RESUMEN
We performed a phase I trial to evaluate the safety and tolerability of repeated skin injections of IL-2-transfected autologous melanoma cells into patients with advanced disease. Cell suspensions, propagated from excised metastases, were IL-2 gene transfected by adenovirus-enhanced transferrinfection and X-irradiated prior to injection. Vaccine production was successful in 54% of the patients. Fifteen patients (37%) received two to eight skin vaccinations of either 3 x 10(6) (intradermal) or 1 x 10(7) (half intradermal, half subcutaneous) transfected melanoma cells per vaccination (secreting 140-17,060 biological response modifier program units of IL-2/10(6) cells/24 hr). Analyses of safety and efficacy were carried out in 15 and 14 patients, respectively. Overall, the vaccine was well tolerated. All patients displayed modest local reactions (erythema, induration, and pruritus) and some experienced flu-like symptoms. Apart from newly appearing (4 of 14) and increasing (5 of 14) anti-adenovirus and newly detectable anti-nuclear antibody titers (1 of 15), recipients developed de novo or exhibited increased melanoma cell-specific delayed-type hypersensitivity (DTH) reactions (8 of 15) and vitiligo (3 of 15) and showed signs of tumor regression (3 of 15). This supports the idea of a vaccine-induced or -amplified anti-cancer immune response. None of the patients exhibited complete or partial regressions, but five of them experienced periods of disease stabilization. Three of these individuals received more than the four planned vaccinations and their mean survival time was 15.7 +/- 3.5 months as compared to 7.8 +/- 4.6 months for the entire patient cohort. These data indicate that IL-2-producing, autologous cancer cells can be safely administered to stage IV melanoma patients and could conceivably be of benefit to patients with less advanced disease.
Asunto(s)
Vacunas contra el Cáncer/uso terapéutico , Melanoma/terapia , Neoplasias Cutáneas/terapia , Adulto , Anciano , Anticuerpos Antineoplásicos/biosíntesis , Anticuerpos Antineoplásicos/inmunología , Vacunas contra el Cáncer/administración & dosificación , Vacunas contra el Cáncer/efectos adversos , Femenino , Humanos , Hipersensibilidad Tardía , Inyecciones Intralesiones , Masculino , Melanoma/patología , Persona de Mediana Edad , Metástasis de la Neoplasia , Neoplasias Cutáneas/patología , Resultado del TratamientoRESUMEN
Primary human fibroblasts and a series of cell lines (A549, BNL CL.2, H225, NIH 3T3 and Rat-1) are efficiently transfected by using positively charged complexes of plasmid DNA and transferrin-polylysine or polylysine in the presence of glycerol (1 molar to 1.8 molar, depending on the cell type). An increase in gene expression of up to several-hundredfold (compared to complexes without glycerol) is obtained if the transfection mixture is incubated with the cells for 3-4 h at 37 degrees C. This simple method has been used for transient expression of luciferase, beta-galactosidase and interleukin-2, and also for the generation of stably transfected cells.
Asunto(s)
Glicerol/química , Ligandos , Macrólidos , Polilisina/química , Transfección/métodos , Animales , Antibacterianos/farmacología , Adhesión Celular/efectos de los fármacos , Línea Celular , Cloroquina/farmacología , Células Clonales/efectos de los fármacos , ADN/química , Inhibidores Enzimáticos/farmacología , Glicol de Etileno , Glicoles de Etileno/farmacología , Fibroblastos/citología , Regulación de la Expresión Génica , Genes Reporteros , Humanos , Interleucina-2/biosíntesis , Plásmidos , Regiones Promotoras Genéticas , beta-Galactosidasa/análisisAsunto(s)
Surfactantes Pulmonares/uso terapéutico , Síndrome de Dificultad Respiratoria del Recién Nacido/tratamiento farmacológico , Síndrome de Dificultad Respiratoria/tratamiento farmacológico , Adulto , Niño , Cuidados Críticos , Humanos , Recién Nacido , Síndrome de Dificultad Respiratoria/fisiopatología , Síndrome de Dificultad Respiratoria del Recién Nacido/fisiopatologíaRESUMEN
The nuclear autoantigen RA33, which is identical to the A2 protein of the heterogeneous nuclear ribonucleoprotein (hnRNP-A2), is a nucleic acid binding protein of 341 amino acids. The N-terminal part contains two RNA binding domains whereas the C-terminal part consists of a long glycine-rich region starting around amino acid 192. Autoantibodies to hnRNP-A2/RA33 can be detected in 20-40% of sera from RA, SLE and MCTD patients. So far, it has not been known which regions of A2/RA33 are recognized by these autoantibodies. To address this issue, tryptic fragments of natural A2/RA33 were investigated by immunoblotting using 14 sera from anti-RA33 positive patients with RA (n = 5), SLE (n = 5) and MCTD (n = 4). Most sera reacted with a 22 kD fragment comprising the N-terminal part of the protein. However, a smaller 18 kD fragment was recognized only by 3 RA and 3 MCTD sera whereas two of five SLE sera were reactive with two larger fragments of 26 and 29 kD. In order to further characterize the epitope(s) C-terminal deletion mutants of recombinant A2/RA33 were investigated by immunoblotting employing 27 sera from anti-RA33 positive patients with RA (n = 10), SLE (n = 8), and MCTD (n = 9). All sera recognized a fragment terminating at amino acid 212 which contained the complete N-terminal region as well as 20 amino acids of the glycine-rich section. Thus, these data indicate that the N-terminal part of A2/RA33 contains epitopes for antiA2/RA33 autoantibodies.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Autoanticuerpos/inmunología , Autoantígenos/inmunología , Enfermedades Autoinmunes/inmunología , Epítopos/análisis , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B , Proteínas Nucleares/inmunología , Ribonucleoproteínas/inmunología , Antígenos Nucleares , Eliminación de Gen , Ribonucleoproteínas Nucleares Heterogéneas , Humanos , Immunoblotting , Ribonucleoproteínas/genéticaRESUMEN
RA33 is a nuclear autoantigen with an apparent molecular mass of 33 kD. Autoantibodies against RA33 are found in about 30% of sera from RA patients, but only occasionally in sera from patients with other connective tissue diseases. To characterize RA33, the antigen was purified from HeLa cell nuclear extracts to more than 90% homogeneity by affinity chromatography on heparin-Sepharose and by chromatofocusing. Sequence analysis of five tryptic peptides revealed that their sequences matched corresponding sequences of the A2 protein of the heterogeneous nuclear ribonucleoprotein (hnRNP) complex. Furthermore, RA33 was shown to be present in the 40S hnRNP complex and to behave indistinguishably from A2 in binding to single stranded DNA. In summary, these data strongly indicate that RA33 and A2 are the same protein, and thus identify on a molecular level a new autoantigen.