RESUMEN
Using direct sequencing of complementary DNA products, the sequences of human CD31 from exon 1 through exon 16 of 179 individuals (139 unrelated) were systematically examined. Of the 14 biallelic single nucleotide polymorphic sites detected, 7 polymorphic sites involved amino acid substitution. These 14 polymorphic sites yielded 18 observed CD31 alleles and 9 predicted CD31 polypeptide sequences. Based on molecular haplotyping and family pedigree analysis, linkage disequilibrium among some single nucleotide polymorphic sites was observed. Single nucleotide polymorphism frequencies between populations were also measured using dot-blot hybridization with DNA or peptide nucleic acid probes.
Asunto(s)
Variación Genética , Genética de Población , Haplotipos , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/genética , Femenino , Genotipo , Humanos , Masculino , LinajeAsunto(s)
Negro o Afroamericano/genética , Tipificación y Pruebas Cruzadas Sanguíneas/métodos , Sistema del Grupo Sanguíneo de Kell/genética , Polimorfismo Genético , Población Blanca/genética , Humanos , Sistema del Grupo Sanguíneo de Kell/clasificación , Polimorfismo de Longitud del Fragmento de Restricción , Reproducibilidad de los Resultados , Reacción a la TransfusiónRESUMEN
Fourteen DRB alleles, DRB1*0705, DRB1*11014, DRB1*1134, DRB1*1136, DRB1*1141, DRB1*1335, DRB1*1337, DRB1*1338, DRB1*1342, DRB1*1343, DRB1*1349, DRB1*1510, DRB3*0105, and DRB5*0103, are described. Among them, eleven are variants which differ by only one nucleotide from previously described alleles, including one silent variant (DRB1*11014). Alleles, DRB1*0705, DRB1*1335 and DRB3*0105, display unique sequence motifs that have never been observed in DRB alleles.
Asunto(s)
Alelos , Antígenos HLA-DR/genética , Células Madre , Cadenas HLA-DRB1 , Cadenas HLA-DRB3 , Cadenas HLA-DRB5 , Humanos , Sistema de Registros , Análisis de Secuencia de ADN , Donantes de TejidosAsunto(s)
Antígenos HLA-DR/genética , Secuencia de Bases , Femenino , Antígenos HLA-DR/aislamiento & purificación , Cadenas HLA-DRB1 , Haplotipos/inmunología , Prueba de Histocompatibilidad/normas , Humanos , Masculino , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/normas , Polimorfismo Conformacional Retorcido-Simple , Valor Predictivo de las PruebasRESUMEN
A human CTL epitope located in a region of the HIV-1 envelope protein gp41 that is highly conserved among various HIV-1 strains was identified. This epitope was recognized by CD4+ CTL clones that were induced in seronegative humans by immunization with recombinant gp160. Fusion proteins carrying portions of the HIV-1 env gene and synthetic peptides were used to localize this epitope to amino acids 584-595 of the HIV-1 BRU env sequence. Only two positions within this epitope showed variation among North American HIV-1 isolates, and the substitutions were conservative in nature. The Lys to Arg substitution at position 593 abolished recognition, probably by interfering with the peptide-MHC interactions. This epitope was recognized in association with at least one subtype of the widely distributed human class II MHC specificity DPw4, namely DPw4.2. The relatively high frequency of this allele (27.2% among Caucasians) makes it likely that a larger fraction of the population would generate a response directed at this epitope than would be the case for epitopes recognized in the context of gene products of most other class II and class I loci. Interestingly, the closely related DP beta-chain allele types 4.1 and 2.1, which differ from 4.2 by 3 and 1 amino acids, respectively, were unable to present this gp41 peptide to DPw4.2-restricted clones. Comparison of the structure of this epitope with that of other peptides recognized in the context of DPw4.2 led to the identification of a consensus sequence for DPw4.2 binding peptides. Because the gp41 CTL epitope 584-595 identified here is highly conserved and is recognized in the context of a common DP allele, it may represent an important target region for vaccine development. Our results indicate that vaccines containing this epitope may induce in a significant fraction of those immunized CTL active against at least half of all HIV-1 strains.
Asunto(s)
Proteína gp41 de Envoltorio del VIH/inmunología , Linfocitos T Citotóxicos/inmunología , Secuencia de Aminoácidos , Linfocitos T CD4-Positivos/inmunología , Células Clonales , Epítopos/genética , Epítopos/inmunología , Variación Genética , Humanos , Terapia de Inmunosupresión , Datos de Secuencia MolecularRESUMEN
The existing estimates of the recombination fraction between DR and DP are quite variable and often based on anecdotal observations. We have estimated the DR/DP crossover frequency on the basis of families typed for HLA markers and GLO. The frequency of DR/GLO crossing over was 8.7% (23/264 informative meioses), maternal recombinations being about twice as frequent as paternal ones. Of 17 DR/GLO recombinant families typed for DPw1-6, DP was informative in 11 (13 recombinations) but only one of these gave rise to a DR/DP crossover. According to these data the DR/DP recombination fraction is below 1%, in contrast to some earlier published materials. HLA-DR/DP haplotypic associations on 127 informative Caucasoid haplotypes have been evaluated. In agreement with previous studies, DR3 was positively associated with DPw1 and, in addition, DR7 was found to be positively associated with DP-blank (not DPw1-6). The rare DPw6 allele is possibly associated with the DR4, Dw14 allele. The DR-DP haplotype profiles suggest other associations which might become significant if larger materials are tested. The frequency of DP alleles in a random material (N = 201) was found to be in accordance with most of the previously published frequences on European Caucasoids with DPw4 as the predominating frequency (gene frequency 40%) and a blank frequency of 27%.
Asunto(s)
Antígenos HLA-DP/genética , Antígenos HLA-DR/genética , Lactoilglutatión Liasa/genética , Alelos , Francia , Haplotipos , Humanos , Recombinación GenéticaRESUMEN
The polymorphism of HLA class II molecules in man is particularly evident when comparisons between population groups are made. This study describes a DR3 haplotype commonly present in the American black population. Unlike the Northern European population, in which almost all DR3 individuals are DQw2, approximately 50% of DR3-positive American blacks express a DQw4 allelic product. This study characterizes the DR subregion of that haplotype. cDNA sequence analysis has revealed a DR beta gene which differs at several positions from previously described DR3 beta 1 genes. It is postulated that a gene-conversion-like event with a DRw52 beta gene as donor has generated some of these differences. The haplotype carries a DRw52a allele as defined by oligonucleotide hybridization studies. DNA restriction fragment analysis using a family and several unrelated individuals has allowed us to identify DR alpha and beta fragments associated with the DR3(w18),DQw4 haplotype. The most striking observation is that the DR3(w18),DQw4 haplotype differs from DR3(w17),DQw2 haplotypes at multiple class II loci. Several genetic mechanisms including reciprocal recombination, gene conversion, and point mutation were involved in generating the differences between these haplotypes. Once established, the DR3(w18),DQw4 haplotype appears to be relatively stable in the population.
Asunto(s)
Antígenos HLA-DR/genética , Complejo Mayor de Histocompatibilidad , Secuencia de Aminoácidos , Secuencia de Bases , Southern Blotting , ADN/análisis , ADN/aislamiento & purificación , Haplotipos , Antígenos de Histocompatibilidad Clase II/análisis , Prueba de Histocompatibilidad , Humanos , Hibridación Genética , Datos de Secuencia Molecular , Polimorfismo de Longitud del Fragmento de Restricción , Mapeo RestrictivoRESUMEN
Immunity to the typhus group of rickettsiae is largely dependent on the effector function of several classes of T lymphocytes, including those which produce gamma interferon. Since the surface protein antigen (SPA) derived from typhus group rickettsiae has been shown to be an effective immunogen in animal models, human T-cell clones specific for the SPA of Rickettsia typhi were isolated and tested for their antigenic specificity, as well as for their ability to produce gamma interferon. Eighteen CD4-positive clones specific for the SPA of R. typhi exhibited considerable diversity in their response to the SPAs derived from two strains of Rickettsia prowazekii and from Rickettsia canada. The vast majority of clones also recognized the SPAs from R. prowazekii but not from R. canada. Two heteroclitic clones demonstrated significantly higher proliferative responses to the SPAs derived from one or both of the R. prowazekii strains than to the SPA of R. typhi, and one clone demonstrated a significantly higher response to the SPA of R. typhi than to the other SPAs. All 18 clones produced gamma interferon in response to SPA stimulation. We conclude that the SPAs from typhus group rickettsiae can elicit both a diverse T-cell response in humans and the efficient stimulation of gamma interferon-mediated immunity.
Asunto(s)
Antígenos Bacterianos/inmunología , Antígenos de Diferenciación de Linfocitos T , Proteínas de la Membrana Bacteriana Externa/inmunología , Rickettsia typhi/inmunología , Linfocitos T/clasificación , Antígenos de Diferenciación de Linfocitos T/inmunología , Células Clonales/clasificación , Células Clonales/microbiología , Epítopos/inmunología , Humanos , Interferón gamma/biosíntesis , Activación de Linfocitos , Fenotipo , Linfocitos T/metabolismo , Linfocitos T/microbiologíaRESUMEN
An enzyme that hydrolyzes soman (1,2,2-trimethylpropyl methylphosphonofluoridate) and two other phosphonofluoridates, but does not hydrolyze DFP (diisopropylphosphorofluoridate), has been partially purified from a rod-shaped spore-forming gram-positive OT (obligate thermophilic) bacterium. The enzyme shows a marked Mn2+ stimulation, and in this and its substrate preference does not resemble the organophosphorus acid anhydrolase (sometimes termed DFPase) found in squid. Like the squid enzyme, it is not inhibited by mipafox (N,N'-diisopropylphosphordiamidofluoridate), is not inactivated by ammonium sulfate, and does hydrolyze the acetylcholinesterase-inhibitory pair of diastereoisomers of soman as well as the relatively noninhibitory pair, thus detoxifying soman. In these three properties the OT enzyme does not resemble the ubiquitous organophosphorus acid anhydrolase often purified from mammalian and bacterial sources by cold ethanol fractionation. Thus this phosphono-specific OT enzyme may have a natural substrate and a physiological role distinct from other organophosphorus acid anhydrolases.
Asunto(s)
Bacterias/enzimología , Monoéster Fosfórico Hidrolasas/metabolismo , Soman/farmacocinética , Inhibidores de la Colinesterasa , Electrodos , Hidrólisis , Inactivación Metabólica , Isoflurofato/metabolismo , Soman/metabolismoRESUMEN
LB-Q1 and LB-Q4 are two subtypes of DRw52, defined by proliferative T-cell clones. These subtypes represent a polymorphism of the DR beta III gene. Similar subtypes of DRw52 can be defined by oligonucleotide typing, serology and RFLP analysis. In the present study we compared these typing techniques on a panel of 22 HLA-D homozygous, DRw52-positive typing cells. All typing techniques correlated very well. Three subtypes of DRw52 could be identified. Our results show that typing for cellularly defined structures can be done with a variety of noncellular techniques. This observation has important implications for matching in unrelated bone marrow transplantation and for disease association studies.
Asunto(s)
Antígenos HLA-DR/genética , Polimorfismo Genético , ADN/genética , Genes MHC Clase II , Subtipos Serológicos HLA-DR , Haplotipos , Prueba de Histocompatibilidad , Humanos , Isoanticuerpos , Oligonucleótidos , Polimorfismo de Longitud del Fragmento de Restricción , Proteínas/genética , Linfocitos T/inmunologíaRESUMEN
Cytolytic human T cell clones generated in response to the intracellular bacterium Rickettsia typhi were characterized. Growing clones were tested for their ability to proliferate specifically in response to antigens derived from typhus group rickettsiae or to lyse targets infected with R. typhi or Rickettsia prowazekii. Two clones were able to lyse targets infected with typhus group rickettsiae. One of these clones was more fully characterized because of its rapid growth characteristics. This cytolytic clone was capable of lysing an autologous infected target as well as a target matched for class I and II histocompatibility leukocyte antigens (HLA). It was not capable, however, of lysing either a target mismatched for both class I and II HLA or a target partially matched for class I HLA. In addition, the clone exhibited specificity in that it was able to lyse an autologous target infected with typhus group rickettsiae, but did not lyse an autologous target infected with an antigenically distinct rickettsial species, Rickettsia tsutsugamushi. These results demonstrate, for the first time, that cells infected with intracellular bacteria can be lysed by human cytotoxic T lymphocytes.
Asunto(s)
Rickettsia typhi/inmunología , Linfocitos T Citotóxicos/inmunología , Antígenos Bacterianos/inmunología , Células Clonales/inmunología , Citotoxicidad Inmunológica , Antígenos HLA/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Activación de Linfocitos , Orientia tsutsugamushi/inmunología , Rickettsia prowazekii/inmunología , Especificidad de la EspecieRESUMEN
Lymphocytes from highly selected donors were primed for 10 days and subsequently bulk-expanded in IL 2 (TCGF) containing cultures. Two well-discriminatory PLT (CDP = Copenhagen DP) reagents against each of the DPw1-w6 specificities and one against each of the two "new" specificities, CDP4s and CDPHEI, were selected for further studies. Three combinations made in two recombinant families and four of ten HLA-A, B, and DR compatible combinations discriminated well in contrast to seven of 46 DR compatible, but HLA-A or B incompatible combinations. All reagents gave highly reproducible results, and high correlations (r-values between 0.73-1.00) for DP assignments were obtained with CDP and GNN reagents. No triplets were found for the DPw1-w6 and CDP HEI specificities. The "new" specificity CDP HEI defined in an HLA-DR/GLO recombinant family gave a coefficient of correlation with GNN 8 of 0.91. Another "new" specificity, CDP4s constitutes a subgroup ("split") of DPw4. The gene frequencies of DPw1-w6 estimated in 102 unrelated randomly selected Danes agreed with those reported for other Caucasoid populations. The gene frequencies CDP HEI and CDP4s were 0.03 and 0.08, respectively. The associations between DR3-DPw1, DR2-DPw4, and DRw6-DPw2 were confirmed. It is concluded that DP-typing with bulk-expanded reagents is a reliable and so far the only technique which can reveal the polymorphism of the DP gene products.
Asunto(s)
Antígenos de Histocompatibilidad Clase II/inmunología , Prueba de Histocompatibilidad/normas , Células Cultivadas , Dinamarca , Epítopos , Frecuencia de los Genes , Ligamiento Genético , Antígenos HLA-DP , Antígenos de Histocompatibilidad Clase II/clasificación , Antígenos de Histocompatibilidad Clase II/genética , Humanos , Polimorfismo Genético , SerotipificaciónRESUMEN
HLA typing of an HLA-B/GLO recombinant family using bulk-expanded DP(SB) alloreactive T-cells (GNN1 to 6) as well as conventional HLA-ABC, D, DR, and DQ typing showed that a paternal recombination had taken place between HLA-DR and DQ on the one hand and HLA-DP(SB) on the other. The recombinant child was DPw4,6-heterozygous and differed in terms of class II determinants only for DPw6 from two otherwise HLA identical siblings, who were probably DPw4/4-homozygous. These siblings did not stimulate the recombinant in MLC whereas they responded to his cells indicating that DPw6 can stimulate in primary MLC. Moreover, it was possible to generate and bulk-expand DPw6-reactive lymphocytes (PLs) between MT and MA as responders and BN as stimulator. The correlation coefficient (r) between the reaction of these PLs and the GNN6 (anti-DPw6) reagents was 1.0 when tested against a panel of 71 individuals. This is the first report demonstrating that the DPw6 gene, like the DPw1-w5 genes, is located between DR/DQ and GLO. The frequency of the DPw6 antigen in 41 unrelated, randomly selected Danes was 7%. HLA-DP typing of the BN family revealed that the DPw6 positive but not DPw6 negative family members gave intermediate responses with one (GNN2B) but not with another (GNN2A) anti-DPw2-reagent. Studies of 63 healthy individuals (unrelated to the BN family) revealed seven similar discrepancies between GNN2A and GNN2B. Strikingly, all four DPw6 positive stimulators gave rise to such discrepancies indicating that the GNN2B reagent is cross-reactive and recognize a common part of the DPw2 and DPw6 molecules. No cases of GNN2A+/GNN2B- stimulators were found.
Asunto(s)
Genes MHC Clase II , Antígenos de Histocompatibilidad Clase II/análisis , Linfocitos T/inmunología , Alelos , Células Cultivadas , Niño , Reacciones Cruzadas , Femenino , Congelación , Ligamiento Genético , Antígenos HLA/genética , Antígenos HLA-DP , Antígenos HLA-DR , Humanos , Masculino , Recombinación GenéticaRESUMEN
Human lymphocytes, 10 days after treatment with phytohemagglutinin P (PHA-P), show PLT (secondary in vitro restimulation) reaction patterns which correspond to their cellular (HLA-D) type. Lymphocytes with shared cellular type stimulate PHA primed cells to a lesser degree than the majority of lymphocytes with unshared HLA-D type, which stimulate a strong response. Using a standard 3H thymidine incorporation technique, it is possible to detect responses in as early as 12 hr total incubation time, although usually a 22-48 hr total incubation time is normally required. This method is simple, gives results that correspond to the primary mixed leukocyte response, and thus may be useful as a cross-matching technique for cadaveric renal transplantation.