RESUMO
Chronic granulomatous disease (CGD) is a primary immunodeficiency disorder which results from absence or malfunction of the respiratory burst oxidase normally expressed in neutrophils and other phagocytic leukocytes. Two-thirds of the patients are males hemizygous for mutations in the X-linked gene coding for gp91-phox. As a therapeutic approach towards the X-linked form of CGD bicistronic retroviral vectors containing the gp91-phox gene and a selectable marker gene were constructed. The ability of these vectors to restore NADPH oxidase activity was tested in a human myeloid leukemic cell line that is defective in superoxide production, as well as in primary CD34+ cells obtained from X-CGD patients. Under optimal conditions 80% of the CD34+ cells derived from bone marrow of one X-CGD patient were transduced. The level of superoxide production, in phagocytes derived from transduced cells was 68.9% of normal levels. Considering that low levels of superoxide generating activity are sufficient for normal host defense, the present experiments provide the basis for the development of a gene replacement therapy for the X-linked form of CGD.
Assuntos
Terapia Genética/métodos , Doença Granulomatosa Crônica/terapia , Animais , Antígenos CD34/metabolismo , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Células da Medula Óssea/virologia , Transplante de Medula Óssea , Linhagem Celular , DNA Complementar/genética , Expressão Gênica , Vetores Genéticos , Doença Granulomatosa Crônica/genética , Doença Granulomatosa Crônica/metabolismo , Humanos , Masculino , Glicoproteínas de Membrana/genética , Camundongos , NADPH Oxidase 2 , NADPH Oxidases/genética , Explosão Respiratória , Retroviridae/genética , Superóxidos/metabolismo , Transdução Genética , Cromossomo X/genéticaRESUMO
Stable gene transfer to human pluripotent hematopoietic stem cells (PHSCs) is an attractive strategy for the curative treatment of many genetic hematologic disorders. In clinical trials, the levels of gene transfer to this cell population have generally been low, reflecting deficiencies in both the vector systems and transduction conditions. In this study, we have used a pseudotyped murine retroviral vector to transduce human CD34(+) cells purified from bone marrow (BM) and umbilical cord blood (CB) under optimized conditions. After transduction, 71% to 97% of the hematopoietic cells were found to express a low-affinity nerve growth factor receptor (LNGFR) marker gene. Six weeks after transplantation into immunodeficient NOD/LtSz-scid/scid (NOD/SCID) mice, LNGFR expression was detected in 6% to 57% of CD45(+) cells in eight of nine engrafted animals. Moreover, proviral DNA was detected in 8.3% to 45% of secondary colonies derived from BM cells of engrafted NOD/SCID mice. Our data show consistent transduction of SCID-repopulating cells (SRCs) and suggest that the efficiency of gene transfer to human hematopoietic repopulating cells can be improved using existing retroviral vector systems and carefully optimized transduction conditions.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Transfecção , Animais , Transplante de Medula Óssea , Células Cultivadas/transplante , Ensaio de Unidades Formadoras de Colônias , Meios de Cultura Livres de Soro , Sangue Fetal/citologia , Genes Reporter , Vetores Genéticos/genética , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Receptor de Fator de Crescimento Neural , Receptores de Fator de Crescimento Neural/biossíntese , Receptores de Fator de Crescimento Neural/genética , Proteínas Recombinantes de Fusão/biossíntese , Retroviridae/genéticaRESUMO
In patients with habitual dislocation of the shoulder and post-fracture malrotation the measurement of humeral torsion is important in preoperative diagnostics. An easy method of ultrasonic measurement of humeral torsion is proposed. The correlation with anthropometric, radiological and CT-measurement is good, what could be shown in 20 macerated humeri. In 111 patients with normal glenohumeral joints the average of humeral retrotorsion was 61 degrees. 86% had a torsion between 40 and 80 degrees.
