RESUMEN
We investigated the effects of hyperosmolality on survival and proliferation of subconfluent cultures of mIMCD3 mouse renal collecting duct cells. High NaCl and/or urea (but not glycerol) reduces the number of viable cells, as measured with 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). Raising osmolality from a normal level (300 mosmol/kg) to 550-1,000 mosmol/kg by adding NaCl and/or urea greatly increases the proportion of cells in the G(2)M phase of the cell cycle within 8 h, as measured by flow cytometry. Up to 600 mosmol/kg the effect is only transient, and by 12 h at 550 mosmol/kg the effect reverses and most cells are in G(1). Flow cytometry with 5-bromodeoxyuridine (BrdU) pulse-chase demonstrates that movement through the S phase of the cell cycle slows, depending on the concentrations of NaCl and/or urea, and that the duration of G(2)M increases greatly (from 2.5 h at 300 mosmol/kg to more than 16 h at the higher osmolalities). Addition of NaCl and/or urea to total osmolality of 550 mosmol/kg or more also induces apoptosis, as demonstrated by characteristic electron microscopic morphological changes, appearance of a subdiploid peak in flow cytometry, and caspase-3 activation. The number of cells with subdiploid DNA and activated caspase-3 peaks at 8-12 h. Caspase-3 activation occurs in all phases of the cell cycle, but to a disproportionate degree in G(0)/G(1) and S phases. We conclude that elevated NaCl and/or urea reduces the number of proliferating mIMCD3 cells by slowing the transit through the S phase, by cell cycle delay in the G(2)M and G(1), and by inducing apoptotic cell death.
Asunto(s)
Apoptosis/efectos de los fármacos , Fase G2/efectos de los fármacos , Túbulos Renales Colectores/efectos de los fármacos , Cloruro de Sodio/farmacología , Urea/farmacología , Animales , Recuento de Células/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Células Cultivadas , Humanos , Médula Renal , Túbulos Renales Colectores/citología , RatonesRESUMEN
BACKGROUND: Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematopoietic stem-cell disorder in which the affected cells are deficient in glycosylphosphatidylinositol (GPI)-anchored proteins. Paroxysmal nocturnal hemoglobinuria is frequently associated with aplastic anemia, although the basis of this relation is unknown. OBJECTIVE: To assess the PNH status of patients with diverse marrow failure syndromes. DESIGN: Correlation of cytofluorometric data with clinical features. SETTING: Hematology Branch, National Heart, Lung, and Blood Institute, Bethesda, Maryland. PATIENTS: 115 patients with aplastic anemia, 39 patients with myelodysplasia, 28 patients who had recently undergone bone marrow transplantation, 18 patients with cancer that was treated with chemotherapy, 13 patients with large granular lymphocytosis, 20 controls who had received renal allografts, and 21 healthy participants. INTERVENTION: Patients with aplastic anemia, myelodysplasia, or renal allografts received antithymocyte globulin. MEASUREMENTS: Flow cytometry was used to assess expression of GPI-anchored proteins on granulocytes. RESULTS: Evidence of PNH was found in 25 of 115 (22%) patients with aplastic anemia. No patient with normal GPI-anchored protein expression at presentation developed PNH after therapy (n = 16). Nine of 39 (23%) patients with myelodysplasia had GPI-anchored protein-deficient cells. Abnormal cells were not detected in patients with constitutional or other forms of bone marrow failure or in renal allograft recipients who had received antithymocyte globulin. Aplastic anemia is known to respond to immunosuppressive therapy; in myelodysplasia, the presence of a PNH population was strongly correlated with hematologic improvement after administration of antithymocyte globulin (P = 0.0015). CONCLUSIONS: Flow cytometric analysis is superior to the Ham test and permits concomitant diagnosis of PNH in about 20% of patients with myelodysplasia (a rate similar to that seen in patients with aplastic anemia). The presence of GPI-anchored protein-deficient cells in myelodysplasia predicts responsiveness to immunosuppressive therapy. Early emergence of GPI-anchored protein-deficient hematopoiesis in a patient with marrow failure may point to an underlying immune pathogenesis.
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Enfermedades de la Médula Ósea/complicaciones , Hemoglobinuria Paroxística/complicaciones , Suero Antilinfocítico/efectos adversos , Suero Antilinfocítico/uso terapéutico , Enfermedades de la Médula Ósea/sangre , Enfermedades de la Médula Ósea/inmunología , Distribución de Chi-Cuadrado , Ritmo Circadiano , Citometría de Flujo , Glicosilfosfatidilinositoles/deficiencia , Granulocitos/metabolismo , Hemoglobinuria Paroxística/sangre , Hemoglobinuria Paroxística/diagnóstico , Hemoglobinuria Paroxística/tratamiento farmacológico , Humanos , Inmunosupresores/efectos adversos , Inmunosupresores/uso terapéutico , FenotipoRESUMEN
Simian immunodeficiency virus (SIV) infection of nonhuman primates is one of the most relevant animals models of HIV infection in humans. To test a potential anti-HIV gene therapy strategy in this model, CD4-enriched lymphocytes from three rhesus macaques were subjected to retrovirally mediated gene transfer with a vector expressing an antisense tat/rev gene. This group of animals and three control macaques were subsequently infected with SIVmac239. Blood and lymph nodes from all macaques were sampled for more than a year to monitor the progress of infection. Although all animals became infected, the animals that received the lymphocytes engineered with the antisense vector demonstrated a significant reduction in viral load in both peripheral blood and lymph nodes, had sustained numbers of CD4+ cells, and exhibited little disruption of lymph node architecture.
Asunto(s)
Linfocitos T CD4-Positivos/virología , Vectores Genéticos/farmacología , Macaca mulatta/virología , Virus de la Inmunodeficiencia de los Simios/genética , Replicación Viral/efectos de los fármacos , Animales , Antivirales/farmacología , Linfocitos T CD4-Positivos/efectos de los fármacos , Productos del Gen rev , Productos del Gen tat , Técnicas de Transferencia de Gen , Ganglios Linfáticos/virología , Oligonucleótidos Antisentido/genética , Síndrome de Inmunodeficiencia Adquirida del Simio/genética , Síndrome de Inmunodeficiencia Adquirida del Simio/terapia , Replicación Viral/genéticaRESUMEN
In an attempt to improve our gene transfer efficiency into hematopoietic stem cells and to evaluate the capacity of immunoselected CD34+Thy-1+(CDw90) cells to reconstitute hematopoiesis following myeloablation, bone marrow (BM) transplantation was performed using autologous, immunoselected CD34+Thy-1+ cells in rhesus macaques. BM samples were positively selected for cells that express CD34, further subdivided using high gradient immunomagnetic selection for cells that express Thy-1, and transduced using a 7-day supernatant transduction protocol with a replication-defective retroviral vector that contained the human glucocerebrosidase (GC) gene. Circulating leukocytes were evaluated using a semiquantitative polymerase chain reaction (PCR) assay for the human GC gene, with the longest surviving animal evaluated at day 558. Provirus was detected at all time points in both CD20+ B cells and CD2+ dim T cells, but long-term gene transfer was not observed in the granulocyte population. The CD2+ dim population was phenotypically identified as being CD8+ natural killer cells. By day 302 and day 330, both the CD2+ bright and dim cell populations and sorted CD4+ and CD8+ cells had detectable provirus. Vector-derived GC mRNA was detected by reverse transcriptase (RT)-PCR analysis as far out as day 588. Thus, CD34+Thy-1+ cells isolated using high gradient magnetic separation techniques can engraft, be transduced with a replication-defective retroviral vector, and contribute to CD20+ B lymphocytes, CD8+ T lymphocytes, and CD4+ T lymphocytes; making them a suitable cell population to target for gene therapies involving lymphocytes.
Asunto(s)
Células de la Médula Ósea , Técnicas de Transferencia de Gen , Glucosilceramidasa/genética , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/metabolismo , Antígenos Thy-1/análisis , Adenovirus Humanos/genética , Adenovirus Humanos/aislamiento & purificación , Animales , Antígenos CD34/análisis , Antígenos CD2/análisis , Antígenos CD8/análisis , ADN Recombinante/análisis , Virus Defectuosos/genética , Virus Defectuosos/aislamiento & purificación , Vectores Genéticos/genética , Vectores Genéticos/aislamiento & purificación , Supervivencia de Injerto , Humanos , Separación Inmunomagnética , Subgrupos Linfocitarios/química , Subgrupos Linfocitarios/inmunología , Subgrupos Linfocitarios/virología , Macaca mulatta , Reacción en Cadena de la Polimerasa , Provirus/genética , Provirus/aislamiento & purificación , Trasplante AutólogoRESUMEN
Granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF) have been shown to stimulate the circulation of hematopoietic progenitor cells in both mice and nonhuman primates. We evaluated the immunophenotype and cell cycle status of CD34+ cells isolated from the bone marrow (BM) and leukapheresis product of cytokine-mobilized nonhuman primates. CD34+ cells were isolated from rhesus macaques that had received no cytokine therapy, 100 micrograms/kg/d G-CSF, 200 micrograms/kg/d SCF, or a combination of both 100 micrograms/kg/d G-CSF and 200 micrograms/kg/d SCF as a subcutaneous injection for 5 days. BM was aspirated before (day 0) and on the last day (day 5) of cytokine administration. On days 4 and 5, peripheral blood (PB) mononuclear cells were collected using a novel method of leukapheresis. Threefold more PB mononuclear cells were collected from animals receiving G-CSF alone or G-CSF and SCF than from animals that had received either SCF alone or no cytokine therapy. CD34+ cells were positively selected using an immunoadsorptive system from the BM, PB, and/or leukapheresis product. Threefold and 10-fold more CD34+ cells were isolated from the leukapheresis product of animals receiving G-CSF or G-CSF and SCF, respectively, than from animals receiving no cytokine therapy or SCF alone. The isolated CD34+ cells were immunophenotyped using CD34-allophycocyanin, CD38-fluorescein isothiocyanate, and Thy-1-phycoerythrin. These cells were later stained with 4', 6-diamidino-2-phenylindole for simultaneous DNA analysis and immunophenotyping. BM-derived CD34+ cells did not differ significantly in cell cycle status and Thy-1 or CD38 phenotype before or after G-CSF and/or SCF administration. Similarly, CD34+ cells isolated from the leukapheresis product did not differ significantly in immunophenotype or cell cycle status before or after G-CSF and/or SCF administration. However, there were consistent differences in both immunophenotype and cell cycle status between BM- and PB-derived CD34+ cells. CD34+ cells isolated from the PB consistently had a smaller percentage of cells in the S+G2/M phase of the cell cycle and had a higher percentage of cells expressing Thy-1 than did CD34+ cells isolated from the BM. A greater proportion of PB-derived CD34+ cells were in the S+G2/M phase of the cell cycle after culture in media supplemented with interleukin-6 and SCF, However, culturing decreased the proportion of CD34+ cells expressing Thy-1.
Asunto(s)
Antígenos CD , Células Sanguíneas/inmunología , Médula Ósea/inmunología , Antígenos Thy-1/metabolismo , ADP-Ribosil Ciclasa , ADP-Ribosil Ciclasa 1 , Animales , Antígenos CD34/análisis , Antígenos de Diferenciación/análisis , Células Sanguíneas/citología , Células de la Médula Ósea , Ciclo Celular , Separación Celular , Células Cultivadas , Citometría de Flujo , Factor Estimulante de Colonias de Granulocitos/farmacología , Inmunofenotipificación , Leucaféresis , Recuento de Leucocitos , Subgrupos Linfocitarios , Macaca mulatta , N-Glicosil Hidrolasas/análisis , Factor de Células Madre/farmacologíaAsunto(s)
Antígenos CD/análisis , Células de la Médula Ósea , Médula Ósea/inmunología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/inmunología , Antígenos CD34 , Biopsia con Aguja , Ciclo Celular , Citometría de Flujo/métodos , Fluoresceína-5-Isotiocianato , Antígenos HLA-DR/análisis , Humanos , Valores de ReferenciaRESUMEN
In pulmonary sarcoidosis, the marked expansion of CD4+ (helper/inducer) T cells in the alveolar structures of the lung is maintained by local IL-2 release by activated CD4+ HLA-DR+ T cells without concomitant expansion and activation of CD8+ (suppressor/cytotoxic) T cells, suggesting that sarcoid may be associated with a generalized abnormality of CD8+ T cells. Consistent with this concept, evaluation of the expression of the IL-2R on fresh lung T cells from individuals with active sarcoidosis demonstrated that 7 +/- 1% of sarcoid lung CD4+ T cells are spontaneously expressing the IL-2R compared with only 1 +/- 1% lung CD8+ T cells (p less than 0.01). However, stimulation of purified sarcoid blood CD8+ T cells with the anti-T3/TCR complex mAb OKT3 was followed by the normal expression of IL-2R (p greater than 0.1) and proliferation (p greater than 0.1). In addition, lung sarcoid CD8+ T cells responded to OKT3 similarly to normal lung CD8+ T cells and to autologous blood CD8+ T cells as regards expression of IL-2R (p greater than 0.1) and proliferation (p greater than 0.1). Finally, using CD4+ cells activated with allogenic Ag to induce, in coculture, fresh autologous CD8+ cells to suppress proliferation of fresh autologous CD4+ cells to the same Ag, sarcoid CD8+ T cells suppressed CD4+ cell proliferation in a normal fashion (p greater than 0.1). These results demonstrate that sarcoid CD8+ (suppressor/cytotoxic) T cells are competent to respond to a proliferation signal normally and can be induced to normally suppress CD4+ T cell proliferation to Ag, suggesting that the expansion of activated CD4+ T cells in pulmonary sarcoidosis is not due to a generalized abnormality of CD8+ T cells or of their suppressor T cell function.