RESUMEN
Royal College of Surgeons rats are genetically predisposed to undergo significant visual loss caused by a primary dysfunction of retinal pigment epithelial (RPE) cells. By using this model, we have examined the efficacy of subretinal transplantation of two independent human RPE cell lines each exhibiting genetic modifications that confer long-term stability in vitro. The two cell lines, a spontaneously derived cell line (ARPE19) and an extensively characterized genetically engineered human RPE cell line (h1RPE7), which expresses SV40 large T (tumor) antigen, were evaluated separately. Both lines result in a significant preservation of visual function as assessed by either behavioral or physiological techniques. This attenuation of visual loss correlates with photoreceptor survival and the presence of donor cells in the areas of rescued photoreceptors at 5 months postgrafting (6 months of age). These results demonstrate the potential of genetically modified human RPE cells for ultimate application in therapeutic transplantation strategies for retinal degenerative diseases caused by RPE dysfunction.
Asunto(s)
Proteínas del Ojo/fisiología , Epitelio Pigmentado Ocular/trasplante , Proteínas Proto-Oncogénicas , Proteínas Tirosina Quinasas Receptoras/deficiencia , Degeneración Retiniana/terapia , Animales , Antígenos Transformadores de Poliomavirus/genética , Línea Celular Transformada/trasplante , Supervivencia Celular , Transformación Celular Viral , Proteínas del Ojo/genética , Movimientos de la Cabeza/fisiología , Humanos , Fagocitosis , Epitelio Pigmentado Ocular/citología , Ratas , Ratas Mutantes , Proteínas Tirosina Quinasas Receptoras/genética , Degeneración Retiniana/genética , Degeneración Retiniana/patología , Segmento Externo de la Célula en Bastón/metabolismo , Segmento Externo de la Célula en Bastón/patología , Umbral Sensorial , Virus 40 de los Simios/genética , Colículos Superiores/fisiopatología , Trasplante Heterólogo , Pruebas de Visión , Campos Visuales , Percepción Visual , Tirosina Quinasa c-MerRESUMEN
Disulone (dapsone + ferrous oxalate) is a sulphone marketed in France since 1958 and authorized in P. Carinii prophylaxis in HIV+ cotrimoxazole intolerant patients, bullous dermatosis, leprosy and polychondritis. Between 1983 and 1998, 249 adverse reactions were reported to French pharmacovigilance centres and Aventis, the manufacturer. Every side-effect was reviewed and the causal relationship was assessed on the basis of the French method for causality assessment. Main side-effects were divided as follows: 117 blood dyscrasias (generally neutropenia and agranulocytosis, rarely methaemoglobinaemia, haemolysis, macrocytosis, anaemia, aplastic anaemia, haemochromatosis and sulphaemoglobinaemia); 29 hypersensitivity syndrome; 39 cutaneous reactions, generally rash; 27 liver injuries (cholestatic, cytolytic and mixed hepatitis); 27 neurological and psychiatric side-effects including 7 axonal neuropathy; 10 gastrointestinal effects, generally nausea and vomiting. Five deaths were reported (4 septicaemia including one case not due to dapsone and 1 digestive bleeding due to underlying disease). In the other cases the outcome was favourable. The results were compared with the published references. It would seem to be important to reinforce information to prescribers about the possible serious adverse reactions with dapsone, particularly hypersensitivity syndrome and agranulocytosis, that can cause death if the drug is not stopped in time.
Asunto(s)
Infecciones Oportunistas Relacionadas con el SIDA/tratamiento farmacológico , Sistemas de Registro de Reacción Adversa a Medicamentos , Antiinfecciosos/efectos adversos , Neumonía por Pneumocystis/tratamiento farmacológico , Sistemas de Registro de Reacción Adversa a Medicamentos/estadística & datos numéricos , Francia , HumanosRESUMEN
Backbone modifications have been introduced into the melanoma derived peptide MART-1(27-35) to increase its binding to class I major histocompatibility complex HLA-A2 molecule, and ultimately to enhance its immunogenicity. Each analogue was obtained by replacing one peptide bond at a time in the natural epitope by the aminomethylene (CH2-NH) surrogate. All analogues displayed an increased resistance to proteolysis. Interestingly, the comparative results showed that five analogues bound more efficiently to HLA-A2 than the parent peptide. On the other hand, two pseudopeptide/HLA-A2 complexes were recognized by one melanoma-specific T cell clone. Close examination of the impact of such modifications at the molecular level provides useful supports for the rational design of stable compounds with applications in anti-tumour specific immunotherapy and in vaccine development.
Asunto(s)
Epítopos/química , Proteínas de Neoplasias/química , Proteínas de Neoplasias/síntesis química , Secuencia de Aminoácidos , Aminoácidos/química , Línea Celular , Cromatografía Líquida de Alta Presión , Relación Dosis-Respuesta a Droga , Epítopos/inmunología , Fluorenos/química , Antígeno HLA-A2/metabolismo , Humanos , Melanoma/inmunología , Melanoma/metabolismo , Datos de Secuencia Molecular , Proteínas de Neoplasias/inmunología , Péptidos/química , Unión Proteica , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
The design of heteroclytic antigens with high MHC binding capacity is of particular interest to overcome the weak immunogenicity of peptide epitopes derived from tissue antigens expressed by tumors. In the present study, double-substituted peptide analogues of the tumor-associated antigen MART-1(27-35) incorporating a substitution at a primary anchor residue and a beta-amino acid residue at different positions in the sequence were synthesized and evaluated for binding to the human histocompatibility class I molecule HLA-A2 and for recognition by tumor-infiltrating lymphocytes. Interestingly, by combining a Leu for Ala substitution at P2 (which alone is deleterious for antigenic activity) with a beta-amino acid substitution at a putative TCR contact residue, recognition by tumor-infiltrating lymphocytes was partially restored. The analogue [Leu(28),beta-HIle(30)]MART-1(27-35) displays both a higher affinity to HLA-A2 and a more prolonged complex stability compared to [Leu(28)]MART-1(27-35). Overall, these results suggest that double-substitution strategies and beta-amino acid replacements at putative TCR contact residues might prove useful for the design of epitope mimics with high MHC binding capacity.
Asunto(s)
Epítopos/química , Antígeno HLA-A2/metabolismo , Linfocitos Infiltrantes de Tumor/metabolismo , Melanoma/química , Proteínas de Neoplasias/química , Fragmentos de Péptidos/síntesis química , Cromatografía Líquida de Alta Presión , Electroforesis Capilar , Humanos , Isoantígenos/química , Isoantígenos/metabolismo , Espectroscopía de Resonancia Magnética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Unión Proteica , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Células Tumorales CultivadasRESUMEN
Chronic myeloid leukemia (CML) is characterized cytogenetically by a t(9;22) translocation which generates a hybrid bcr-abl gene, encoding a p210(bcr-abl) fusion protein. The induction in vitro of leukemia-specific T cells reactive with p210(bcr-abl) is a strategy developed for an immunological therapeutic approach in CML. Peptides from the junction region of this chimeric protein have been considered as potential targets for a cytotoxic response against leukemic cells. However, only a few peptides encompassing the two p210(bcr-abl) breakpoints have been shown to bind to the most common HLA class I molecules, which limits the number of patients who could benefit from this approach. We assume that the presence of chimeric BCR-ABL protein in leukemic cells may affect processing and delivery of peptides, possibly giving rise to new epitopes at the cell surface. We selected 162 peptides from the whole sequence of this protein, including 14 peptides of the b2a2 and b3a2 junctions, which had an anchor motif for a common HLA class I molecule. We tested their ability to bind to eight HLA class I molecules (HLA-A1, -A2, -A3, -A11, -B7, -B8, -B27, -B44). We identified 48 peptides from outside the junction region, with intermediate or strong binding capacities to these HLA class I molecules contrasting with only six junction peptides with a moderate binding capacity to HLA-A3/A11, -B8, or -B44 molecules. Moreover, cytotoxic T lymphocyte lines specific for various peptides outside the junction were generated from peripheral blood mononuclear cells of HLA-A2 or -B7 healthy donors and from one CML patient. These results contribute to evaluation of immunity to the BCR-ABL chimeric protein. Further studies are required to investigate whether such epitopes are correctly processed and presented by leukemic cells.
Asunto(s)
Proteínas de Fusión bcr-abl/inmunología , Antígenos HLA/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Linfocitos T Citotóxicos/inmunología , Secuencia de Aminoácidos , Sitios de Unión/genética , Línea Celular , Línea Celular Transformada , Proteínas de Fusión bcr-abl/genética , Antígeno HLA-B7/metabolismo , Herpesvirus Humano 4 , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/inmunología , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Unión Proteica , Células Tumorales CultivadasRESUMEN
Chemokine receptors are molecules involved in the fusion of immunodeficiency viruses after their attachment. As chimpanzees are the animal model for infection by HIV-1, we cloned and sequenced chimpanzee CXCR4 and CCR5 from PBMCs. Chimpanzee CXCR4 was found to be identical to human CXCR4, which provides an explanation for the sensitivity of chimpanzees to lymphotropic isolates of HIV-1. Chimpanzee CCR5 showed two substitutions with respect to human CCR5. However, we show that the macrophage-tropic isolate HIV-1-Ba-L can use chimpanzee CCR5 as a fusion receptor. Therefore, the resistance of chimpanzee PBMCs to infection by macrophage-tropic isolates of HIV-1 is unlikely to be due to substitutions in CCR5.
Asunto(s)
VIH-1/metabolismo , Receptores CCR5/metabolismo , Receptores CXCR4/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Complementario , Humanos , Datos de Secuencia Molecular , Pan troglodytesRESUMEN
The structure-function of the CD4-class II MHC interaction was investigated. Two functional assays were used to assess the responses of the 3DT52.5.8 murine T cell hybridoma expressing human CD4 (h-CD4) or murine CD4 (m-CD4). First, we determined the responses of the CD4+ and CD4-effector cells toward DAP-3 cells co-expressing the cognate alloantigen H-2Dd together with several human (DRw52b, DR4-Dw4, DR2A, and DPw2) and murine (I-Ab, I-Ak, IA alpha b I-A beta k and I-Ek) class II alleles and isotypes. We found that h-CD4 and m-CD4 strongly enhance the T cell response to H-2Dd, demonstrating that interspecies CD4/class II interactions occur efficiently. Furthermore, mutations in h-CD4 at positions 19, 89, and 165 markedly reduced the interaction with both human class II and mouse class II, indicating that the structural features of this cross-species interaction are strongly conserved. This was further supported by the finding that a h-CD4 deletion mutant (deletion F43-S49) interacted with both human and murine class II. Moreover, as 3DT cells express the responsive V beta element for the bacterial superantigen staphylococcal enterotoxin B, a co-receptor assay was conducted. DAP-3 cells expressing only class II molecules were used as APCs to present staphylococcal enterotoxin B to h-CD4+ and m-CD4+ T cells. h-CD4 and m-CD4 were able to enhance the T cell response to staphylococcal enterotoxin B, further demonstrating the conservation of the CD4-class II MHC interaction.
Asunto(s)
Alelos , Antígenos CD4/genética , Antígenos CD4/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Mutación/inmunología , Animales , Enterotoxinas/metabolismo , Antígenos de Histocompatibilidad Clase II/clasificación , Antígenos de Histocompatibilidad Clase II/genética , Humanos , Ratones , Especificidad de la Especie , Staphylococcus aureus/inmunología , Relación Estructura-Actividad , Superantígenos/inmunologíaRESUMEN
CD4 is the coreceptor molecule expressed on the surface of T cells specific for or restricted by class II molecules of the major histocompatibility complex (MHC). Its expression on T cells is required for an optimal response to antigen (Ag). Three mechanisms have been invoked for the involvement of CD4 in T cell activation. First, it was shown that CD4 binds to MHC class II molecules on antigen presenting cells (APCs) thereby favoring an adhesion between effector cells and APCs. Association of CD4 to the T cell receptor and to the tyrosine kinase p56lck have also been shown to be critically involved in the positive function of CD4. Here, we demonstrate that the interaction of CD4 with p56lck is not required to enhance the response of two CD4-dependent, Ag-specific T cell hybridomas. Mutant forms of CD4 (TCD4), which lose association to p56lck, were expressed in these T cells and were shown to enhance the Ag-specific response as efficiently as the wild-type CD4. Moreover both CD4-dependent and independent T cell responses were inhibited by CD4-specific mAbs even when CD4 was not associated with p56lck. These results indicate that mechanisms distinct from sequestration of p56lck and/or negative signaling operate in these inhibitions. Results demonstrating enhancement of TCR-mediated signaling by the coaggregation of TCD4 mutant to the TCR further confirm that the association of p56lck to CD4 is not absolutely required for the regulatory functions of CD4. Our results suggest that the mechanisms implicated in the enhancement of T cell stimulation via CD4 depend solely on the extracellular and transmembrane domains of CD4.
Asunto(s)
Antígenos CD4/metabolismo , Activación de Linfocitos , Proteínas Tirosina Quinasas/metabolismo , Linfocitos T/inmunología , Animales , Anticuerpos Monoclonales , Citometría de Flujo , Antígenos de Histocompatibilidad Clase II/biosíntesis , Humanos , Hibridomas/inmunología , Insulina/farmacología , Interleucina-2/biosíntesis , Células L , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito , Ratones , Porcinos , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , TransfecciónRESUMEN
Insulin and pancreatic somatostatin secretions were studied after stimulation with an arginine infusion (5 mmol/l) in isolated perfused pancreata of adult streptozotocin-diabetic rats. In the presence of 2.8 mmol/l glucose, arginine clearly stimulated insulin and somatostatin secretions in diabetic rats, whereas it was ineffective in normal rats. Thus, not only the B-cells, but also the D-cells of the pancreas from streptozotocin-diabetic rats are hypersensitive to arginine. The infusion of insulin (4 U/l) did not modify this hypersensitivity of the D-cells to arginine in pancreata of streptozotocin-diabetic rats.
Asunto(s)
Arginina/administración & dosificación , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Somatostatina/metabolismo , Animales , Diabetes Mellitus Experimental/fisiopatología , Secreción de Insulina , Masculino , Ratas , Ratas EndogámicasRESUMEN
We have investigated restriction fragment length polymorphism (RFLP) of HLA and non-HLA regions of the genome in the homogeneous Danish population. Insulin-dependent diabetes mellitus (IDDM) patients and healthy individuals were selected for being HLA-DR 3/4 heterozygous to evaluate the influence of genes other than DR on disease susceptibility. Five different probes were used: HLA alpha and beta DQ (chromosome 6), the Ins 310 genomic fragment which detects a polymorphic region 5' to the insulin gene (chromosome 11), and cDNA for the constant regions of the T cell receptor alpha and beta genes (chromosomes 14 and 7). Fifteen cells homozygous for the HLA-D region were used to obtain reference DNA patterns. This allowed us to describe four splits among the HLA-DQw3 haplotypes (DQw3.1 to DQw3.4). The two new haplotypes DQw3.3 and DQw3.4 do not code for the TA10 serological marker which is found on DQw3.1 positive cells. One-hundred per cent of IDDM patients were typed as DQw3.2 versus 68 per cent for controls (p = 0.003). However, our results do not indicate a role for the Ins 310 or for the alpha DX locus region in IDDM susceptibility, in contrast to previous reports by others. The restriction enzymes that we have used did not reveal significant differences between DNA patterns of patients and controls with probes for the constant region of the T cell receptor genes.
Asunto(s)
Diabetes Mellitus Tipo 1/genética , Genes MHC Clase I , Antígenos HLA-DR/genética , Polimorfismo Genético , Polimorfismo de Longitud del Fragmento de Restricción , Biomarcadores , Sondas de ADN , Dinamarca , Diabetes Mellitus Tipo 1/sangre , Genotipo , Antígenos HLA-D , Antígenos HLA-DQ , Haplotipos , HumanosRESUMEN
In the genetically homogeneous Danish population, 27 HLA-DR3,4 heterozygous patients with insulin-dependent diabetes mellitus (IDDM) and 19 DR3,4 heterozygous controls without family history of IDDM were investigated for HLA-region markers and Gm and Km immunoglobulin allotypes. The aim was to define susceptibility factors for IDDM development other than HLA-DR using a number of techniques: lymphocytotoxicity (HLA-DR and DQ antigens), cellular methods (Dw and DP typing), restriction fragment length polymorphism (DQ alleles), electrophoresis and immunofixation (BF and C4 allotypes), and passive hemagglutination inhibition (Gm and Km immunoglobulin allotypes). The complement allotype C4A3 and the HLA-DQw8 (DQw3.2) antigen were found in all of the patients, whereas this was the case for only 8 of the 19 controls (P = 6 x 10(-6)): five lacked C4A3, five others lacked DQw8, and one of the controls lacked both of these factors. Fourteen of the patients had the complement allotype C4B3 versus three of the controls (P = 0.01). Previously reported family studies suggest that these alleles are part of the following haplotype: B15, BFS, C4A3, C4B3, DR4, Dw4, DQw8, and these factors were found together in ten of the patients versus one of the controls (P = 0.01). The markers usually associated with DR3 did not show significant differences between IDDM patients and controls, and the non-HLA markers studied showed no significant deviation from what was expected. In addition to the susceptibility factor DQw8, the study suggests the existence of susceptibility genes for IDDM near the complement C4 genes on DR4-carrying haplotypes.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Complemento C4/genética , Diabetes Mellitus Tipo 1/genética , Antígenos HLA-DQ/genética , Frecuencia de los Genes , Genes de Inmunoglobulinas , Genes MHC Clase II , Haplotipos , Heterocigoto , Humanos , Alotipos de Inmunoglobulinas/genéticaRESUMEN
This work was designed to study the effects of sodium 2-chloropropionate (2CP) alone or combined with insulin, in vitro, on glucagon secretion from pancreas isolated from rats, made diabetic by streptozotocin (66 mg/kg i.p.). The pancreata were perfused with a physiological solution containing 2.8 mM glucose (0.5 g/l) and glucagon secretion was stimulated by an arginine infusion (5 mM) for 30 min. When 2CP (1 mM) and/or insulin (4 IU/l) were applied, they were infused from the start of the organ perfusion. In the presence of glucose alone, a marked decrease in glucagon output was observed in diabetic rat pancreas. The arginine perfusion induced a biphasic glucagon secretion both in normal and diabetic rat pancreas; this response was however clearly reduced in diabetic rat pancreas. In diabetic rat pancreas, the infusion of either 2CP or insulin had no effect on glucagon output in presence of glucose alone, nor did it modify the response to arginine. In contrast, the combined infusion of insulin and 2CP induced different effects depending on the conditions: whereas in presence of glucose alone it restored a glucagon output close to that recorded in normal rat pancreas, it did not modify the response to arginine.
Asunto(s)
Diabetes Mellitus Experimental/metabolismo , Glucagón/metabolismo , Insulina/farmacología , Páncreas/metabolismo , Propionatos/farmacología , Animales , Arginina/farmacología , Combinación de Medicamentos , Glucosa/farmacología , Hidrocarburos Clorados , Ratas , Ratas EndogámicasRESUMEN
The effects of adenosine, adenosine triphosphate (ATP) and structural analogues have been studied on glucagon secretion from the isolated perfused pancreas of the rat in the presence of glucose (2.8 mM). Adenosine induced a transient increase of glucagon secretion. This effect was concentration-dependent in the range of 0.165 to 165 microM. ATP also induced an increase, but the effect was no greater at 165 microM than at 16.5 microM. 2-Chloroadenosine, an analogue more resistant to metabolism or uptake systems than adenosine, was more effective. Among the three structural analogues of ATP or ADP studied, beta, gamma-methylene ATP which can be hydrolyzed into AMP and adenosine had an effect similar to adenosine or ATP at the same concentrations (1.65 and 16.5 microM); in contrast alpha, beta-methylene ATP and alpha, beta-methylene ADP (resistant to hydrolysis into AMP and adenosine) were ineffective. Theophylline (50 microM) a specific blocker of the adenosine receptor, suppressed the glucagon peak induced by adenosine, 2-chloroadenosine, ATP and beta, gamma-methylene ATP (1.65 microM). An inhibitor of 5' nucleotidase, alpha, beta-methylene ADP (16.5 microM), reduced the glucagon increase induced by ATP and did not affect the response to adenosine (1.65 microM). These results support the hypothesis of adenosine receptors (P1-purinoceptors) on the pancreatic glucagon secretory cells and indicate that ATP acts after hydrolysis to adenosine.