Biomedical subjects

Alexis Styche

Publications and source records attributed to Alexis Styche.

7 recordsLinked to original sources

PubMed2007

Pyrosequencing-based strategies for improved allele typing of human leukocyte antigen loci.

Successful transplantation of tissue during solid organ and bone marrow transplantation relies on accurate determination of the human leukocyte antigen (HLA) phenotype of the potential donor(s) and recipient. Matching donor with recipient for a kidney transplant generally means finding a six-antigen match by looking at each of two alleles at HLA-A, -B, and -DR loci. For bone marrow transplantation the HLA-C and -DQ alleles are also considered. Molecular techniques, including sequencing, are capable of precisely defining HLA alleles. Because of the large number of possible allelic combinations there are numerous ambiguities associated with heterozygous genotypes even when sequence-based typing protocols are used. Sequencing-by-synthesis methodology employed by Pyrosequencing represents an improvement when applied to HLA genotyping that allows resolution of many ambiguous allelic pairs. Out-of-phase sequencing of HLA alleles by Pyrosequencing can resolve cis/trans ambiguities that would otherwise require the sequencing of isolated cloned DNAs. Single-nucleotide polymorphism typing of HLA for the presence of specific variants is also beneficial for monitoring HLA-encoded genetic risk to autoimmune diseases, such as celiac disease, rheumatoid arthritis, and type 1 diabetes mellitus.

Alleles↗

PubMed2007

Web-based primer design software for genome-scale genotyping by pyrosequencing.

Design of locus-specific primers for use during genetic analysis requires combining information from multiple sources and can be a time-consuming process when validating large numbers of assays. Data warehousing of genomic DNA sequences and genetic variations when coupled with software applications for optimizing the generation of locus-specific primers can increase the efficiency of assay development. Selection of oligonucleotide primers for PCR and Pyrosequencing (SOP3) software allows user-directed queries of warehoused data collected from the human and mouse genome sequencing projects. The software automates collection of DNA sequence flanking single-nucleotide polymorphisms (SNPs) as well as the incorporation of locus-associated functional information, such as whether the SNP occurs in an exon, intron, or untranslated region. SOP3 software accepts three types of user-directed input consisting of gene locus symbols, SNP reference sequence numbers, or chromosomal physical location. For human polymorphisms, SOP3 incorporates haplotype, ethnicity, and SNP validation attributes. The output is a list of oligonucleotide primers recommended for Pyrosequencing-based typing of genetic variations. SOP3 is available at the Division of Immunogenetics computational server found at http://imgen.ccbb.pitt.edu.

Animals↗

PubMed2006

Interleukin-7 is a survival factor for CD4+ CD25+ T-cells and is expressed by diabetes-suppressive dendritic cells.

Dendritic cells can facilitate allograft survival and prevent autoimmunity via direct and indirect cell-mediated mechanisms. Recent studies demonstrate that immunoregulatory dendritic cells (iDCs) confer immune hyporesponsiveness in part through CD4(+) CD25(+) T regulatory cells (Tregs). Herein, we provide evidence to support the hypothesis that dendritic cells derived from NOD mice and engineered ex vivo to exhibit suppressed expression of the CD40, CD80, and CD86 costimulatory molecules motivate an increase in the prevalence of regulatory CD4(+) CD25(+) T-cells via interleukin (IL)-7. Unlike control dendritic cells, these dendritic cells expressed significant levels of IL-7. Exogenous addition of IL-7 to NOD T-cells did not promote expansion or proliferation, but instead selectively maintained the number of CD4(+) CD25(+) T-cells by inhibiting activation of apoptosis in these cells. In vitro, IL-7 receptor alpha-chain (IL-7Ralpha) was expressed at significantly higher levels on CD4(+) CD25(+) T-cells compared with CD4(+) CD25(-) T-cells irrespective of resting or stimulated state. In vivo, CD4(+) CD25(+) T-cells obtained from NOD-scid mice reconstituted with ex vivo engineered iDCs and NOD splenocytes expressed significantly higher levels of IL-7Ralpha compared with levels in the CD4(+) CD25(-) subset, especially in diabetes-suppressive dendritic cell-administered NOD-scid recipients. Taken together, our data suggest a novel mechanism by which iDCs delay autoimmunity through the CD4(+) CD25(+) Treg pathway and suggest IL-7 as a survival factor for these putative Tregs, which express the alpha-chain of its receptor at considerably higher levels than CD4(+) CD25(-) T-cells.

Animals↗

PubMed2005

SOP3v2: web-based selection of oligonucleotide primer trios for genotyping of human and mouse polymorphisms.

SOP3v2 is a database-driven graphical web-based application for facilitating genotyping assay design. SOP3v2 accepts data input in numerous forms, including gene names, reference sequence numbers and physical location. For each entry, the application presents a set of recommended forward and reverse PCR primers, along with a sequencing primer, which is optimized for sequence-based genotyping assays. SOP3v2-generated oligonucleotide primer trios enable analysis of single nucleotide polymorphisms (SNPs) as well as insertion/deletion polymorphisms found in genomic DNA. The application's database was generated by warehousing information from the National Center for Biotechnology Information (NCBI) dbSNP database, genomic DNA sequences from human and mouse, and LocusLink gene attribute information. Query results can be sorted by their biological relevance, such as nonsynonymous coding changes or physical location. Human polymorphism queries may specify ethnicity, haplotype and validation status. Primers are developed using SOP3v2's core algorithm for evaluating primer candidates through stability tests and are suitable for use with sequence-based genotyping methods requiring locus-specific amplification. The method has undergone laboratory validation. Of the SOP3v2-designed primer trios that were tested, a majority (>80%) have successfully produced genotyping data. The application may be accessed via the web at http://imgen.ccbb.pitt.edu/sop3v2.

Animals↗

PubMed2005

SOP3: a web-based tool for selection of oligonucleotide primers for single nucleotide polymorphism analysis by Pyrosequencing.

SOP3 is a web-based software tool for designing oligonucleotide primers for use in the analysis of single nucleotide polymorphisms (SNPs). Accessible via the Internet, the application is optimized for developing the PCR and sequencing primers that are necessary for Pyrosequencing. The application accepts as input gene name, SNP reference sequence number, or chromosomal nucleotide location. Output can be parsed by gene name, SNP reference number, heterozygosity value, location, chromosome, or function. The location of an individual polymorphism, such as an intron, exon, or 5' or 3' untranslated region is indicated, as are whether nucleotide changes in an exon are associated with a change in an amino acid sequence. SOP3 presents for each entry a set of forward and biotinylated reverse PCR primers as well as a sequencing primer for use during the analysis of SNPs by Pyrosequencing. Theoretical pyrograms for each allele are calculated and presented graphically. The method has been tested in the development of Pyrosequencing assays for determining SNPs and for deletion/insertion polymorphisms in the human genome. Of the SOP3-designed primer sets that were tested, a large majority of the primer sets have successfully produced PCR products and Pyrosequencing data.

Algorithms↗

PubMed2004

Antisense oligonucleotides down-regulating costimulation confer diabetes-preventive properties to nonobese diabetic mouse dendritic cells.

Phenotypically "immature" dendritic cells (DCs), defined by low cell surface CD40, CD80, and CD86 can elicit host immune suppression in allotransplantation and autoimmunity. Herein, we report the most direct means of achieving phenotypic immaturity in NOD bone marrow-derived DCs aiming at preventing diabetes in syngeneic recipients. CD40, CD80, and CD86 cell surface molecules were specifically down-regulated by treating NOD DCs ex vivo with a mixture of antisense oligonucleotides targeting the CD40, CD80, and CD86 primary transcripts. The incidence of diabetes was significantly delayed by a single injection of the engineered NOD DCs into syngeneic recipients. Insulitis was absent in diabetes-free recipients and their splenic T cells proliferated in response to alloantigen. Engineered DC promoted an increased prevalence of CD4(+)CD25(+) T cells in NOD recipients at all ages examined and diabetes-free recipients exhibited significantly greater numbers of CD4(+)CD25(+) T cells compared with untreated NOD mice. In NOD-scid recipients, antisense-treated NOD DC promoted an increased prevalence of these putative regulatory T cells. Collectively, these data demonstrate that direct interference of cell surface expression of the major costimulatory molecules at the transcriptional level confers diabetes protection by promoting, in part, the proliferation and/or survival of regulatory T cells. This approach is a useful tool by which DC-mediated activation of regulatory T cells can be studied as well as a potential therapeutic option for type 1 diabetes.

Adoptive Transfer↗

PubMed2004

HLA class II DRB high resolution genotyping by pyrosequencing: comparison of group specific PCR and pyrosequencing primers.

Sequencing of alleles of the highly polymorphic, multiple loci HLA-DRB gene family was performed by pyrosequencing using purified DNA from the 11(th) International Histocompatibility Workshop human lymphoblastiod cell lines as well as genomic DNA isolated from blood samples obtained from healthy adult volunteers. Genomic DNA was prepared from donors whose blood had been stored either frozen or as dried blood spots. Pyrosequence-based typing was optimized for identifying alleles of the HLA-DRB1, -3, -4, and -5 genes. The procedure should be applicable to other HLA loci including the class I genes HLA-A and -B that, along with HLA-DRB, are crucial for histocompatibility matching of tissue antigens during transplantation. Computer simulation of pyrosequencing data suggest that alleles of HLA-DRB1, -3, -4, and -5 were readily identifiable by pyrosequencing as were their heterozygous allelic combinations. Pyrosequencing primers were designed to specifically sequence HLA loci of interest even in a background of other amplified, closely related sequences such as alleles of the pseudogene HLA-DRB6, -7, -8, and -9. Polymorphic residues of HLA-DRB genes were identified within each pyrosequencing reaction, obtained by 50 to 70 nucleotide read lengths. Heterozygous allelic combinations of HLA genes were analyzed and compared successfully to genotyping of alleles by sequence-specific oligonucleotide probe hybridization as well as allele specific polymerase chain reaction protocols. Pyrosequence-based typing is compatible with genotyping of allelic combinations expected from heterozygous individuals, resulting in nucleotide resolution of the highly polymorphic HLA system. Using a single pyrosequence instrument, complete typing of HLA-DRB genes can be performed daily on hundreds of individuals for high resolution histocompatibility genotyping studies.

Base Sequence↗