RESUMO
AIM: Validate the Roche, MagNAPure96 (MP96) nucleic acid extraction platform for Seegene Anyplex II HPV28 (Anyplex28) detection of Human Papillomavirus. METHODS AND RESULTS: Comparisons were made for Anyplex28 genotyping from 115 cervical samples extracted on the Hamilton, STARlet and the MP96. Two DNA concentrations were used for the MP96, one matched for sample input to the STARlet and another 5× concentration (laboratory standard). Agreement of HPV detection was 89·8% (κ = 0·798; P = 0·007), with HPV detected in 10 more samples for the MP96. There was a high concordance of detection for any oncogenic HPV genotype (κ = 0·77; P = 0·007) and for any low-risk HPV genotype (κ = 0·85; P = 0·008). DNA extracted at laboratory standard had a lower overall agreement 85·2% (κ = 0·708; P < 0·001), with 17/115 discordant positive samples that tested negative after STARlet extraction. Of the discordant genotypes, 72·7% were detected in the lowest signal range for Anyplex28 ('+'). CONCLUSIONS: MP96 performed with high concordance to STARlet, although produced DNA with a higher analytical sensitivity on the Anyplex28. SIGNIFICANCE AND IMPACT OF THE STUDY: This analysis supports the use of samples extracted on the MP96 for HPV genotyping using the Anyplex28. Furthermore, an increase in DNA concentration increased analytical sensitivity of the Anyplex28, particularly appropriate for prevalence studies.
Assuntos
Ácidos Nucleicos , Infecções por Papillomavirus , DNA Viral/genética , Genótipo , Técnicas de Genotipagem , Humanos , Papillomaviridae/genética , Sensibilidade e EspecificidadeRESUMO
The IL-2/IL-2R signaling pathway has an important role in autoimmunity. Several genes identified in genome-wide association (GWA) studies encode proteins in the IL-2/IL-2R signaling cascade that are associated with autoimmune diseases. One of these, PTPN2, encodes a protein tyrosine phosphatase that is highly expressed in T cells and regulates cytokine signaling. An intronic risk allele in PTPN2, rs1893217(C), correlated with decreased IL-2R signaling in CD4(+) T cells as measured by phosphorylation of STAT5 (phosphorylated STAT5 (pSTAT5)). We modeled an additive single nucleotide polymorphism (SNP) genotype, in which each copy of the risk allele conferred a decrease in IL-2R signaling (P=4.4 × 10(-8)). Decreased pSTAT5 impacted IL-2Rß chain signaling resulting in reduced FOXP3 expression in activated cells. This phenotype was not due to overt differences in expression of the IL-2R, molecules in the IL-2R signaling cascade or defects in STAT5. However, the rs1893217(C) risk variant did correlate with decreased PTPN2 expression in CD4(+)CD45RO T cells (P=0.0002). Thus, the PTPN2rs1893217(C) risk allele associated with reduced pSTAT5 in response to IL-2 and reduced PTPN2 expression. Together, these data suggest that decreased expression of PTPN2 may indirectly modulate IL-2 responsiveness. These findings, identified through genotype/phenotype relationships, may lead to identification of novel mechanisms underlying dysregulation of cytokine signaling in autoimmunity.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 2/genética , Proteína Tirosina Fosfatase não Receptora Tipo 2/metabolismo , Receptores de Interleucina-2/imunologia , Receptores de Interleucina-2/metabolismo , Adulto , Alelos , Autoimunidade/imunologia , Feminino , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Estudo de Associação Genômica Ampla , Genótipo , Humanos , Interleucina-2/genética , Interleucina-2/imunologia , Interleucina-2/metabolismo , Masculino , Fenótipo , Fosforilação , Polimorfismo de Nucleotídeo Único , Fator de Transcrição STAT5/metabolismo , Transdução de SinaisRESUMO
Immune responses to autoantigens are in part controlled by deletion of autoreactive cells through genetically regulated selection mechanisms. We have directly analyzed peripheral CD4+ proinsulin (PI) 76-90 (SLQPLALEGSLQKRG)-specific T cells using soluble fluorescent major histocompatibility complex class II tetramers. Subjects with type I diabetes and healthy controls with high levels of peripheral proinsulin-specific T cells were characterized by the presence of a disease-susceptible polymorphism in the insulin variable number of tandem repeats (INS-VNTR) gene. Conversely, subjects with a 'protective' polymorphism in the INS-VNTR gene had nearly undetectable levels of proinsulin tetramer-positive T cells. These results strongly imply a direct relationship between genetic control of autoantigen expression and peripheral autoreactivity, in which proinsulin genotype restricts the quantity and quality of the potential T-cell response. Using a modified tetramer to isolate low-avidity proinsulin-specific T cells from subjects with the susceptible genotype, transcript arrays identified several induced pro-apoptotic genes in the control, but not diabetic subjects, likely representing a second peripheral mechanism for maintenance of tolerance to self antigens.
Assuntos
Autoimunidade/imunologia , Genótipo , Insulina/genética , Repetições Minissatélites/genética , Proinsulina/imunologia , Autoantígenos/imunologia , Diabetes Mellitus Tipo 1/genética , Suscetibilidade a Doenças/imunologia , Antígenos de Histocompatibilidade Classe II/genética , Humanos , Fenótipo , Polimorfismo Genético , Proinsulina/genética , Linfócitos T/imunologiaRESUMO
KIR3DL1 and KIR3DL2 are NK cell receptors for polymorphic HLA-B and -A determinants. The proportion of NK cells that bind anti-KIR3DL1-specific Ab DX9 and their level of binding vary between individuals. To determine whether these differences are due to KIR polymorphism, we assessed KIR3D gene diversity in unrelated individuals and families. Both KIR3DL1 and KIR3DL2 are highly polymorphic genes, with KIR3DS1 segregating like an allele of KIR3DL1. A KIR haplotype lacking KIR3DL1 and KIR3DS1 was defined. The two KIR3DL1 alleles of a heterozygous donor were expressed by different, but overlapping, subsets of NK cell clones. Sequence variation in KIR3DL1 and KIR3DL2 appear distinct; recombination is more evident in KIR3DL1, and point mutation is more evident in KIR3DL2. The KIR3DL1 genotype correlates well with levels of DX9 binding by NK cells, but not with the frequency of DX9-binding cells. Different KIR3DL1 alleles determine high, low, and no binding of DX9 Ab. Consequently, heterozygotes for high and low binding KIR3DL1 alleles have distinct subpopulations of NK cells that bind DX9 at high and low levels, giving characteristic bimodal distributions in flow cytometry. The Z27 Ab gave binding patterns similar to those of DX9. Four KIR3DL1 alleles producing high DX9 binding phenotypes were distinguished from four alleles producing low or no binding phenotypes by substitution at one or more of four positions in the encoded protein: 182 and 283 in the extracellular Ig-like domains, 320 in the transmembrane region, and 373 in the cytoplasmic tail.
Assuntos
Anticorpos Monoclonais/metabolismo , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Polimorfismo Genético/imunologia , Receptores Imunológicos/genética , Receptores Imunológicos/imunologia , Alelos , Sítios de Ligação de Anticorpos/genética , Células Clonais , Triagem de Portadores Genéticos , Variação Genética/imunologia , Haplótipos , Teste de Histocompatibilidade , Humanos , Imunofenotipagem , Dados de Sequência Molecular , Família Multigênica/imunologia , Receptores KIR , Receptores KIR3DL1 , Receptores KIR3DL2 , Receptores KIR3DS1Assuntos
Células Matadoras Naturais/imunologia , Receptores Imunológicos/genética , Recombinação Genética , Sequência de Aminoácidos , Sequência de Bases , DNA Complementar , Humanos , Dados de Sequência Molecular , Conformação Proteica , Receptores Imunológicos/química , Receptores KIR , Homologia de Sequência do Ácido NucleicoRESUMO
NK cells express receptors that bind to polymorphic determinants of MHC class I heavy chains. MHC ligands vary greatly between mammalian species, and the use of distinct molecular families of NK cell receptors by humans and mice suggests that the receptors too can be evolving rapidly. The KIR (killer cell inhibitory receptor) family of receptors are found in primates and recognize class I epitopes that are of relatively recent origin in primate evolution. Therefore, KIR molecules have probably evolved class I receptor function more recently than C-type lectins, which are represented in both humans and mice. Individual humans express NK cell receptors for which they have no class I ligand, demonstrating a looseness in the coupling of expression between the receptors and their ligands. However, study of a single donor suggests that every NK cell expresses at least one inhibitory receptor for a self-HLA class I allotype, consistent with the missing self hypothesis. Thus the NK-cell receptor-class I interaction appears to control the NK-cell repertoire during ontogeny of the individual and has the potential to be a selective factor influencing both MHC class I and NK cell receptor diversity in the evolution of populations and species.
Assuntos
Evolução Molecular , Antígenos de Histocompatibilidade Classe I/fisiologia , Células Matadoras Naturais/metabolismo , Receptores Imunológicos/fisiologia , Sequência de Aminoácidos , Animais , Humanos , Dados de Sequência MolecularRESUMO
The expression of KIR and CD94:NKG2 receptors was determined for more than 100 natural killer (NK) cell clones obtained from two blood donors who differ in their HLA class I and KIR genes. More than 98% of the clones were inhibited by individual autologous class I allotypes, and every clone was inhibited by the combination of autologous allotypes. The patterns of inhibition correlate with expression of inhibitory receptors of defined specificity. One donor possesses three class I ligands for KIR, and a majority of NK cells use KIR as their inhibitory receptor; the second donor possesses only a single ligand for KIR, and a majority of NK cells use the more broadly reactive CD94:NKG2a as their inhibitory receptor. Because of these differences, the first donor has subpopulations of NK cells that kill cells of the second donor, whereas the NK cells of the second donor are universally tolerant of cells from the first donor.
Assuntos
Antígenos CD/imunologia , Células Matadoras Naturais/imunologia , Lectinas Tipo C , Glicoproteínas de Membrana/imunologia , Receptores Imunológicos/imunologia , Antígenos CD/genética , Sequência de Bases , Doadores de Sangue , DNA Complementar , Antígenos HLA-A/imunologia , Antígenos HLA-B/imunologia , Antígenos HLA-C/imunologia , Humanos , Células Matadoras Naturais/citologia , Lectinas/genética , Lectinas/imunologia , Leucócitos Mononucleares/imunologia , Glicoproteínas de Membrana/genética , Dados de Sequência Molecular , Subfamília C de Receptores Semelhantes a Lectina de Células NK , Subfamília D de Receptores Semelhantes a Lectina de Células NK , Receptores Imunológicos/genética , Receptores KIR , Receptores de Células Matadoras NaturaisRESUMO
The presence and expression of killer inhibitory receptor (KIR) and CD94:NKG2 genes from 68 donors were analyzed using molecular typing techniques. The genes encoding CD94:NKG2 receptors were present in each person, but KIR gene possession varied. Most individuals expressed inhibitory KIR for the three well-defined HLA-B and -C ligands, but noninhibitory KIR genes were more variable. Twenty different KIR phenotypes were defined. Two groups of KIR haplotypes were distinguished and occurred at relatively even frequency. Group A KIR haplotypes consist of six genes: the main inhibitory KIR, one noninhibitory KIR, and a structurally divergent KIR. Allelic polymorphism within five KIR genes was detected. Group B comprises more noninhibitory KIR genes and contains at least one additional gene not represented in group A. The KIR locus therefore appears to be polygenic and polymorphic within the human population.
Assuntos
Antígenos CD/genética , Variação Genética , Células Matadoras Naturais/imunologia , Lectinas Tipo C , Glicoproteínas de Membrana/genética , Receptores Imunológicos/genética , Expressão Gênica , Genótipo , Humanos , Subfamília C de Receptores Semelhantes a Lectina de Células NK , Subfamília D de Receptores Semelhantes a Lectina de Células NK , RNA Mensageiro , Receptores KIR , Receptores de Células Matadoras NaturaisRESUMO
Elk is a member of the eph family of receptor-like tyrosine kinases. Although its function is unknown, elk is postulated to play a role in nervous system development. Using Northern analysis, we examined the developmental regulation of RNAs encoding elk, and several ligands for the eph family of RTKs, the LERKs. Expression of elk, LERK-1, and LERK-2 RNAs is high in all regions examined in the embryonic and postnatal rat brain and decreases to low levels with age. One exception is the adult olfactory bulb which continues to express a moderate level of LERK-2. In contrast, moderate LERK-4 expression was limited to the developing hippocampus and cerebral cortex. These data indicate that elk and some of the LERKs may play a role in nervous system development, maintenance, and/or regeneration.
Assuntos
Encéfalo/enzimologia , Proteínas de Ligação a DNA , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Membrana/biossíntese , Proteínas do Tecido Nervoso/metabolismo , Biossíntese de Proteínas , Receptores Proteína Tirosina Quinases/metabolismo , Fatores de Transcrição , Animais , Encéfalo/embriologia , Encéfalo/crescimento & desenvolvimento , Encéfalo/ultraestrutura , Indução Enzimática , Efrina-A1 , Efrina-A2 , Efrina-A3 , Efrina-A4 , Efrina-B1 , Efrina-B2 , Glicosilfosfatidilinositóis/metabolismo , Ligantes , Proteínas de Membrana/genética , Família Multigênica , Proteínas/genética , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , Ratos , Ratos Sprague-Dawley , Receptores Proteína Tirosina Quinases/classificação , Organismos Livres de Patógenos Específicos , Proteínas Elk-1 do Domínio etsRESUMO
We attempted to isolate novel receptor tyrosine kinase, which may play a role in hematopoietic development by screening for expressed sequences with conserved tyrosine kinase catalytic domains. Among the known tyrosine kinases identified in this screen, we found a gene with characteristics of a receptor tyrosine kinase but unusual motifs in the catalytic domain. This gene is identical to ryk described independently by other investigators. Chromosomal fluorescence in situ hybridization localization of human ryk was clarified by using monochromosomal hybrids and placing it as a single locus in 3q22. Although Northern analysis reveals widespread expression in adult mouse tissues, we have found that ryk expression is not ubiquitous. Expression increased in bone marrow cells from mice treated with 5-fluorouracil. Northern analysis on cell lines indicates expression in CD3-, CD4-, CD8- T cells (at a low level), pre-T cells, thymic epithelial cells, and mature myeloid cells, but not myeloid precursors or B cell precursors. Expression analysis with the use of RT-PCR on mouse bone marrow cells separated on the basis of cell surface markers (B220, CD4, CD8, Gr-1, Mac-1) reveals that this receptor is expressed in differentiated cells (Lin+) but is not expressed in the precursor cells (Lin-). Flow cytometric analysis with a monospecific anti-Ryk Ab demonstrates that Ryk+ cells constitute 36.7% and Lin+/Ryk+ cells constitute 33.7% of low density bone marrow cells whereas Ryk+ cells represent only 0.3% of the Lin- population. We conclude that ryk expression is regulated during hematopoietic development by lineage commitment and stage of maturation.
Assuntos
Células-Tronco Hematopoéticas/fisiologia , Receptores Proteína Tirosina Quinases/biossíntese , Receptores de Superfície Celular/biossíntese , Sequência de Aminoácidos , Animais , Sequência de Bases , Células da Medula Óssea , Linhagem Celular , Mapeamento Cromossômico , Feminino , Citometria de Fluxo , Hibridização in Situ Fluorescente , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , Reação em Cadeia da PolimeraseRESUMO
We have isolated and characterized cDNA clones that encode the rat homologue of a binding protein, LERK-2, for the receptor tyrosine kinase, elk. The cDNAs contain an open reading frame of 1527 nucleotides capable of encoding a protein 345 amino acid residues in length. The nucleotide sequence of the present clones is > 90% identical to the previously identified human LERK-2 cDNA, and the predicted proteins encoded by the rat and human clones are identical at 95% of amino acid residues. Recombinant proteins expressed from the rat cDNAs bind to elk with high affinity, similar to recombinant human LERK-2 and an endogenously-expressed rat elk-binding protein. Expression of the rat LERK-2 mRNA was detected in embryonic brain, kidney, lung, skeletal muscle, thymus, liver, and heart, and diminished in the early post-natal period. Significant LERK-2 mRNA expression in the young adult rat was restricted to the lung, kidney, heart and testes.
Assuntos
Sequência Conservada , Proteínas de Ligação a DNA , Regulação da Expressão Gênica , Proteínas/genética , Proteínas Proto-Oncogênicas , Receptores Proteína Tirosina Quinases/metabolismo , Proteínas Oncogênicas de Retroviridae/metabolismo , Fatores de Transcrição , Sequência de Aminoácidos , Animais , Sequência de Bases , Evolução Biológica , Clonagem Molecular , DNA Complementar , Efrina-B1 , Humanos , Masculino , Dados de Sequência Molecular , Fases de Leitura Aberta , Ligação Proteica , Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Homologia de Sequência de Aminoácidos , Distribuição Tecidual , Células Tumorais Cultivadas , Proteínas Elk-1 do Domínio etsRESUMO
A family of tyrosine kinase receptors related to the product of the eph gene has been described recently. One of these receptors, elk, has been shown to be expressed only in brain and testes. Using a direct expression cloning technique, a ligand for the elk receptor has been isolated by screening a human placenta cDNA library with a fusion protein containing the extracellular domain of the receptor. This isolated cDNA encodes a transmembrane protein. While the sequence of the ligand cDNA is unique, it is related to a previously described sequence known as B61. Northern blot analysis of human tissue mRNA showed that the elk ligand's mRNA is 3.5 kb long and is found in placenta, heart, lung, liver, skeletal muscle, kidney and pancreas. Southern blot analysis showed that the gene is highly conserved in a wide variety of species. Both elk ligand and B61 mRNAs are inducible by tumour necrosis factor in human umbilical vein endothelial cells. In addition, both proteins show promiscuity in binding to the elk and the related hek receptors. Since these two ligand sequences are similar, and since elk and hek are members of a larger family of eph-related receptor molecules, we refer to these ligands as LERKs (ligands for eph-related kinases).