RESUMO
Introduction: There is a continuing worldwide shortage of organs from deceased human donors for transplantation into patients with end-stage organ failure. Genetically engineered pigs could resolve this problem, and could also provide tissues and cells for the treatment of conditions such as diabetes, Parkinson's disease and corneal blindness. Sources of data: The current literature has been reviewed. Areas of agreement: The pathobiologic barriers are now largely defined. Research progress has advanced through the increasing availability of genetically engineered pigs and novel immunosuppressive agents. Life-supporting pig kidneys and islets have functioned for months or years in nonhuman primates. Areas of controversy: The potential risk of transfer of a pig infectious microorganism to the recipient continues to be debated. Growing points: Increased attention is being paid to selection of patients for initial clinical trials. Areas timely for developing research: Most of the advances required to justify a clinical trial have now been met.
Assuntos
Xenoenxertos , Coleta de Tecidos e Órgãos/métodos , Transplante Heterólogo , Animais , Animais Geneticamente Modificados , Sobrevivência de Enxerto , Humanos , Suínos , Transplante Heterólogo/efeitos adversos , Transplante Heterólogo/métodosRESUMO
We have characterized swine leucocyte antigen (SLA) classes I and II molecules of a domestic pig as a model for use in our xenotransplant program. Molecular characterization of the SLA classes I and II genes is critical to understanding the adaptive immune responses between swine and humans in the event of xenotransplantation. Seven swine leucocyte antigen genes (SLA-1, SLA-2, SLA-3, DQB1, DRB1, DQA and DRA) were analyzed and 15 alleles were identified. A novel DRA*w04re01 is reported for this limited polymorphic class II gene. The heterozygous haplotypes, Hp-32.0/35.0 and Hp-0.13/0.23 were deduced for our IU-pig model, for SLA classes I and II regions, respectively.
Assuntos
Alelos , Haplótipos , Heterozigoto , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe I/genética , Suínos/genética , Imunidade Adaptativa , Animais , Cruzamentos Genéticos , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Suínos/imunologiaRESUMO
Xenotransplantation using genetically modified pig organs could solve the donor organ shortage problem. Two inactivated genes that make humans unique from pigs are GGTA1 and CMAH, the products of which produce the carbohydrate epitopes, aGal and Neu5Gc that attract preformed human antibody. When the GGTA1 and CMAH genes were deleted in pigs, human antibody binding was reduced in preliminary analysis. We analyzed the binding of human IgM and IgG from 121 healthy human serum samples for binding to GGTA1 KO and GGTA1/CMAH KO peripheral blood mononuclear cells (PBMCs). We analyzed a sub population for reactivity toward genetically modified pig PBMCs as compared to chimpanzee and human PBMCs. Deletion of the GGTA1 and CMAH genes in pigs improved the crossmatch results beyond those observed with chimpanzees. Sorting the 121 human samples tested against the GGTA1/CMAH KO pig PBMCs did not reveal a distinguishing feature such as blood group, age or gender. Modification of genes to make pig carbohydrates more similar to humans has improved the crossmatch with human serum significantly.
Assuntos
Anticorpos/química , Galactosiltransferases/genética , Transplante Heterólogo , Adolescente , Adulto , Idoso , Animais , Animais Geneticamente Modificados , Carboidratos/química , Epitopos/química , Feminino , Deleção de Genes , Rejeição de Enxerto , Humanos , Imunoglobulina G/química , Imunoglobulina M/química , Leucócitos Mononucleares/citologia , Masculino , Pessoa de Meia-Idade , Pan troglodytes , Ligação Proteica , SuínosRESUMO
The cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel with 12 membrane-spanning sequences, undergoes inefficient maturation in the endoplasmic reticulum (ER). Potentially charged residues in transmembrane segments may contribute to this defect in biogenesis. We demonstrate that transmembrane segment 6 of CFTR, which contains three basic amino acids, is extremely unstable in the lipid bilayer upon membrane insertion in vitro and in vivo. However, two distinct mechanisms counteract this anchoring deficiency: (i) the ribosome and the ER translocon co-operate to prevent transmembrane segment 6 from passing through the membrane co- translationally; and (ii) cytosolic domains of the ion channel post-translationally maintain this segment of CFTR in a membrane-spanning topology. Although these mechanisms are essential for successful completion of CFTR biogenesis, inefficiencies in their function retard the maturation of the protein. It seems possible that some of the disease-causing mutations in CFTR may reduce the efficiency of proper membrane anchoring of the protein.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/química , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Sequência de Aminoácidos , Animais , Membrana Celular/metabolismo , Clonagem Molecular , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Microssomos/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Reação em Cadeia da Polimerase , Biossíntese de Proteínas , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Reticulócitos/metabolismoRESUMO
Class I histocompatibility proteins fold and assemble with beta 2-microglobulin (beta 2m) into heterodimers before binding short peptides in the endoplasmic reticulum. Here, we show that class I proteins rapidly form disulfide bonds, and that the process is highly reversible in Daudi cells lacking beta 2m. Three distinct class I protein conformations are present in equal amounts in these cells, each associated with the molecular chaperone calnexin. When binding of calnexin is inhibited by the glucosidase inhibitor castanospermine, fully oxidized class I proteins are no longer detected, suggesting that calnexin is required for completion of folding. However, in Daudi cells transfected to express beta 2m, castanospermine decreases only slightly the levels of fully oxidized class I proteins, indicating that folding is much less dependent on calnexin in the presence of beta 2m. Furthermore, calreticulin, a chaperone with functional similarities to calnexin, associates with class I molecules in beta 2m-positive cells. but not in Daudi cells, consistent with completion of folding and disulfide bond formation of class I heavy chains before binding to calreticulin occurs. This study demonstrates that calnexin and beta 2m can function independently to promote folding of class I heavy chains prior to formation of stable class I dimers.
Assuntos
Proteínas de Ligação ao Cálcio/fisiologia , Dissulfetos/metabolismo , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/metabolismo , Chaperonas Moleculares/fisiologia , Dobramento de Proteína , Microglobulina beta-2/fisiologia , Linfoma de Burkitt/química , Linfoma de Burkitt/imunologia , Linfoma de Burkitt/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Calnexina , Calreticulina , Ciclo Celular/imunologia , Antígenos de Histocompatibilidade Classe I/biossíntese , Humanos , Oxirredução , Peptídeos/química , Peptídeos/metabolismo , Ribonucleoproteínas/metabolismo , Células Tumorais CultivadasRESUMO
Class I major histocompatibility complex heavy chains bind to calnexin before associating with beta 2-microglobulin (beta 2m) and peptides. Calnexin has been shown to retain in the endoplasmic reticulum those class I heavy chains which have not assembled properly and, thus, to serve as a quality control mechanism. In addition, calnexin may direct the folding of class I subunits or their subsequent assembly. We asked whether calnexin plays a role in the initial folding of HLA-B*0702 heavy chains by assessing disulfide bond formation in vivo. Our results show that class I heavy chains form intrachain disulfide bonds very soon after translation, and that calnexin is bound to both reduced and oxidized forms during this process. When a cell-permeable reducing agent, dithiothreitol, was added to cells, disulfide bond formation in newly synthesized heavy chains was substantially blocked, as was their association with calnexin. The reducing agent appeared to affect calnexin directly, since binding was similarly abolished to a subset of proteins which do not contain internal disulfide bonds. Addition of the glucosidase inhibitor castanospermine to cells, shown previously to disrupt calnexin binding to ligands, slowed formation of disulfide bonds but did not decrease the amount of assembled heavy chain-beta 2m complexes that formed. Our data suggest that calnexin can promote disulfide bond formation in class I heavy chains but does not directly facilitate subsequent binding to beta 2m.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Antígenos HLA-B/metabolismo , Dobramento de Proteína , Microglobulina beta-2/metabolismo , Proteínas de Ligação ao Cálcio/química , Calnexina , Linhagem Celular , Dissulfetos/química , Humanos , OxirreduçãoRESUMO
Proper folding of nascent polypeptides is essential for their function and is monitored by intracellular "quality control" elements. The molecular chaperone calnexin participates in this process by retaining in the endoplasmic reticulum a variety of unfolded proteins, including class I major histocompatibility complex molecules. We transfected human B cell lines with genes encoding either wild-type HLA-A2 heavy chains or mutant heavy chains lacking sites for glycosylation or deficient in binding to beta 2-microglobulin (beta 2m). In CIR cells, calnexin did not associate detectably with wild-type heavy chains but bound strongly to mutant heavy chains unable to bind beta 2m. Removal of the glycosylation addition site by further mutagenesis prevented binding of mutant heavy chains to calnexin. In Daudi cells, deficient in synthesis of beta 2m, wild-type HLA-A2 heavy chains, but not a non-glycosylated mutant, bound calnexin. Castanospermine, which blocks trimming of glucose residues from asparagine-linked glycans, inhibited association of calnexin with heavy chains encoded by a second class I gene, HLA-B*0702. Although initiation of calnexin binding appears to depend on the presence of oligosaccharide on the substrate, removal of the glycan from calnexin-associated heavy chains by digestion with endoglycosidase H did not disrupt the interaction. These results suggest that calnexin first recognizes carbohydrate on substrate proteins and then binds more stably to peptide determinants, which disappear upon folding.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Metabolismo dos Carboidratos , Antígeno HLA-A2/metabolismo , Proteínas/metabolismo , Calnexina , Linhagem Celular , Genes MHC Classe I , Antígeno HLA-A2/química , Antígeno HLA-A2/genética , Antígeno HLA-B7/efeitos dos fármacos , Antígeno HLA-B7/genética , Antígeno HLA-B7/metabolismo , Humanos , Indolizinas/farmacologia , Mutação , Polissacarídeos/metabolismo , Ligação Proteica , TemperaturaRESUMO
Class I major histocompatibility complex (MHC) molecules are heterotrimers of heavy chains, beta 2-microglobulin, and 8-10 amino acid-long peptides. Assembly of class I MHC molecules into complexes which are stable and can be transported to the cell surface occurs soon after insertion of individual subunits into the endoplasmic reticulum (ER). To identify subcellular compartments required for class I MHC assembly, we studied class I biosynthesis in human cell lines treated with several inhibitors of intracellular transport. We found that HLA-B701 molecules do not assemble in CIR transfectants in which a block in protein transport from the ER is established by treatment with phosphatase inhibitors. In contrast, stable HLA-B701 complexes form in cells in which the ER becomes mixed with the Golgi after treatment with brefeldin A. Neither treatment impaired binding of HLA-B701 to the ER-resident protein calnexin, and unassembled heavy chains in phosphatase-inhibited cells showed prolonged association with calnexin. In addition, the mouse class I molecule H-2Db, which binds beta 2-microglobulin in human T2 cells in the absence of transporter of antigenic peptides, formed complexes in CIR cell transfectants treated with phosphatase inhibitors. Taken together, these data demonstrate that phosphatase inhibitors do not prevent assembly of class I heavy chain beta 2-microglobulin dimers, but instead interfere with peptide loading. These results are consistent with the possibility that class I MHC molecules are transported from their initial site of insertion into the rough ER before binding peptides, or alternatively that peptide loading mediated by transporter of antigenic peptides is blocked by phosphatase inhibitors.
Assuntos
Linfócitos B/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Peptídeos/metabolismo , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Animais , Transporte Biológico/efeitos dos fármacos , Brefeldina A , Cantaridina/farmacologia , Compartimento Celular , Ciclopentanos/farmacologia , Retículo Endoplasmático/metabolismo , Éteres Cíclicos/farmacologia , Antígenos H-2/metabolismo , Antígenos HLA-B/metabolismo , Humanos , Camundongos , Ácido Okadáico , Polissacarídeos/biossíntese , Ligação Proteica/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , alfa-Macroglobulinas/metabolismoRESUMO
A novel class I-peptide-binding assay was developed and used to identify a series of peptides derived from the human p53 tumor-suppressor gene product capable of binding the HLA-A2 class I allele. Brief pH 3.3 acid treatment of human cell lines rapidly denatures pre-existing class I complexes, as detected by loss of binding of conformation-dependent mAbs, leaving only free class I heavy chains associated with the viable cell surface. These heavy chains may be induced to refold and be recognized by antibodies (in 2-4 hours) when acid-treated cells are coincubated with exogenous beta 2-microglobulin and peptides capable of binding the relevant class I allele examined. This assay, with a detection limit of 1-10 nM peptide, was used to screen the capacity of a panel of nine peptides bearing HLA-A2-binding motifs and derived from the human p53 tumor-suppressor protein sequence. Eight of the nine peptides bound to, and reconstituted, HLA-A2 on acid-treated cells. This assay system will enable the rapid identification of peptides binding to any class I allele, which is the initial prerequisite for elucidating potential CD8+ T-cell epitopes.
Assuntos
Antígeno HLA-A2/análise , Linfócitos T/imunologia , Proteína Supressora de Tumor p53/imunologia , Sequência de Aminoácidos , Linhagem Celular , Citometria de Fluxo , Humanos , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Proteína Supressora de Tumor p53/análiseRESUMO
Previous reports have described an association between cytomegalovirus infection and increased donor-specific alloreactivity of bronchoalveolar lavage (BAL) lymphocytes in transplanted lungs and a higher risk of bronchiolitis obliterans due to chronic rejection. We have postulated that during infection, intragraft CMV-specific lymphocytes are activated and release lymphokines that augment cellular rejection. This study deals with an analysis of CMV antigen induced proliferation of 28 BAL lymphocyte and 27 peripheral blood lymphocytes samples from 17 lung transplant patients with or without CMV infection. Kinetic studies of lymphocyte proliferation have shown that CMV infection of the lung allograft is associated with an accelerated response of BAL lymphocytes but not PBL, following in vitro exposure to CMV antigen. These findings indicate an accumulation of primed CMV-specific lymphocytes within the lung allograft during CMV infection. Evidence has also been obtained that primed CMV-specific lymphocytes may persist for months in BAL. We conclude that the CMV antigen induced proliferation assay is useful for studies of CMV infection in transplant patients.
Assuntos
Antígenos Virais/análise , Líquido da Lavagem Broncoalveolar/microbiologia , Citomegalovirus/imunologia , Transplante de Pulmão/patologia , Linfócitos/microbiologia , Líquido da Lavagem Broncoalveolar/citologia , Infecções por Citomegalovirus/imunologia , Humanos , Memória Imunológica , Cinética , Transplante de Pulmão/imunologia , Ativação Linfocitária , MasculinoRESUMO
Glutathione S-transferase activity has been measured in erythrocytes from 101 subjects. Specific activity in the female subjects was significantly greater than that in the males, but there was no relationship in either sex between enzyme activity and age or between enzyme activity and the most common erythrocyte antigens. Separation of erythrocytes by age using isopycnic centrifugation showed that enzyme activity was constant during the life of the cell.
Assuntos
Eritrócitos/enzimologia , Glutationa Transferase/metabolismo , Adolescente , Adulto , Idoso , Centrifugação Isopícnica , Criança , Envelhecimento Eritrocítico , Feminino , Glutationa Transferase/análise , Humanos , Cinética , Masculino , Pessoa de Meia-Idade , Valores de Referência , Fatores SexuaisRESUMO
A case of congenital dyserythropoietic anaemia is described which appears to be at variance with the accepted classification. The morphological findings are of normoblastic hyperplasia with marked cytoplasmic vacuolation. Electron microscopy shows these to be myelin figures in all stages of erythroid differentiation.