Assuntos
Transplante de Células-Tronco Hematopoéticas/métodos , Imunotoxinas/uso terapêutico , Depleção Linfocítica/métodos , Transfusão de Linfócitos , Receptores de Interleucina-2/imunologia , Linfócitos T/transplante , Antígenos CD/imunologia , Infecções por Citomegalovirus/terapia , Humanos , Sensibilidade e Especificidade , Doadores de TecidosRESUMO
We have attempted to improve retrovirus-mediated gene transfer efficacy into hematopoietic progenitor cells (HPCs) without causing them to lose their lymphoid potential. Highly purified CD34(+) cells on CH-296 fibronectin fragments have been transduced with three different cytokine combinations. Murine CD2 was used as a marker gene. Transgene expression was assayed by FACS analysis shortly after transduction of CD34(+) cells and after long-term culture (LTC) extended by differentiation of various lymphoid lineages: NK cells, B cells, and dendritic cells. Compared with the historical cytokine mix, i.e., SCF (stem cell factor) + IL-3 (interleukin 3) + IL-6, the combination SCF + FL (Flt-3 ligand) + M-GDF (megakaryocyte growth and differentiation factor) + IL-3 significantly improved the total number of viable cells and CD34(+) cells after transduction and the long term-cultured progenitors after 6 weeks. In addition, the combination of SCF + FL + M-GDF + IL-3 maintained more efficiently the lymphoid potential of the progeny of transduced long term-cultured CD34(+) cells, as attested by the significantly higher number of CD56(+), CD19(+), and CD1a(+) cells recovered when FL and M-GDF were added to SCF + IL-3. Thus, even though additional improvements may still be needed in transduction of HPCs, these conditions were adopted for a clinical trial of gene therapy for X-linked severe combined immunodeficiency.
Assuntos
Técnicas de Transferência de Genes , Linfócitos/citologia , Retroviridae/genética , Células-Tronco/citologia , Animais , Antígenos CD1/metabolismo , Antígenos CD19/metabolismo , Antígenos CD34/metabolismo , Linfócitos B/metabolismo , Antígenos CD2/metabolismo , Antígeno CD56/metabolismo , Separação Celular , Citocinas/metabolismo , Células Dendríticas/metabolismo , Sangue Fetal/metabolismo , Fibronectinas/metabolismo , Citometria de Fluxo , Interleucina-3/metabolismo , Interleucina-6/metabolismo , Células Matadoras Naturais/metabolismo , Linfócitos/fisiologia , Proteínas de Membrana/metabolismo , Camundongos , Fenótipo , Fator de Células-Tronco/metabolismo , Células-Tronco/fisiologia , Trombopoetina/metabolismo , Fatores de Tempo , Transdução Genética , TransgenesRESUMO
Using a reporter plasmid containing the luciferase gene under the control of the insulin-like growth factor 1 (IGF-1) promoter region [including its 5' untranslated region (UTR)], we demonstrate that a 17-mer oligophosphorothioate containing C-5 propyne pyrimidines is able to inhibit luciferase gene expression in the nanomolar concentration range when the anti-sense oligonucleotide is targeted either to a coding sequence in the luciferase gene or to the 5' UTR of the gene for IGF-1. Inhibition was obtained independently of whether the plasmid and the anti-sense oligonucleotide were co-transfected or transfected separately into hepatocarcinoma cells. However, the efficiency of inhibition by the anti-sense oligonucleotides was 10-fold greater in the first case. The unmodified oligophosphorothioate targeted to the 5' UTR of IGF-1 did not inhibit luciferase gene expression at a 100-fold higher concentration unless its length was increased from 17 to 21 nt, in which case an inhibition of gene expression was obtained and an IC50 of 200 nM was observed.
Assuntos
Alcinos/metabolismo , Regulação da Expressão Gênica , Genes Reporter/genética , Oligonucleotídeos Antissenso/genética , Regiões 5' não Traduzidas/genética , Animais , Sequência de Bases , Cátions/metabolismo , Éxons/genética , Humanos , Concentração Inibidora 50 , Fator de Crescimento Insulin-Like I/genética , Metabolismo dos Lipídeos , Luciferases/genética , Peso Molecular , Ácidos Nucleicos Heteroduplexes/genética , Ácidos Nucleicos Heteroduplexes/metabolismo , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/metabolismo , Regiões Promotoras Genéticas/genética , Pirimidinas/metabolismo , Ratos , Homologia de Sequência do Ácido Nucleico , Temperatura , Tionucleotídeos/genética , Tionucleotídeos/metabolismo , Transfecção , Células Tumorais CultivadasRESUMO
A new technic using the principle of enzyme-linked immunoassay (ELISA) has been developed for the detection of complement components on red blood cells sensitized in vivo or in vitro. Using a double-antibody technic, anticomplement antisera (anti-C3c or anti-C3c/C3d) produced in rabbits was incubated with the red blood cells, followed by incubation with antirabbit alkaline phosphatase conjugated antiglobulin. The amount of the enzyme fixed was measured spectrophotometrically by the enzymatic hydrolysis of the substrate PNPP. A calibration curve was made from red blood cells on which complement was deposited by the method of Fruitstone . The technic showed a greater sensitivity than the standard antiglobulin tests and allowed simultaneous qualitative and semiquantitative estimates. The technic can be performed in any laboratory equipped with the standard equipment found in a blood bank, including a spectrophotometer. The authors made a modification of Alsever 's solution, which allowed the safe and stable preservation of complement coated red blood cells for 15 days. Significant positive results were obtained clinically using this technic, while negative or weakly positive reactions were obtained by the conventional antiglobulin tests.