RESUMO
BACKGROUND/AIMS: Haemangiomas are common benign tumours of infancy that consist of rapidly proliferating endothelial cells. A locus for an autosomal dominant predisposition to haemangioma has been identified recently on chromosome 5q. This study aimed to investigate loss of heterozygosity on chromosomes 5 and 9 in haemangiomas. METHODS: Sporadic proliferative phase haemangiomas were microdissected. Polymerase chain reaction amplification and analysis of microsatellite markers on chromosomes 5 and 9 was carried out. RESULTS: There was a significant loss of heterozygosity for markers on chromosome 5q in haemangioma tissue, when compared with either markers from chromosome 5p (p < 0.05) or markers from chromosome 9 (p < 0.05). CONCLUSIONS: These results suggest that haemangioma formation might be associated with somatic mutational events, and provides evidence that a locus on 5q is involved in the formation of sporadic haemangiomas.
Assuntos
Cromossomos Humanos Par 5/genética , Hemangioma/genética , Perda de Heterozigosidade , Cromossomos Humanos Par 9/genética , Feminino , Humanos , Lactente , Masculino , Repetições de MicrossatélitesRESUMO
CD94/NKG2 receptors on mouse NK cells recognize the nonclassical class I molecule Qa1 and can deliver inhibitory signals that prevent NK cells from lysing Qa1-expressing cells. However, the exact circumstances under which Qa1 protects cells from NK lysis and, in particular, the role of the dominant Qa1-associated peptide, Qdm, are unclear. In this study, we examined in detail the lysis of Qa1-expressing cells by fetal NK cells that express CD94/NKG2 receptors for Qa1 but that lack receptors for classical class I molecules. Whereas mouse L cells and human C1R cells transfected with Qa1 were resistant to lysis by these effectors, Qa1-transfected TAP-deficient human T2 cells showed no resistance despite expressing high levels of surface Qa1. However, these cells could be efficiently protected by exposure to low concentrations of Qdm peptide or certain Qdm-related peptides. By contrast, even prolonged exposure of TAP-deficient RMA/S cells to high doses of Qdm peptide failed to induce levels of surface Qa1 detectable with a Qa1-specific mAb or to protect them from NK lysis, although such treatment induced sensitivity to lysis by Qa1-specific CTL. Collectively, these findings indicate that high surface expression of Qa1 is necessary but not sufficient for protection, and that effective protection requires the expression of sufficient levels of suitable Qa1-peptide complexes to overcome activatory signals. Results obtained with a series of substituted Qdm peptides suggest that residues at positions 3, 4, 5, and 8 of the Qdm sequence, AMAPRTLLL, are important for recognition of Qa1-Qdm complexes by inhibitory CD94/NKG2 receptors.
Assuntos
Testes Imunológicos de Citotoxicidade , Citotoxicidade Imunológica , Antígenos de Histocompatibilidade Classe I/imunologia , Células Matadoras Naturais/imunologia , Lectinas Tipo C , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Antígenos CD/metabolismo , Linhagem Celular , Testes Imunológicos de Citotoxicidade/métodos , Citotoxicidade Imunológica/genética , Feto , Antígenos de Histocompatibilidade Classe I/biossíntese , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Células Matadoras Naturais/metabolismo , Células L , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Subfamília C de Receptores Semelhantes a Lectina de Células NK , Subfamília D de Receptores Semelhantes a Lectina de Células NK , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Peptídeos/imunologia , Peptídeos/metabolismo , Peptídeos/farmacologia , Ligação Proteica/imunologia , Sinais Direcionadores de Proteínas , Receptores de Antígenos/biossíntese , Receptores Imunológicos/metabolismo , Receptores de Células Matadoras Naturais , Especificidade da Espécie , Temperatura , Transfecção , Células Tumorais CultivadasRESUMO
Endoglin (CD105) is expressed on the surface of endothelial and haematopoietic cells in mammals and binds TGFbeta isoforms 1 and 3 in combination with the signaling complex of TGFbeta receptors types I and II. Endoglin expression increases during angiogenesis, wound healing, and inflammation, all of which are associated with TGFbeta signaling and alterations in vascular structure. The importance of endoglin for normal vascular architecture is further indicated by the association of mutations in the endoglin gene with the inherited disorder Hereditary Haemorrhagic Telangiectasia Type 1 (HHT1), a disease characterised by bleeding from vascular malformations. In order to study the role of endoglin in vivo in more detail and to work toward developing an animal model of HHT1, we have derived mice that carry a targeted nonsense mutation in the endoglin gene. Studies on these mice have revealed that endoglin is essential for early development. Embryos homozygous for the endoglin mutation fail to progress beyond 10.5 days postcoitum and fail to form mature blood vessels in the yolk sac. This phenotype is remarkably similar to that of the TGFbeta1 and the TGFbeta receptor II knockout mice, indicating that endoglin is needed in vivo for TGFbeta1 signaling during extraembryonic vascular development. In addition, we have observed cardiac defects in homozygous endoglin-deficient embryos, suggesting endoglin also plays a role in cardiogenesis. We anticipate that heterozygous mice will ultimately serve as a useful disease model for HHT1, as some individuals have dilated and fragile blood vessels similar to vascular malformations seen in HHT patients.
Assuntos
Coração/embriologia , Neovascularização Fisiológica/fisiologia , Molécula 1 de Adesão de Célula Vascular/fisiologia , Animais , Antígenos CD , Sequência de Bases , Códon de Terminação , Primers do DNA , Endoglina , Endotélio Vascular/metabolismo , Genes Letais , Hematopoese/genética , Heterozigoto , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Superfície Celular , Telangiectasia Hemorrágica Hereditária/genética , Telangiectasia Hemorrágica Hereditária/fisiopatologia , Molécula 1 de Adesão de Célula Vascular/genéticaAssuntos
Artrite Reumatoide/imunologia , Proteínas de Bactérias , Citocinas/genética , Exotoxinas/farmacologia , Proteínas de Membrana , Pirogênios/farmacologia , Células Th1/imunologia , Citocinas/imunologia , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/imunologia , Humanos , RNA Mensageiro/análise , Células Th1/efeitos dos fármacosRESUMO
Cytotoxic T lymphocytes have been implicated in the control of hepatitis C virus (HCV) infection. Recognition by cytotoxic T lymphocytes of epitopes within HCV core protein has been defined previously by in vitro stimulation with synthetic peptides. The aim of this study has been to examine cytotoxic T-lymphocyte responses generated against peptides produced naturally following intracellular processing of viral protein. Antigen-specific cytotoxic T-lymphocyte lines were generated from both HCV uninfected and infected individuals by culturing CD8+ T cells with autologous dendritic cells loaded intracytoplasmically with recombinant HCV core protein. Analysis of the epitopes recognized by core protein-specific cytotoxic T lymphocytes used synthetic peptides that were selected based on their predicted binding to HLA-A*0201 molecules. Core protein-specific cytotoxic T lymphocytes derived from HCV uninfected and infected individuals were able to lyse autologous target cells pulsed with each of 5 predicted epitopes. Generation of HCV-specific cytotoxic T lymphocytes using dendritic cells as antigen presenting cells provides a method of comparing the potential repertoire of cytotoxic T-lymphocyte responses to the responses that occur in chronically infected individuals. No evidence of a qualitatively different response by patient cytotoxic T lymphocytes was apparent which might explain persistence of the virus.
Assuntos
Hepacivirus/imunologia , Hepatite C/imunologia , Linfócitos T Citotóxicos/imunologia , Proteínas do Core Viral/imunologia , Testes Imunológicos de Citotoxicidade , Epitopos/química , Epitopos/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/imunologia , Linfócitos T Citotóxicos/virologia , Proteínas do Core Viral/químicaRESUMO
PBC is characterized by T cell-mediated destruction of the biliary epithelial cells lining the small intrahepatic bile ducts. The E2 and E3 binding protein (E3BP (protein X)) components of pyruvate dehydrogenase complex (PDC) are disease-specific autoantigens in PBC. Attempts to localize the T cell autoepitopes within PDC-E2 have, however, generated contradictory results. One study has suggested the presence of T cell epitopes throughout PDC-E2, whilst another has identified a single dominant 14 amino acid T cell epitope (p163) spanning the lipoic acid binding lysine residue in the inner lipoyl domain (ILD) of PDC-E2. The aim of the current study was to determine the prevalence of T cell responses to p163 and PDC-E2 ILD, and the role played by lipoylation of these antigens in their immunogenicity, in a UK PBC population. We found that the majority of the PBC patients showing a 6-day peripheral blood T cell proliferative response to native human PDC also responded, in a MHC class II-restricted fashion, to biochemically purified PDC-E2 and E3BP (which co-purify) (9/10 positive (SI > 2.76), mean SI 5.74 +/- 5.04 (PDC-E2/E3BP) versus 6.67 +/- 3.84 (PDC), P = NS), implying that the important PBC-specific T cell epitopes are contained within the PDC-E2 or E3BP components of PDC. Only a minority of patients responsive to PDC, however, responded to either lipoylated recombinant PDC-E2 ILD (4/10 positive, mean SI 1.98 +/- 1.24, P < 0.005 versus PDC response) or lipoylated p163 (4/12 positive, mean SI 1.90 +/- 1.58, P < 0.001). The lipoylation state did not affect the T cell response to either ILD or p163. Our findings suggest that in some UK patients with PBC there are immunodominant T cell autoepitopes within PDC-E2/E3BP which are outside the ILD of PDC-E2.
Assuntos
Autoantígenos , Epitopos Imunodominantes , Cirrose Hepática Biliar/imunologia , Complexo Piruvato Desidrogenase/imunologia , Linfócitos T/imunologia , Autoimunidade , Di-Hidrolipoil-Lisina-Resíduo Acetiltransferase , Epitopos , Humanos , Lisina/imunologia , Fragmentos de Peptídeos/imunologia , Proteínas Recombinantes/imunologia , Ácido Tióctico/imunologiaRESUMO
BACKGROUND & AIMS: Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease characterized by the presence of antimitochondrial autoantibodies in patients' serum. The major autoantigen, recognized by antibodies from > 95% of patients with PBC, has been identified as the E2 component (E2p) of the pyruvate dehydrogenase multienzyme complex. Immunodominant sites on E2p have been localized to the inner of the two lipoyl domains, where the essential cofactor lipoic acid is attached covalently. The aim of this study was to determine the three-dimensional structure of the inner lipoyl domain of human E2p. METHODS: The domain was expressed in Escherichia coli; after purification, its structure was analyzed using nuclear magnetic resonance spectroscopy. RESULTS: The structure of the lipoyl domain from human E2p was determined, and the implications of the structure for autoimmune recognition were assessed. CONCLUSIONS: Knowledge of the structure further defines the major epitope and may help in the design of antigen-specific immunotherapy for treatment of PBC.
Assuntos
Autoantígenos/química , Cirrose Hepática Biliar/imunologia , Estrutura Secundária de Proteína , Sequência de Aminoácidos , Humanos , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Proteínas Recombinantes/químicaRESUMO
Streptococcus pyogenes (group A Streptococcus) cell extracts (CE) have a remarkably powerful and dose-dependent inhibitory effect on antigen, superantigen, or mitogen-stimulated human peripheral blood mononuclear cell (PBMC) proliferation in vitro. Purification of the inhibitory component present in S. pyogenes type M5 (Manfredo strain) CE by anion-exchange chromatography followed by gel filtration chromatography showed that the inhibitor had an approximate native molecular mass of 100 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified inhibitory fractions followed by silver staining gave a single band with an approximate molecular mass of 47 kDa, indicating that the inhibitor is composed of two identical subunits. NH2-terminal sequencing of the protein revealed that it was identical to the previously characterized streptococcal acid glycoprotein (SAGP); this protein possesses between 31.5 and 39.0% amino acid identity with arginine deiminase (AD) from Mycoplasma hominis, Mycoplasma arginini, Pseudomonas putida, and Pseudomonas aeruginosa. AD enzyme activity was present in unfractionated CE prepared from a range of streptococcal strains, and partially purified inhibitory fractions of Manfredo CE also had high levels of activity. The inhibitory effect of Manfredo CE was overcome by the addition of L-arginine to proliferation assays in which human PBMC were stimulated with phytohemagglutinin. We conclude that SAGP, or its homolog, possesses AD activity and that the potent inhibition of proliferation of human T cells by streptococcal CE is due to activity of this enzyme.
Assuntos
Hidrolases/fisiologia , Imunossupressores , Ativação Linfocitária , Streptococcus pyogenes/fisiologia , Linfócitos T/imunologia , Sequência de Aminoácidos , Arginina/farmacologia , Autoimunidade , Humanos , Dados de Sequência Molecular , Peso Molecular , Streptococcus pyogenes/química , Streptococcus pyogenes/imunologiaAssuntos
Citocinas/biossíntese , Proteínas da Matriz Extracelular , Proteoglicanas/imunologia , Linfócitos T/imunologia , Agrecanas , Sequência de Aminoácidos , Animais , Cartilagem , Bovinos , Células Cultivadas , Proteoglicanas de Sulfatos de Condroitina/imunologia , Concanavalina A , Humanos , Interferon gama/biossíntese , Interleucina-10/biossíntese , Interleucina-2/biossíntese , Interleucina-4/biossíntese , Lectinas Tipo C , Linfonodos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/farmacologia , Proteoglicanas/química , Proteoglicanas/farmacologia , Linfócitos T/efeitos dos fármacosRESUMO
The study of T cell responses to autoantigens in human autoimmunity has been hampered by difficulties, firstly in identifying significant autoantigens, and secondly in the purification of authentic human proteins in sufficient quantities to allow characterization of antigen-specific T cell responses. In this study we have purified a human autoantigen, pyruvate dehydrogenase, retaining its enzymatic activity, and characterized autoreactive T cell responses to it in a human autoimmune disease, primary biliary cirrhosis. T cell responses to a mixture of the E2 and protein X subunits of human pyruvate dehydrogenase complex are seen in most affected patients, but in only a small minority of normal and chronic liver disease controls. By contrast, responses to whole pyruvate dehydrogenase complex occur with equal frequency in both groups. This suggests that responses to the E2 component/protein X of pyruvate dehydrogenase complex play a role in the pathogenesis of primary biliary cirrhosis. The availability of significant quantities of the human autoantigen in primary biliary cirrhosis makes this condition an interesting model in which to study true autoreactive human T cell responses.
Assuntos
Autoantígenos/imunologia , Cirrose Hepática Biliar/imunologia , Proteínas/imunologia , Complexo Piruvato Desidrogenase/imunologia , Linfócitos T/imunologia , Adulto , Idoso , Di-Hidrolipoil-Lisina-Resíduo Acetiltransferase , Feminino , Humanos , Pessoa de Meia-Idade , Peptídeos/imunologiaRESUMO
BACKGROUND: Primary biliary cirrhosis (PBC) has an autoimmune aetiology, although little is known regarding the mechanisms of breakdown of self tolerance. One postulated mechanism of control of self tolerance is through interacting T cell subsets, a phenomenon explored in this study. AIMS: To characterise and compare T cell subset responses to an antigen (tuberculin purified protein derivative derived from mycobacteria) in PBC patients and controls. Cross reactive responses to mycobacteria have recently been implicated in the aetiology of PBC. SUBJECTS: 58 PBC patients, 25 normal controls, and 34 chronic liver disease controls. METHODS: Responses to antigen were measured in terms of primary T cell proliferation and cytokine secretion (by ELISA). Responding cells were phenotyped by FACS analysis. RESULTS: Similar CD4+ T cell proliferative responses were seen in PBC patients (mean (SD) stimulation index (SI) 22.6 (27.2), 42 of 58 (72.4%) positive response), normal controls (46.5 (88.0), 17 of 25 (68%) positive), and chronic liver disease controls (24.8 (49.8), 27 of 34 (79.4%) positive)). Secretion of both interferon gamma and IL10 was significantly lower in PBC patients than controls (IFN gamma: PBC 822.7 (1100) pg/ml, controls 2929 (3402) pg/ml, p < 0.05: IL10: PBC 11.1 (15.6) pg/ml, controls 34.7 (63.4) pg/ml, p < 0.05). CONCLUSIONS: In PBC unimpaired T cell proliferation is seen with reduced secretion of both Th-1 (interferon gamma) and Th-2 type (IL10) cytokines. These findings may result from differential subset responses and may help explain the defects of functional immunity seen in PBC.
Assuntos
Antígenos de Bactérias/farmacologia , Citocinas/metabolismo , Cirrose Hepática Biliar/imunologia , Ativação Linfocitária/efeitos dos fármacos , Linfócitos T/imunologia , Tuberculina , Complexo CD3/imunologia , Linfócitos T CD4-Positivos/imunologia , Doença Crônica , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Imunofluorescência , Humanos , Interferon gama/metabolismo , Interleucina-10/metabolismo , Hepatopatias/imunologia , Masculino , Pessoa de Meia-IdadeRESUMO
Currently, the limited supply and stability of some human autoantigens pose formidable difficulties in characterizing patients' T cells specific for them; recombinant preparations may contain bacterial contaminants, and synthetic peptides have arbitrarily chosen start and stop points. In order to provide a stable antigen source with naturally processed epitopes, a full-length acetylcholine receptor (AChR) alpha subunit construct was transfected into B-lymphoblastoid cell lines (B-LCL). Expression was much easier to detect at the mRNA level than the protein level. Nevertheless, this transfectant also stimulated a T-cell line that recognized the alpha 149-156 region in the context of HLA-DR4 at high sensitivity. The responses were specific both for the antigen transfected and for the presenting HLA-DR allele. This study thus confirms the potential of autologous B-LCL expressing natural epitopes in the context of HLA class II molecules for characterizing established T-cell lines, and perhaps also for initiating new ones.
Assuntos
Autoantígenos/genética , Linfócitos B , Linfócitos T CD4-Positivos/imunologia , Receptores Colinérgicos/imunologia , Transfecção , Linfócitos B/imunologia , Northern Blotting , Western Blotting , Linhagem Celular , Humanos , Iodeto Peroxidase/genética , Iodeto Peroxidase/metabolismo , RNA Mensageiro/metabolismo , Receptores Colinérgicos/genéticaRESUMO
Primary biliary cirrhosis (PBC) is an autoimmune condition that results in destruction of the intrahepatic biliary epithelial cells and is characterized by autoantibodies to pyruvate dehydrogenase complex (PDC). The portal tract T-cell infiltrate and up-regulation of HLA class I, HLA class II, and cell adhesion molecules such as intercellular adhesion molecule-1 on the biliary epithelial cells suggest that T cells play a significant role in mediating this damage. The authors have characterized the peripheral blood T-cell proliferative responses of 24 PBC patients and 48 controls (20 normal, 28 non-PBC chronic liver disease) to the dominant autoantigen PDC, and its constituent components E1, E2 and protein X (which co-purify), and E3. A significant proportion of both PBC patients and controls showed T-cell responses to whole PDC (12 of 24 vs. 24 of 48 SI > 2.5 P = NS) and E1 (15 of 24 vs. 25 of 48 P = NS). Responses to PDC and E1 are thus seen in normal individuals and are not limited to PBC patients. T-cell responses to E2/X were seen in most PBC patients (14 of 24), but in only a small number of controls (6 of 48, P < .0001), responses to E2/X being significantly more frequent in pre-cirrhotic PBC patients (stages I to III, 12 of 15) than cirrhotic (stage IV, 2 of 9 P < .05). Peripheral blood T-cell responses to E2/X are thus strongly associated with early PBC. Responses to E3 were low in both PBC patients and controls.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Cirrose Hepática Biliar/imunologia , Complexo Piruvato Desidrogenase/imunologia , Linfócitos T/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Di-Hidrolipoil-Lisina-Resíduo Acetiltransferase , Feminino , Humanos , Imunoglobulina G/sangue , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Peptídeos/imunologia , Toxoide Tetânico/imunologiaRESUMO
Autoimmune diseases represent a failure of control in the immune system. In recent years, our understanding of the mechanisms of action of both the innate and the specific immune responses has increased greatly. In particular, we now know much more about the nature of antigens recognized by lymphocytes, as well as how diversity of antigen receptors is generated, antigens and antigen receptors interact, and the cells of the immune system communicate. It is apparent that an inevitable consequence of the diversity of the potential response to antigen is self-reactivity. However, the relative infrequency with which pathological self-reactivity occurs implies the existence of effective control of immune responses. The conditions under which immune responses can be activated, and the factors which regulate their progression, have been subjected to detailed scrutiny. Several of the mechanisms involved in the removal or inactivation of self-reactive lymphocytes, the process of self-tolerance, are now understood. What is less clear are the conditions under which, and the mechanisms by which, this self-tolerance can break down, giving rise to autoimmunity. Several classes of explanation have been put forward to explain this failure of self-tolerance. Although they are of great theoretical interest, proof of their involvement in the pathogenesis of the major autoimmune diseases is largely lacking. A further expansion of our understanding of the mechanisms by which self-tolerance is normally maintained is still needed, in order to comprehend the pathways of breakdown of this tolerance in autoimmunity. Only then will sites and mechanisms for effective therapeutic intervention be identified.
Assuntos
Autoimunidade , Animais , Autoantígenos , Antígenos de Histocompatibilidade , Humanos , Tolerância Imunológica , Imunidade , Receptores de Antígenos de Linfócitos B , Receptores de Antígenos de Linfócitos T , Linfócitos T/imunologiaRESUMO
Approximately 95% of patients with primary biliary cirrhosis have antimitochondrial antibodies against the E2 component of the pyruvate dehydrogenase complex (E2p). Immunodominant sites on E2p have been localized to the inner lipoyl domain, which serves as a covalent attachment site for the essential cofactor, lipoic acid. However, it is not clear whether the presence of lipoic acid is necessary for autoimmune recognition of human E2p. To facilitate further studies on the inner lipoyl domain and to assess the importance of lipoic acid in antibody binding, we used the previously cloned human E2p cDNA in the construction and high-level expression in Escherichia coli of a subgene encoding the domain. Purification and analysis of the gene product revealed that both lipoylated and unlipoylated forms of the intact domain are generated. Immunoblotting, enzyme-linked immunosorbent assay inhibition experiments and antibody affinity measurements using isolated lipoylated and unlipoylated domains demonstrated that the presence of the lipoyl residue is crucial for effective recognition by primary biliary cirrhosis patients' autoantibodies, which have a higher relative affinity for the lipoylated form. Contrary to some previous suggestions, these results indicate that antibodies in primary biliary cirrhosis patients' sera bind most effectively to a unique peptide-cofactor conformation in the lipoyl domain of the human E2p polypeptide. Moreover, the availability of large amounts of human lipoyl domain will permit further studies into the role of the antigen (if any) in disease pathogenesis.
Assuntos
Acetiltransferases/imunologia , Autoantígenos/imunologia , Cirrose Hepática Biliar/imunologia , Complexo Piruvato Desidrogenase/imunologia , Ácido Tióctico/imunologia , Reações Antígeno-Anticorpo , Autoanticorpos/imunologia , Di-Hidrolipoil-Lisina-Resíduo Acetiltransferase , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Humanos , ImmunoblottingRESUMO
The TSH-receptor (TSH-R) and thyroid peroxidase (TPO) are targets of autoantibody production in the autoimmune thyroid disease, Graves' disease, and are also likely to be the target of T-cell responses. To facilitate the analysis of T-cell responses we have investigated a system that allows expression of these autoantigens as recombinant proteins in autologous cells. Human B-lymphoblastoid cell lines (B-LCL), which are known to present antigen to autologous T cells, were transfected with constructs directing the expression of human TSH-R and TPO. The constructs utilized an expression vector replicating under the control of EBV-derived sequences that is maintained episomally in transfected cells. Both proteins were shown to be expressed by transfected B-LCL and present on the cell surface. Such transfected B-LCL could be used for the derivation, screening and characterization of autologous T-cell clones against thyroid autoantigens.
Assuntos
Autoantígenos/genética , Herpesvirus Humano 4/genética , Iodeto Peroxidase/genética , Receptores da Tireotropina/genética , Linfócitos B/imunologia , Sequência de Bases , Vetores Genéticos , Doença de Graves/imunologia , Humanos , Iodeto Peroxidase/imunologia , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Receptores da Tireotropina/imunologia , Proteínas Recombinantes de Fusão/imunologia , Glândula Tireoide/imunologia , Transfecção , Células Tumorais CultivadasRESUMO
Diabetes-prone (DP) and diabetes-resistant (DR) sublines of the BB rat have been established in Edinburgh, U.K., separately from other existing colonies. In an examination of the lymphoid status of the two lines, BB-DP/Ed and BB-DR/Ed, it has been found that both lines have very low T-cell numbers, depressed B-lymphocyte numbers and a complete absence of peripheral CD8+ T cells, all features characteristic of the previously described genetic lymphopenia lesion. It was also noted that the peripheral T cells of both BB/Ed lines were larger than normal. The DP/Ed and DR/Ed lines were indistinguishable in all these respects, and furthermore, they were both shown to type as RT1u at the major histocompatibility complex (MHC). The genetic combination of lymphopenia and RT1u without expression of diabetes is not present in other extant BB lines and makes BB-DR/Ed a uniquely useful control strain for BB rat studies as well as a valuable genetic resource for the further genetic analysis of diabetes susceptibility in rats.
Assuntos
Diabetes Mellitus/genética , Linfopenia/genética , Ratos Endogâmicos BB/genética , Animais , Diabetes Mellitus/imunologia , Suscetibilidade a Doenças , Feminino , Contagem de Leucócitos , Subpopulações de Linfócitos/imunologia , Linfopenia/imunologia , Complexo Principal de Histocompatibilidade , Masculino , Polimorfismo de Fragmento de Restrição , RatosRESUMO
The dihydrolipoamide acetyltransferase subunit (E2p) of mammalian pyruvate dehydrogenase complex has two highly conserved lipoyl domains each modified with a lipoyl cofactor bound in amide linkage to a specific lysine residue. A sub-gene encoding the inner lipoyl domain of human E2p has been over-expressed in Escherichia coli. Two forms of the domain have been purified, corresponding to lipoylated and non-lipoylated species. The apo-domain can be lipoylated in vitro with partially purified E. coli lipoate protein ligase, and the lipoylated domain can be reductively acetylated by human E1p (pyruvate dehydrogenase). Availability of the two forms will now allow detailed biochemical and structural studies of the human lipoyl domains.
Assuntos
Acetiltransferases/genética , Escherichia coli/genética , Complexo Piruvato Desidrogenase/metabolismo , Acetiltransferases/química , Sequência de Aminoácidos , Sequência de Bases , Cromatografia por Troca Iônica , Clonagem Molecular , DNA Recombinante , Di-Hidrolipoil-Lisina-Resíduo Acetiltransferase , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Glutationa Transferase/química , Humanos , Dados de Sequência Molecular , Peptídeo Sintases/metabolismo , Plasmídeos , Complexo Piruvato Desidrogenase/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genéticaRESUMO
The cDNA sequence of the flavoprotein subunit of bovine heart succinate dehydrogenase is reported. This is the first complete eukaryotic sequence of the flavoprotein subunit to be characterized, and it encodes a 665-amino acid protein that consists of a presequence and a 621-residue mature protein. The deduced bovine sequence shows homology to the corresponding peptides of prokaryotic succinate dehydrogenase and the related fumarate reductases; in particular, there is good overall homology (48%) to the flavoprotein subunit of Escherichia coli succinate dehydrogenase. The conserved sequences comprising the active site and those involved in FAD binding are also found in the bovine protein. The active site of the bovine polypeptide contains a cysteine that confers sensitivity of the enzyme to sulfhydryl reagents; this cysteine is only present in some sequences and thus provides a discriminatory biochemical marker. A putative flavoprotein subunit of human placental succinate dehydrogenase (partial sequence) that lacks this critical cysteine (Malcovati, M., Marchetti, T., Zanelli, T., and Tenchini, M. L. (1991) in Flavins and Flavoproteins 1990 (Curti, B., Ronchi, S., and Zanetti, G., eds) pp. 727-730, Walter de Gruyter & Co., Berlin) has only 16% homology to the bovine heart flavoprotein subunit. However, we show that the enzyme from human placenta is as sensitive to N-ethylmaleimide as that from bovine tissues. In addition, a transcript in human placenta and muscle hybridizes to the bovine heart flavoprotein cDNA and is the same size as that in bovine tissues.
Assuntos
Flavoproteínas/genética , Miocárdio/enzimologia , Succinato Desidrogenase/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Northern Blotting , Bovinos , DNA/genética , Etilmaleimida/farmacologia , Feminino , Flavina-Adenina Dinucleotídeo/metabolismo , Humanos , Dados de Sequência Molecular , Placenta/enzimologia , Gravidez , RNA/genética , Alinhamento de Sequência , Succinato Desidrogenase/antagonistas & inibidoresRESUMO
Little is known about the organization of class I genes in the rat although there is prima facie evidence that it is distinct from that of the mouse. We report the cloning of 61 nonclassical rat class I genes into cosmid clusters with a total mapped length of 1,264 kb. It is certain that the total number of class I genes in the rat must exceed this number. From restriction maps it is possible to identify substantial regions of duplication. By transfection of cosmids into mouse L cells, it has been possible to demonstrate at least seven different nonclassical rat class I genes that are expressible on the cell surface. Crossreaction of a single mouse monoclonal antibody with all of these class I molecules is consistent with sequence homogenization within the rat nonclassical system. Attempts to find rat homologues of the mouse Tla genes by crosshybridization of rat cosmids with a range of different TLa-specific probes were unsuccessful, suggesting that this large group of divergent class I genes is absent or nearly so from the rat. The large number of class I genes in the rat appears to have arisen by expansion of genes more closely related to the classical sequence.