Biomedical subjects

William A Rudert

Publications and source records attributed to William A Rudert.

14 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

Modulatory role of DR4- to DQ8-restricted CD4 T-cell responses and type 1 diabetes susceptibility.

This study addressed an important biological question, namely how certain HLA molecules modulate the disease risk conferred by other HLA molecules. The HLA molecules under investigation were HLA-DQ8 and -DR4, the two most prevalent HLA class II alleles found in Caucasian type 1 diabetic patients. A panel of human GAD (hGAD65)-specific CD4 T-cell lines and hybridomas was generated to serve as detection reagents for evaluating the peptide occupancy of DQ8 and DR4. Results indicated that DQ8 and DR4 (0401) were able to bind the same hGAD65 peptides. The coexpression of DR4 (0401) diminished DQ8-restricted T-cell responses. In addition, we also demonstrated that the diminished T-cell response varied according to the specific DRB1*04 alleles. Taken together, this study provides evidence that DR4 is able to modulate DQ8-restricted T-cell responses, possibly by competing for peptides. Given that DQ8 is a primary genetic determinant of type 1 diabetes, the decreased DQ8-restricted CD4 T-cell activity due to peptide competition may be the mechanism explaining the modulation effect of DR4 to type 1 diabetes susceptibility.

Amino Acid Sequence↗

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

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↗

PubMed2003

Four-color flow cytometric analysis of peripheral blood donor cell chimerism.

Passenger leukocytes have been demonstrated to play significant roles in initiating and also regulating immune reactions after organ transplantation. Reliable techniques to detect donor leukocytes in recipients after organ transplantation are essential to analyze the role, function, and behavior of these leukocytes. In this report we describe a simple, reliable method to detect donor cells with low frequencies using peripheral blood samples. Detection of small numbers of major histocompatibility complex (MHC) mismatched cells was first studied using four-color flow cytometry in artificially created cell mixtures. By selecting the CD45(+) population and simultaneous staining with several leukocyte lineage markers (CD3, CD4, CD8, CD56, and CD19), MHC-mismatched leukocytes were consistently detected in cell suspensions prepared from directly stained whole blood samples with a threshold sensitivity as low as 0.1%-0.2%. When the fresh peripheral blood mononuclear cells were separated by conventional Ficoll gradient purification, similar, but slightly lower levels of donor cells were detected. Blood samples obtained 1-5 months after liver, kidney, and intestine transplants revealed that the kind of organ allograft influenced levels and lineage pattern of the circulating donor cells. This procedure provided a simple and reliable method in determining early chimerism in transplant recipients. However, the detection of MHC-mismatched leukocytes of all lineages was much lower when frozen peripheral blood mononuclear cells were used.

Antibodies, Monoclonal↗

PubMed2003

Body window-enabled in vivo multicolor imaging of transplanted mouse islets expressing an insulin-Timer fusion protein.

Type 1 diabetes results from the selective destruction of insulin-producing beta cells in the islets of Langerhans, and autoimmune T cells are thought to be the mediators of this destruction. T cells are also responsible for allorejection once the islets are transplanted into a patient to reduce the negative consequences of a lack of insulin. To better understand these processes, we have developed a transgenic mouse expressing proinsulin II tagged with a live-cell fluorescent reporter protein, Timer. Timer protein is unique because it changes color from green to red in the first 24 h after synthesis. With this marker, insulin synthesis can be carefully monitored through fluorescent changes over time. To complement this new biotechnological research tool, we designed a body window to allow for in vivo imaging over time of the islets transplanted under the kidney capsule. The window device, which is sutured to replace the underlying skin and body wall over the site of islet transplantation, may be used to simultaneously observe beta cells and T cells that have been labeled with a fluorochrome distinguishable from Timer. The imaging of both insulin-producing cells and T cells may be carried out repeatedly for a week or more with no need for repeated surgery, while preserving the life of the studied animal.

Animals↗

PubMed2002

Production of alpha 1,3-galactosyltransferase-deficient pigs.

The enzyme alpha1,3-galactosyltransferase (alpha1,3GT or GGTA1) synthesizes alpha1,3-galactose (alpha1,3Gal) epitopes (Galalpha1,3Galbeta1,4GlcNAc-R), which are the major xenoantigens causing hyperacute rejection in pig-to-human xenotransplantation. Complete removal of alpha1,3Gal from pig organs is the critical step toward the success of xenotransplantation. We reported earlier the targeted disruption of one allele of the alpha1,3GT gene in cloned pigs. A selection procedure based on a bacterial toxin was used to select for cells in which the second allele of the gene was knocked out. Sequencing analysis demonstrated that knockout of the second allele of the alpha1,3GT gene was caused by a T-to-G single point mutation at the second base of exon 9, which resulted in inactivation of the alpha1,3GT protein. Four healthy alpha1,3GT double-knockout female piglets were produced by three consecutive rounds of cloning. The piglets carrying a point mutation in the alpha1,3GT gene hold significant value, as they would allow production of alpha1,3Gal-deficient pigs free of antibiotic-resistance genes and thus have the potential to make a safer product for human use.

Alleles↗

PubMed2002

Felic (CIP4b), a novel binding partner with the Src kinase Lyn and Cdc42, localizes to the phagocytic cup.

Through its Src homology 3 (SH3) and SH2 domains, the Src kinase Lyn interacts with a small number of phosphoproteins, such as Shc, Cbl, and Vav, which regulate cell cycle and the cytoskeleton. Using Lyn's Unique, SH3, and SH2 domains as bait in a yeast 2-hybrid screen, we isolated a novel gene product with features of a scaffolding protein. We named it Felic because it contains a domain homologous to the tyrosine kinase Fes and the cytoskeletal protein ezrin and forms a Lyn interaction with the GTPase Cdc42 (Felic). Felic was expressed in both hematopoietic and nonhematopoietic tissues. Because it represents an alternative splice product related to the Cdc42-interacting protein 4, CIP4, we also refer to Felic as CIP4b. Felic contains an SH3 recognition site RXPXXP and multiple tyrosine residues. In insulin or serum-stimulated HEK293 cells, Felic became tyrosine phosphorylated. Like CIP4, Felic associated with Cdc42 in its activated form only. Unlike CIP4, Felic does not possess a C-terminal SH3 domain. Coprecipitation studies show that Felic bound to Lyn or activated forms of Cdc42. Overexpression of Felic or CIP4 inhibited NIH 3T3 cell invasiveness in a Matrigel assay. Because Lyn and Cdc42 are involved in phagocytosis, we examined the distribution of Felic in RAW macrophages during particle ingestion. Felic was recruited more efficiently than CIP4 to the phagocytic cups. Altogether, these data suggest that CIP4/Felic constitute a novel family of cytoskeletal scaffolding proteins, integrating Src and Cdc42 pathways. The absence of an SH3 domain in Felic provides a structural basis for functional differences.

Amino Acid Sequence↗

PubMed2002

Immunosuppressive effects of glucosamine.

Glucosamine is a naturally occurring derivative of glucose and is an essential component of glycoproteins and proteoglycans, important constituents of many eukaryotic proteins. In cells, glucosamine is produced enzymatically by the amidation of glucose 6-phosphate and can then be further modified by acetylation to result in N-acetylglucosamine. Commercially, glucosamine is sold over-the-counter to relieve arthritis. Although there is evidence in favor of the beneficial effects of glucosamine, the mechanism is unknown. Our data demonstrate that glucosamine suppresses the activation of T-lymphoblasts and dendritic cells in vitro as well as allogeneic mixed leukocyte reactivity in a dose-dependent manner. There was no inherent cellular toxicity involved in the inhibition, and the activity was not reproducible with other amine sugars. More importantly, glucosamine administration prolonged allogeneic cardiac allograft survival in vivo. We conclude that, despite its documented effects on insulin sensitivity, glucosamine possesses immunosuppressive activity and could be beneficial as an immunosuppressive agent.

Adenosine Triphosphate↗

PubMed2002

Repression of c-Cbl leads to enhanced G-CSF Jak-STAT signaling without increased cell proliferation.

Engagement of the Granulocyte-Colony-Stimulating Factor (G-CSF) receptor activates non-receptor protein tyrosine kinases Lyn and Jak2. We found that Lyn-deficient DT40 cells that express the G-CSF receptor (DT40GR) do not demonstrate G-CSF-induced mitogenic signaling. Lyn associates with and phosphorylates a small set of molecules, including c-Cbl. c-Cbl is an adaptor involved in cell growth and cytoskeletal reorganization, predominantly in hematopoietic cells. Using yeast two-hybrid analysis, we found that c-Cbl directly couples Lyn to PI 3-kinase. We also found that expression of the c-CblY731F mutant, which uncouples PI 3-kinase, resulted in the inhibition of G-CSF-induced proliferative signaling in DT40GR cells. As a complementary strategy, we sought to analyse the effects of c-Cbl deficiency in DT40GR cells. We isolated, cloned and sequenced the full-length cDNA for chicken c-Cbl and constructed antisense vectors. Antisense inhibition of c-Cbl expression in DT40GR cells led to enhanced Jak-STAT activation following G-CSF stimulation. Yet, this enhancement of Jak-STAT activation was associated with decreased G-CSF-induced PI 3-kinase activity and DNA synthesis. PI 3-kinase activity correlated with DNA synthesis and physiological levels of c-Cbl. Together, these data suggest that physiologic level of c-Cbl provides a growth stimulatory pathway for G-CSF and that enhanced Jak-STAT activation is not sufficient for G-CSF-induced growth.

Amino Acid Sequence↗

PubMed2002

Identification and genetic analysis of human and mouse activated Cdc42 interacting protein-4 isoforms.

By yeast two-hybrid screening with the Src kinase Lyn as bait, we identified a novel gene product with features of a scaffolding protein. Reported as Felic ( es-related, with homology to Ezrin, Lyn interactor with Cdc42), it is related to the CIP4 (Cdc42 Interacting Protein-4) gene. Southern blotting for CIP4/Felic of genomic DNA shows a single band, suggesting no gene duplication. Felic differs from CIP4 because of a 29 nucleotide sequence derived from the end of intron 13. Consequently, there is an out-of-frame translation that destroys an SH3 domain. Analysis of various tissues shows that the original CIP4 is the predominant transcript. Therefore, we propose to call that, CIP4a and Felic, CIP4b. During screening of the colorectal CaCo2 cell line, clones corresponding to a third CIP4-related transcript (CIP4c) were identified. CIP4c encodes a premature stop codon, resulting in the loss of the SH3 domain. A fourth, relatively abundant transcript (CIP4h) was isolated from heart, lung, and trachea tissue. CIP4h retains the SH3 domain. CIP4 levels are modified by all-trans-retinoic acid. The presence of alternative splice transcripts, with or without SH3 domains, suggests that CIP4 regulates cytoskeletal organization through structural-functional differences in a tissue-specific manner.

Amino Acid Sequence↗

PubMed2002

Indoleamine 2,3-dioxygenase expression in transplanted NOD Islets prolongs graft survival after adoptive transfer of diabetogenic splenocytes.

Indoleamine 2,3-dioxygenase (IDO) catalyzes the breakdown of the amino acid tryptophan into kyneurenine. It has been shown that IDO production by placental trophoblasts prevents the attack of maternal T-cells activated in response to the paternal HLA alleles expressed by the tissues of the fetus. In this article, we show that adenoviral gene transfer of IDO to pancreatic islets can sufficiently deplete culture media of tryptophan and consequently inhibit the proliferation of T-cells in vitro. Experiments in vivo have also demonstrated that transplantation of IDO-expressing islets from prediabetic NOD mouse donors into NODscid recipient mice is associated with a prolongation in islet graft survival after adoptive transfer of NOD diabetogenic T-cells. This protection is attributed to the depletion of tryptophan at the transplantation site beneath the kidney capsule. These results suggest that local modulation of tryptophan catabolism may be a means of facilitating islet transplantation as a therapy for type 1 diabetes.

Adenoviridae↗