Biotechnology  2011;29(5):415-420.
Here we present a standard developed by the Genomic Standards Consortium (GSC) for reporting marker gene sequences—the minimum information about a marker gene sequence (MIMARKS). We also introduce a system for describing the environment from which a biological sample originates. The ‘environmental packages’ apply to any genome sequence of known origin and can be used in combination with MIMARKS and other GSC checklists. Finally, to establish a unified standard for describing sequence data and to provide a single point of entry for the scientific community to access and learn about GSC checklists, we present the minimum information about any (x) sequence (MIxS). Adoption of MIxS will enhance our ability to analyze natural genetic diversity documented by massive DNA sequencing efforts from myriad ecosystems in our ever-changing biosphere.
doi:10.1038/nbt.1823
PMCID: PMC3367316  PMID: 21552244
6.  The Role of Innate APOBEC3G and Adaptive AID Immune Responses in HLA-HIV/SIV Immunized SHIV Infected Macaques 
PLoS ONE  2012;7(4):e34433.
The AID/APOBEC family (activation induced deaminase/apolipoprotein B mRNA editing cytokine deaminase) in B cells play important roles in adaptive and innate immunity. Whereas APOBEC3G has been studied in CD4+ T cells and myeloid cells its functional potential in B cells has received little attention. AID combines two critical functions of antibodies, class switching and affinity maturation and may serve as a functional surrogate of protection. These functions were studied following systemic immunization of rhesus macaques with recombinant HLA constructs, linked with HIV and SIV antigens and HSP70 to dextran. The results showed significant upregulation of AID in CD20+ B cells, APOBEC 3G in CD27+ memory B cells and CD4+ effector memory T cells. After immunization the upregulated APOBEC 3G and AID were directly correlated in B cells (p<0.0001). Following challenge with SHIV SF162.P4 the viral load was inversely correlated with AID in B cells and APOBEC 3G in B and T cells, suggesting that both deaminases may have protective functions. Investigation of major interactions between DC, T cells and B cells showed significant increase in membrane associated IL-15 in DC and CD40L in CD4+ T cells. IL-15 binds the IL-15 receptor complex in CD4+ T and B cells, which may reactivate the DC, T and B cell interactions. The overall results are consistent with AID inhibiting pre-entry SHIV by eliciting IgG and IgA antibodies, whereas APOBEC 3G may contribute to the post-entry control of SHIV replication and cellular spread.
doi:10.1371/journal.pone.0034433
PMCID: PMC3326050  PMID: 22514633
7.  Immunization with Recombinant HLA Classes I and II, HIV-1 gp140, and SIV p27 Elicits Protection against Heterologous SHIV Infection in Rhesus Macaques▿† 
Journal of Virology  2011;85(13):6442-6452.
Major histocompatibility complex (MHC) molecules expressed on the surface of human immunodeficiency virus (HIV) are potential targets for neutralizing antibodies. Since MHC molecules are polymorphic, nonself MHC can also be immunogenic. We have used combinations of novel recombinant HLA class I and II and HIV/simian immunodeficiency virus (SIV) antigens, all linked to dextran, to investigate whether they can elicit protective immunity against heterologous simian/human immunodeficiency virus (SHIV) challenge in rhesus macaques. Three groups of animals were immunized with HLA (group 1, n = 8), trimeric YU2 HIV type 1 (HIV-1) gp140 and SIV p27 (HIV/SIV antigens; group 2, n = 8), or HLA plus HIV/SIV antigens (group 3, n = 8), all with Hsp70 and TiterMax Gold adjuvant. Another group (group 4, n = 6) received the same vaccine as group 3 without TiterMax Gold. Two of eight macaques in group 3 were completely protected against intravenous challenge with 18 50% animal infective doses (AID50) of SHIV-SF162P4/C grown in human cells expressing HLA class I and II lineages represented in the vaccine, while the remaining six macaques showed decreased viral loads compared to those in unimmunized animals. Complement-dependent neutralizing activity in serum and high levels of anti-HLA antibodies were elicited in groups 1 and 3, and both were inversely correlated with the plasma viral load at 2 weeks postchallenge. Antibody-mediated protection was strongly supported by the fact that transfer of pooled serum from the two challenged but uninfected animals protected two naïve animals against repeated low-dose challenge with the same SHIV stock. This study demonstrates that immunization with recombinant HLA in combination with HIV-1 antigens might be developed into an alternative strategy for a future AIDS vaccine.
doi:10.1128/JVI.00129-11
PMCID: PMC3126489  PMID: 21490092
8.  Major submissions tool developments at the European nucleotide archive 
Nucleic Acids Research  2011;40(D1):D43-D47.
The European Nucleotide Archive (ENA; http://www.ebi.ac.uk/ena), Europe's primary nucleotide sequence resource, captures and presents globally comprehensive nucleic acid sequence and associated information. Covering the spectrum from raw data to assembled and functionally annotated genomes, the ENA has witnessed a dramatic growth resulting from advances in sequencing technology and ever broadening application of the methodology. During 2011, we have continued to operate and extend the broad range of ENA services. In particular, we have released major new functionality in our interactive web submission system, Webin, through developments in template-based submissions for annotated sequences and support for raw next-generation sequence read submissions.
doi:10.1093/nar/gkr946
PMCID: PMC3245037  PMID: 22080548
9.  Hepatitis C Virus Nonstructural Protein 5A: Biochemical Characterization of a Novel Structural Class of RNA-Binding Proteins▿  
Journal of Virology  2010;84(24):12480-12491.
Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) exhibits a preference for G/U-rich RNA in vitro. Biological analysis of the NS5A RNA-binding activity and its target sites in the genome will be facilitated by a description of the NS5A-RNA complex. We demonstrate that the C-4 carbonyl of the uracil base and, by inference, the C-6 carbonyl of the guanine base interact with NS5A. U-rich RNA of 5 to 6 nucleotides (nt) is sufficient for high-affinity binding to NS5A. The minimal RNA-binding domain of NS5A consists of residues 2005 to 2221 (referred to as domain I-plus). This region of the protein includes the amino-terminal domain I as well as the subsequent linker that separates domains I and II. This linker region is the site of adaptive mutations. U-rich RNA-binding activity is not observed for an NS5A derivative containing only residues 2194 to 2419 (domains II and III). Mass spectrometric analysis of an NS5A-poly(rU) complex identified domains I and II as sites for interaction with RNA. Dimerization of NS5A was demonstrated by glutaraldehyde cross-linking. This dimerization is likely mediated by domain I-plus, as dimers of this protein are trapped by cross-linking. Dimers of the domain II-III protein are not observed. The monomer-dimer equilibrium of NS5A shifts in favor of dimer when U-rich RNA is present but not when A-rich RNA is present, consistent with an NS5A dimer being the RNA-binding-competent form of the protein. These data provide a molecular perspective of the NS5A-RNA complex and suggest possible mechanisms for regulation of HCV and cellular gene expression.
doi:10.1128/JVI.01319-10
PMCID: PMC3004340  PMID: 20926572
10.  The association of the PTPN22 620W polymorphism with Behçet's disease 
Annals of the Rheumatic Diseases  2007;66(11):1531-1533.
Objectives
A single nucleotide polymorphism (SNP) of the gene encoding protein tyrosine phosphatase type 22 (PTPN22 620W) has recently been described as a strong common genetic risk factor for human autoimmune disease. We have analysed the association of PTPN22 620W in patients with Behçet's disease (BD).
Methods
Genomic DNA was obtained from 270 patients with BD from the UK and the Middle East. Normal controls (n  =  203) were collected from the same populations. Patients with idiopathic retinal vasculitis from the UK (n  =  136) were used as disease controls. PTPN22 620W was detected by SSP–PCR analysis and agarose gel electrophoresis.
Results
The results showed an inverse correlation between the presence of PTPN22 620W and Behçet's disease in either patient group tested. There was a greatly reduced prevalence in Middle Eastern compared to UK patients and controls. Finally, there was no association with either UK patients with retinal vasculitis compared with UK controls.
Conclusions
The presence of PTPN22 620W was inversely associated with BD and the distribution of the SNP in the Middle East supports previous findings in the global prevalence.
doi:10.1136/ard.2007.073866
PMCID: PMC2111602  PMID: 17660222
11.  The European Nucleotide Archive 
Nucleic Acids Research  2010;39(Database issue):D28-D31.
The European Nucleotide Archive (ENA; http://www.ebi.ac.uk/ena) is Europe’s primary nucleotide-sequence repository. The ENA consists of three main databases: the Sequence Read Archive (SRA), the Trace Archive and EMBL-Bank. The objective of ENA is to support and promote the use of nucleotide sequencing as an experimental research platform by providing data submission, archive, search and download services. In this article, we outline these services and describe major changes and improvements introduced during 2010. These include extended EMBL-Bank and SRA-data submission services, extended ENA Browser functionality, support for submitting data to the European Genome-phenome Archive (EGA) through SRA, and the launch of a new sequence similarity search service.
doi:10.1093/nar/gkq967
PMCID: PMC3013801  PMID: 20972220
12.  Reduced telomere length in rheumatoid arthritis is independent of disease activity and duration 
Annals of the Rheumatic Diseases  2006;66(4):476-480.
Background
Rheumatoid arthritis (RA) is associated with reduced lifespan and shortened telomere length in lymphocytes, but the mechanism underlying this is unclear. Telomere loss in white blood cells (WBC) is accelerated by oxidative stress and inflammation in vitro. It was postulated that the accelerated WBC telomere shortening in RA occurs as a result of exposure to chronic inflammation.
Objectives
To measure telomere terminal restriction fragment (TRF) length in a large cohort of RA cases and healthy controls, to explore associations of TRF length with features of disease and with RA‐associated HLA‐DRB1 alleles.
Methods
WBC and TRF length were measured by Southern blot in DNA from 176 hospital‐based RA cases satisfying the 1987 American College of Rheumatology criteria and from 1151 controls. TRF length was compared between cases and controls, and the effects of disease duration, severity and HLA‐DRB1 alleles encoding the shared epitope (SE) were assessed.
Results
Age‐ and sex‐adjusted TRF length was significantly shorter in RA cases compared with controls (p<0.001). There was no association between age‐ and sex‐adjusted TRF length and disease duration, C reactive protein or Larsen score. The presence of one or more SE‐encoding alleles was associated with reduced adjusted TRF length in RA cases (SE positive vs SE negative cases, p = 0.038), but not in controls.
Conclusion
The reduced TRF length in a large group of patients with RA compared with controls has been shown. The reduction is apparently independent of disease duration and markers of disease severity, but is influenced by HLA‐DRB1 genotype.
doi:10.1136/ard.2006.059188
PMCID: PMC1856061  PMID: 17114192
13.  Heterosexual and Homosexual Partners Practising Unprotected Sex May Develop Allogeneic Immunity and to a Lesser Extent Tolerance 
PLoS ONE  2009;4(11):e7938.
Background
Epidemiological studies suggest that allogeneic immunity may inhibit HIV-1 transmission from mother to baby and is less frequent in multiparous than uniparous women. Alloimmune responses may also be elicited during unprotected heterosexual intercourse, which is associated ex vivo with resistance to HIV infection.
Methodology/Principal Findings
The investigation was carried out in well-defined heterosexual and homosexual monogamous partners, practising unprotected sex and a heterosexual cohort practising protected sex. Allogeneic CD4+ and CD8+ T cell proliferative responses were elicited by stimulating PBMC with the partners' irradiated monocytes and compared with 3rd party unrelated monocytes, using the CFSE method. Significant increase in allogeneic proliferative responses was found in the CD4+ and CD8+ T cells to the partners' irradiated monocytes, as compared with 3rd party unrelated monocytes (p≤0.001). However, a significant decrease in proliferative responses, especially of CD8+ T cells to the partners' compared with 3rd party monocytes was consistent with tolerization, in both the heterosexual and homosexual partners (p<0.01). Examination of CD4+CD25+FoxP3+ regulatory T cells by flow cytometry revealed a significantly greater proportion of these cells in the homosexual than heterosexual partners practising unprotected sex (p<0.05). Ex vivo studies of infectivity of PBMC with HIV-1 showed significantly greater inhibition of infectivity of PBMC from heterosexual subjects practising unprotected compared with those practising protected sex (p = 0.02).
Conclusions/Significance
Both heterosexual and homosexual monogamous partners practising unprotected sex develop allogeneic CD4+ and CD8+ T cell proliferative responses to the partners' unmatched cells and a minority may be tolerized. However, a greater proportion of homosexual rather than heterosexual partners developed CD4+CD25FoxP3+ regulatory T cells. These results, in addition to finding greater inhibition of HIV-1 infectivity in PBMC ex vivo in heterosexual partners practising unprotected, compared with those practising protected sex, suggest that allogeneic immunity may play a significant role in the immuno-pathogenesis of HIV-1 infection.
doi:10.1371/journal.pone.0007938
PMCID: PMC2775923  PMID: 19956755
14.  Improvements to services at the European Nucleotide Archive 
Nucleic Acids Research  2009;38(Database issue):D39-D45.
The European Nucleotide Archive (ENA; http://www.ebi.ac.uk/ena) is Europe’s primary nucleotide sequence archival resource, safeguarding open nucleotide data access, engaging in worldwide collaborative data exchange and integrating with the scientific publication process. ENA has made significant contributions to the collaborative nucleotide archival arena as an active proponent of extending the traditional collaboration to cover capillary and next-generation sequencing information. We have continued to co-develop data and metadata representation formats with our collaborators for both data exchange and public data dissemination. In addition to the DDBJ/EMBL/GenBank feature table format, we share metadata formats for capillary and next-generation sequencing traces and are using and contributing to the NCBI SRA Toolkit for the long-term storage of the next-generation sequence traces. During the course of 2009, ENA has significantly improved sequence submission, search and access functionalities provided at EMBL–EBI. In this article, we briefly describe the content and scope of our archive and introduce major improvements to our services.
doi:10.1093/nar/gkp998
PMCID: PMC2808951  PMID: 19906712
15.  RNA Binding by the Brome Mosaic Virus Capsid Protein and the Regulation of Viral RNA Accumulation 
Journal of molecular biology  2009;391(2):314-326.
Viral capsid proteins (CPs) can regulate gene expression and encapsulate viral RNAs. Low-level expression of the brome mosaic virus (BMV) CP was found to stimulate viral RNA accumulation, while higher levels inhibited translation and BMV RNA replication. Regulation of translation acts through an RNA element named the B box, which is also critical for the replicase assembly. The BMV CP has also been shown to preferentially bind to an RNA element named SLC that contains the core promoter for genomic minus-strand RNA synthesis. To further elucidate CP interaction with RNA, Available online we used a reversible cross-linking–peptide fingerprinting assay to identify 27 May 2009 peptides in the capsid that contact the SLC, the B-box RNA, and the encapsidated RNA. Transient expression of three mutations made in residues within or close by the cross-linked peptides partially released the normal inhibition of viral RNA accumulation in agroinfiltrated Nicotiana benthamiana. Interestingly, two of the mutants, R142A and D148A, were found to retain the ability to down-regulate reporter RNA translation. These two mutants formed viral particles in inoculated leaves, but only R142Awas able to move systemically in the inoculated plant. The R142A CP was found to have higher affinities for SLC and the B box compared with those of wild-type CP and to alter contacts to the RNA in the virion. These results better define how the BMV CP can interact with RNA and regulate different viral processes.
doi:10.1016/j.jmb.2009.05.065
PMCID: PMC2774812  PMID: 19481091
positive-strand RNA virus; brome mosaic virus; capsid protein; RNA replication; translation
16.  B-cell diversity decreases in old age and is correlated with poor health status 
Aging Cell  2009;8(1):18-25.
Older people suffer from a decline in immune system, which affects their ability to respond to infections and to raise efficient responses to vaccines. Effective and specific antibodies in responses from older individuals are decreased in favour of non-specific antibody production. We investigated the B-cell repertoire in DNA samples from peripheral blood of individuals aged 86–94 years, and a control group aged 19–54 years, using spectratype analysis of the IGHV complementarity determining region (CDR)3. We found that a proportion of older individuals had a dramatic collapse in their B-cell repertoire diversity. Sequencing of polymerase chain reaction products from a selection of samples indicated that this loss of diversity was characterized by clonal expansions of B cells in vivo. Statistical analysis of the spectratypes enabled objective comparisons and showed that loss of diversity correlated very strongly with the general health status of the individuals; a distorted spectratype can be used to predict frailty. Correlations with survival and vitamin B12 status were also seen. We conclude that B-cell diversity can decrease dramatically with age and may have important implications for the immune health of older people. B-cell immune frailty is also a marker of general frailty.
doi:10.1111/j.1474-9726.2008.00443.x
PMCID: PMC2667647  PMID: 18986373
aging; B cells; diversity; elderly; immune frailty
17.  Genotype analysis of polymorphisms in autoimmune susceptibility genes, CTLA-4 and PTPN22, in an acute anterior uveitis cohort 
Molecular Vision  2009;15:208-212.
Purpose
Acute anterior uveitis (AAU) is the most common form of uveitis and is thought to be autoimmune in nature. Recent studies have described genes that act as master controllers of autoimmunity. Protein tyrosine phosphatase type 22 (PTPN22) and Cytotoxic T lymphocyte antigen-4 (CTLA-4) are two of these genes, and single nucleotide polymorphisms (SNPs) in the genes encoding these molecules have been associated with several autoimmune diseases. In this study we have analyzed SNPs in PTPN22 and CTLA-4 in patients with AAU.
Methods
The functional protein tyrosine phosphatase type 22 (PTPN22) SNP (R620W rs2476601, 1858C/T), and two CTLA-4 SNPs (rs5742909, −318C/T and rs231775, 49A/G) were analyzed in 140 patients with AAU and 92 healthy controls by sequence-specific primer -polymerase chain reaction (SSP-PCR). Data was analyzed by χ2 analysis and Fisher’s exact test.
Results
There was no significant association between PTPN22 620W, CTLA-4 −318C/T, or CTLA-4 49A/G and AAU. Similarly, there was no association with the three SNPs when patients were classified by race or gender. Finally, there was no association with the presence of ankylosing spondylitis in the patient cohort.
Conclusions
The data do not support an association between SNPs in PTPN22 and CTLA-4, genes regarded as genetic master switches of autoimmunity. This raises the issue of the etiology of AAU and the possibility that it should be regarded as an autoinflammatory rather than an autoimmune condition.
PMCID: PMC2632733  PMID: 19180256
18.  Single-Stranded Oligonucleotides Can Inhibit Cytokine Production Induced by Human Toll-Like Receptor 3▿ †  
Molecular and Cellular Biology  2008;28(14):4507-4519.
Toll-like receptor 3 (TLR3) can signal the production of a suite of cytokines and chemokines in response to double-stranded RNA (dsRNA) ligands or the dsRNA mimic poly(I-C). Using a human embryonic kidney 293T cell line to express human TLR3, we determined that poly(I-C)-induced signal could be significantly inhibited by single-stranded DNAs (ssDNAs), but not ssRNA or dsDNA. The ssDNA molecules that down-modulated TLR3 signaling did not affect TLR4 and do not require the hypomethylated CpG motif found in TLR9 ligands. The degree of modulation can be altered by the length, base sequence, and modification state of the ssDNAs. An inhibitory ssDNA was found to colocalize with TLR3 in transfected cells and in a cell line that naturally expresses TLR3. The inhibitory ssDNAs can compete efficiently with dsRNA for binding purified TLR3 ectodomains in vitro, while noninhibitory nucleic acids do not. The ssDNAs also decrease the levels of several cytokines produced by the human bronchial epithelial cell line BEAS-2B and by human peripheral blood mononuclear cells in response to poly(I-C) stimulation of native TLR3. These activities indicate that ssDNAs could be used to regulate the inflammatory response through TLR3.
doi:10.1128/MCB.00308-08
PMCID: PMC2447120  PMID: 18490443
19.  Petabyte-scale innovations at the European Nucleotide Archive 
Nucleic Acids Research  2008;37(Database issue):D19-D25.
Dramatic increases in the throughput of nucleotide sequencing machines, and the promise of ever greater performance, have thrust bioinformatics into the era of petabyte-scale data sets. Sequence repositories, which provide the feed for these data sets into the worldwide computational infrastructure, are challenged by the impact of these data volumes. The European Nucleotide Archive (ENA; http://www.ebi.ac.uk/embl), comprising the EMBL Nucleotide Sequence Database and the Ensembl Trace Archive, has identified challenges in the storage, movement, analysis, interpretation and visualization of petabyte-scale data sets. We present here our new repository for next generation sequence data, a brief summary of contents of the ENA and provide details of major developments to submission pipelines, high-throughput rule-based validation infrastructure and data integration approaches.
doi:10.1093/nar/gkn765
PMCID: PMC2686451  PMID: 18978013
20.  Priorities for nucleotide trace, sequence and annotation data capture at the Ensembl Trace Archive and the EMBL Nucleotide Sequence Database 
Nucleic Acids Research  2007;36(Database issue):D5-D12.
The Ensembl Trace Archive (http://trace.ensembl.org/) and the EMBL Nucleotide Sequence Database (http://www.ebi.ac.uk/embl/), known together as the European Nucleotide Archive, continue to see growth in data volume and diversity. Selected major developments of 2007 are presented briefly, along with data submission and retrieval information. In the face of increasing requirements for nucleotide trace, sequence and annotation data archiving, data capture priority decisions have been taken at the European Nucleotide Archive. Priorities are discussed in terms of how reliably information can be captured, the long-term benefits of its capture and the ease with which it can be captured.
doi:10.1093/nar/gkm1018
PMCID: PMC2238915  PMID: 18039715
21.  eGenomics: Cataloguing Our Complete Genome Collection III 
This meeting report summarizes the proceedings of the “eGenomics: Cataloguing our Complete Genome Collection III” workshop held September 11–13, 2006, at the National Institute for Environmental eScience (NIEeS), Cambridge, United Kingdom. This 3rd workshop of the Genomic Standards Consortium was divided into two parts. The first half of the three-day workshop was dedicated to reviewing the genomic diversity of our current and future genome and metagenome collection, and exploring linkages to a series of existing projects through formal presentations. The second half was dedicated to strategic discussions. Outcomes of the workshop include a revised “Minimum Information about a Genome Sequence” (MIGS) specification (v1.1), consensus on a variety of features to be added to the Genome Catalogue (GCat), agreement by several researchers to adopt MIGS for imminent genome publications, and an agreement by the EBI and NCBI to input their genome collections into GCat for the purpose of quantifying the amount of optional data already available (e.g., for geographic location coordinates) and working towards a single, global list of all public genomes and metagenomes.
doi:10.1155/2007/47304
PMCID: PMC1872051
22.  EMBL Nucleotide Sequence Database in 2006 
Nucleic Acids Research  2006;35(Database issue):D16-D20.
The EMBL Nucleotide Sequence Database () at the EMBL European Bioinformatics Institute, UK, offers a large and freely accessible collection of nucleotide sequences and accompanying annotation. The database is maintained in collaboration with DDBJ and GenBank. Data are exchanged between the collaborating databases on a daily basis to achieve optimal synchrony. Webin is the preferred tool for individual submissions of nucleotide sequences, including Third Party Annotation, alignments and bulk data. Automated procedures are provided for submissions from large-scale sequencing projects and data from the European Patent Office. In 2006, the volume of data has continued to grow exponentially. Access to the data is provided via SRS, ftp and variety of other methods. Extensive external and internal cross-references enable users to search for related information across other databases and within the database. All available resources can be accessed via the EBI home page at . Changes over the past year include changes to the file format, further development of the EMBLCDS dataset and developments to the XML format.
doi:10.1093/nar/gkl913
PMCID: PMC1897316  PMID: 17148479
23.  EMBL Nucleotide Sequence Database: developments in 2005 
Nucleic Acids Research  2005;34(Database issue):D10-D15.
The EMBL Nucleotide Sequence Database () at the EMBL European Bioinformatics Institute, UK, offers a comprehensive set of publicly available nucleotide sequence and annotation, freely accessible to all. Maintained in collaboration with partners DDBJ and GenBank, coverage includes whole genome sequencing project data, directly submitted sequence, sequence recorded in support of patent applications and much more. The database continues to offer submission tools, data retrieval facilities and user support. In 2005, the volume of data offered has continued to grow exponentially. In addition to the newly presented data, the database encompasses a range of new data types generated by novel technologies, offers enhanced presentation and searchability of the data and has greater integration with other data resources offered at the EBI and elsewhere. In stride with these developing data types, the database has continued to develop submission and retrieval tools to maximise the information content of submitted data and to offer the simplest possible submission routes for data producers. New developments, the submission process, data retrieval and access to support are presented in this paper, along with links to sources of further information.
doi:10.1093/nar/gkj130
PMCID: PMC1347492  PMID: 16381823
24.  The EMBL Nucleotide Sequence Database 
Nucleic Acids Research  2004;32(Database issue):D27-D30.
The EMBL Nucleotide Sequence Database (http://www.ebi.ac.uk/embl/), maintained at the European Bioinformatics Institute (EBI), incorporates, organizes and distributes nucleotide sequences from public sources. The database is a part of an international collaboration with DDBJ (Japan) and GenBank (USA). Data are exchanged between the collaborating databases on a daily basis to achieve optimal synchrony. The web-based tool, Webin, is the preferred system for individual submission of nucleotide sequences, including Third Party Annotation (TPA) and alignment data. Automatic submission procedures are used for submission of data from large-scale genome sequencing centres and from the European Patent Office. Database releases are produced quarterly. The latest data collection can be accessed via FTP, email and WWW interfaces. The EBI’s Sequence Retrieval System (SRS) integrates and links the main nucleotide and protein databases as well as many other specialist molecular biology databases. For sequence similarity searching, a variety of tools (e.g. FASTA and BLAST) are available that allow external users to compare their own sequences against the data in the EMBL Nucleotide Sequence Database, the complete genomic component subsection of the database, the WGS data sets and other databases. All available resources can be accessed via the EBI home page at http://www.ebi.ac.uk.
doi:10.1093/nar/gkh120
PMCID: PMC308854  PMID: 14681351
25.  The EMBL Nucleotide Sequence Database: major new developments 
Nucleic Acids Research  2003;31(1):17-22.
The EMBL Nucleotide Sequence Database (http://www.ebi.ac.uk/embl/) incorporates, organizes and distributes nucleotide sequences from all available public sources. The database is located and maintained at the European Bioinformatics Institute (EBI) near Cambridge, UK. In an international collaboration with DDBJ (Japan) and GenBank (USA), data are exchanged amongst the collaborating databases on a daily basis to achieve optimal synchronization. Webin is the preferred web-based submission system for individual submitters, while automatic procedures allow incorporation of sequence data from large-scale genome sequencing centres and from the European Patent Office (EPO). Database releases are produced quarterly. Network services allow free access to the most up-to-date data collection via FTP, Email and World Wide Web interfaces. EBI's Sequence Retrieval System (SRS) integrates and links the main nucleotide and protein databases plus many other specialized molecular biology databases. For sequence similarity searching, a variety of tools (e.g. Fasta, BLAST) are available which allow external users to compare their own sequences against the latest data in the EMBL Nucleotide Sequence Database and SWISS-PROT. All resources can be accessed via the EBI home page at http://www.ebi.ac.uk.
PMCID: PMC165468  PMID: 12519939

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