RESUMEN
Extracorporeal photopheresis (ECP) is an established therapy for the treatment of graft-versus-host-disease (GVHD) following an allogeneic stem cell transplant. We performed a prospective analysis of patients receiving ECP treatment for GVHD to identify a clinical pathway and resource utilization of this process. The cohort included consecutive allogeneic stem cell recipients with GVHD. ECP was performed using the CELLEX Photopheresis System or the UVAR XTS Photopheresis System (Therakos, Inc, Exton, PA). A clinical pathway was developed and a time and motion study was conducted to define the resource utilization and costs associated with ECP. Patients were treated with either CELLEX (n = 18 procedures) or UVAR (n = 4 procedures). Total time commitment for each procedure for the 2 machines differed. The time for ECP was 117 min (median, range: 91-164 min) using CELLEX and 161 min (median; range: 140-210) using the UVAR-XTS machine. Total costs of each ECP procedure were $3420.50. There is a considerable time commitment of the patient and the clinical staff when employing ECP to treat GVHD. ECP costs are significant considering this is a prolonged therapy continued for several months. With this finalized pathway and costs, we have a standardized clinical pathway for the treatment of GVHD. We are addressing minimizing resource utilization while emphasizing quality care for these patients.
Asunto(s)
Vías Clínicas/normas , Enfermedad Injerto contra Huésped/terapia , Fotoféresis/métodos , Aloinjertos , Vías Clínicas/economía , Humanos , Fotoféresis/economía , Fotoféresis/instrumentación , Trasplante de Células Madre/efectos adversosRESUMEN
BACKGROUND: The NKG2D receptor, one of the natural killer (NK) cell-activating receptors, is expressed on the surface of CD3+CD8+ T cells, γδ+ T cells, NK cells, NKT cells, and a few CD4+ T cells. We show, for the first time, a critical role for the NKG2D receptor on CD3+CD8+ T cells isolated from myeloma patients, in identifying and killing autologous myeloma cells isolated from the same patients' marrow. We also show that blocking NKG2D using anti-NKG2D reverses the cytotoxicity while blocking HLA-I using antibodies does not have the same effect, showing that the autologous cytotoxicity is NKG2D dependent and major histocompatibility complex (MHC)-I independent. We further confirmed the NKG2D specificity by small interfering RNA (siRNA) down regulation of NKG2D receptor. STUDY DESIGN AND METHODS: Using ex vivo expansion methods that enrich for NKG2D+CD3+CD8+ T cells, we investigated whether these ex vivo expanded NKG2D+CD3+CD8+ T cells would recognize and lyse autologous and allogeneic myeloma cells, independent of T-cell receptor or MHC-I expression. RESULTS: Myeloma cell lysis by the NKG2D+CD3+CD8+ T cells correlated with the amount of NKG2D ligand expression. With receptor-ligand interaction, interferon-γ and tumor necrosis factor-α were released. Blocking the NKG2D receptor by using either monoclonal antibodies or siRNAs inhibited the receptor's function and prevented myeloma cell lysis. CONCLUSION: Clinical trials are ongoing to determine a correlation with the number and function of NKG2D+CD3+CD8+ T cells and clinical outcomes in transplanted myeloma patients, including lymphocyte recovery following transplant and overall survival.
Asunto(s)
Complejo CD3/metabolismo , Linfocitos T CD8-positivos/metabolismo , Subfamilia K de Receptores Similares a Lectina de Células NK/metabolismo , Línea Celular Tumoral , Quimiocina CCL5/metabolismo , Citocinas/metabolismo , Humanos , Interleucina-2/metabolismo , Interleucina-4/metabolismo , Interleucina-5/metabolismo , Interleucina-7/metabolismo , ARN Interferente Pequeño , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The number of circulating lymphocytes on day 15 after transplantation correlates with improved survival in patients with myeloma, but the lymphocyte subset responsible is unknown. NKG2D is a natural killer (NK) cell activating receptor that mediates non-MHC restricted and TCR-independent cell lysis. Our preliminary results indicate that CD3(+)CD8(+) T cells expressing NKG2D may be a critical lymphocyte population. A phase II trial examined the feasibility of infusing ex vivo-expanded cells enriched for NKG2D(+)CD3(+)CD8(+) T cells at weeks 1, 2, 4, and 8 after an autologous transplantation. In addition, low-dose IL-2 (6 × 10(5) IU/m(2)/day) was administered for 4 weeks, beginning on the day of transplantation. Twenty-three patients were accrued and 19 patients are evaluable. There were no treatment-related deaths. All patients completed their course of IL-2 and demonstrated normal engraftment. When compared with patients with myeloma who underwent transplantation not receiving posttransplantation immune therapy, the treated patients demonstrated an increase in the number of circulating NKG2D(+)CD3(+)CD8(+) T cells/µL (P < .004), CD3(+)CD8(+) T cells/µL (P < .04), CD3(+)CD8(+)CD56(+) T cells/µL (P < .004), and NKG2D(+)CD3(-)CD56(+) T cells/µL (P < .003). Myeloma cell-directed cytotoxicity by the circulating mononuclear cells increased after transplantation (P < .002). When compared to posttransplantation IL-2 therapy alone in this patient population, the addition of cells enriched for NKG2D(+)CD3(+)CD8(+) T cells increased tumor-specific immunity, as demonstrated by enhanced lysis of autologous myeloma cells (P = .02). We postulate that this regimen that increased the number and function of the NKG2D(+)CD3(+)CD8(+) T cells after transplantation may improve clinical outcomes by eliminating residual malignant cells in vivo.
Asunto(s)
Traslado Adoptivo , Complejo CD3 , Linfocitos T CD8-positivos/trasplante , Mieloma Múltiple/terapia , Subfamilia K de Receptores Similares a Lectina de Células NK , Trasplante de Células Madre de Sangre Periférica , Adolescente , Adulto , Anciano , Linfocitos T CD8-positivos/inmunología , Femenino , Humanos , Interleucina-2/administración & dosificación , Transfusión de Linfocitos , Masculino , Persona de Mediana Edad , Mieloma Múltiple/inmunología , Mieloma Múltiple/mortalidad , Trasplante AutólogoRESUMEN
BACKGROUND AIMS: A phase I trial examined the ability of immunotherapy to mobilize progenitor and activated T cells. METHODS: Interleukin (IL)-2 was administered subcutaneously for 11 days, with granulocyte (G)-colony-stimulating factor (CSF) (5 mcg/kg/day) and granulocyte-macrophage (GM)-CSF (7.5 mcg/kg/day) added for the last 5 days. Leukapheresis was initiated on day 11. Thirteen patients were treated (myeloma n = 11, non-Hodgkin's lymphoma n = 2). RESULTS: Toxicities were minimal. IL-2 was stopped in two patients because of capillary leak (n = 1) and diarrhea (n = 1). Each patient required 2.5 leukaphereses (median; range 1-3) to collect 3.2 x 106 CD34+ cells/kg (median; range 1.9-6.6 x 106/kg). Immune mobilization increased the number of CD3+ CD8+ T cells (P = 0.002), CD56+ natural killer (NK) cells (P = 0.0001), CD8+ CD56+ T cells (P = 0.002) and CD4+ CD25+ cells (P = 0.0001) compared with cancer patients mobilized with G-CSF alone. There was increased lysis of myeloma cells after 7 days (P = 0.03) or 11 days (P = 0.02). The maximum tolerated dose of IL-2 was 1 x 106 IU/m²/day. CONCLUSIONS: Immune mobilization is well tolerated with normal subsequent marrow engraftment. As cells within the graft influence lymphocyte recovery, an increased number of functional lymphocytes may result in more rapid immune reconstitution.
Asunto(s)
Neoplasias Hematológicas/terapia , Movilización de Célula Madre Hematopoyética , Trasplante de Células Madre Hematopoyéticas , Subgrupos de Linfocitos T/metabolismo , Linfocitos T/metabolismo , Adolescente , Adulto , Anciano , Síndrome de Fuga Capilar/etiología , Femenino , Factor Estimulante de Colonias de Granulocitos/administración & dosificación , Factor Estimulante de Colonias de Granulocitos y Macrófagos/administración & dosificación , Neoplasias Hematológicas/patología , Neoplasias Hematológicas/fisiopatología , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Humanos , Interleucina-2/administración & dosificación , Leucaféresis , Activación de Linfocitos , Masculino , Dosis Máxima Tolerada , Persona de Mediana Edad , Subgrupos de Linfocitos T/efectos de los fármacos , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/patología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/patología , Trasplante AutólogoRESUMEN
BACKGROUND: P. aeruginosa chronically colonizes the lung in CF patients and elicits a proinflammatory response. Excessive secretion of IL-6 and IL-8 by CF airway cells in response to P. aeruginosa infection in the CF airway is though to contribute to lung injury. Accordingly, the goal of this study was to test the hypothesis that Corr4a and VRT325, investigational compounds that increase DeltaF508-CFTR mediated Cl(-) secretion in human CF airway cells, reduce the pro-inflammatory response to P. aeruginosa. METHODS: IL-6 and IL-8 secretion by polarized CF human airway epithelial cells (CFBE41o-) were measured by multiplex analysis, and DeltaF508-CFTR Cl- secretion was measured in Ussing chambers. Airway cells were exposed to P. aeruginosa (PAO1 or PA14) and Corr4a or VRT325. RESULTS: Corr4a and VRT325 increased DeltaF508-CFTR Cl(-) secretion but did not reduce either constitutive IL-6 or IL-8 secretion, or IL-6 and IL-8 secretion stimulated by P. aeruginosa (PA14 or PAO1). CONCLUSIONS: Corr4a and VRT325 do not reduce the inflammatory response to P. aeruginosa in human cystic fibrosis airway epithelial cells.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/uso terapéutico , Fibrosis Quística/tratamiento farmacológico , Células Epiteliales/microbiología , Piperazinas/uso terapéutico , Infecciones por Pseudomonas/tratamiento farmacológico , Infecciones por Pseudomonas/inmunología , Quinazolinas/uso terapéutico , Línea Celular , Fibrosis Quística/patología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Piperazinas/farmacología , Quinazolinas/farmacologíaRESUMEN
Runx1 is expressed in skeletal elements, but its role in fracture repair has not been analyzed. We created mice with a hypomorphic Runx1 allele (Runx1(L148A) ) and generated Runx1(L148A/-) mice in which >50% of Runx1 activity was abrogated. Runx1(L148A/-) mice were viable but runted. Their growth plates had extended proliferating and hypertrophic zones, and the percentages of Sox9-, Runx2-, and Runx3-positive cells were decreased. Femoral fracture experiments revealed delayed cartilaginous callus formation, and the expression of chondrogenic markers was decreased. Conditional ablation of Runx1 in the mesenchymal progenitor cells of the limb with Prx1-Cre conferred no obvious limb phenotype; however, cartilaginous callus formation was delayed following fracture. Embryonic limb bud-derived mesenchymal cells showed delayed chondrogenesis when the Runx1 allele was deleted ex vivo with adenoviral-expressed Cre. Collectively, our data suggest that Runx1 is required for commitment and differentiation of chondroprogenitor cells into the chondrogenic lineage.
Asunto(s)
Huesos/metabolismo , Condrocitos/citología , Subunidad alfa 2 del Factor de Unión al Sitio Principal/fisiología , Alelos , Animales , Cartílago/citología , Cartílago/metabolismo , Relación Dosis-Respuesta a Droga , Exones , Femenino , Curación de Fractura , Mesodermo/citología , Ratones , Ratones Transgénicos , Mutación , Osteocalcina/biosíntesis , Fenotipo , Regiones Promotoras Genéticas , Células Madre/citologíaRESUMEN
The immune system plays a critical role determining the outcomes in transplanted multiple myeloma patients, since enhanced lymphocyte recovery results in improved survival. Since mobilization regimens influence the cellular subsets collected and infused for transplant, these regimens may determine immune recovery following transplant. We hypothesized that a mobilized stem cell product harboring an increased number of lymphocytes would enhance immune recovery following autologous stem cell infusion, increase lymphocyte recovery, and improve clinical outcomes. We designed a phase I immune mobilization trial using IL-2 and growth factors to increase the number of lymphocytes within the stem cell product. This regimen efficiently mobilized CD34+ progenitor cells (median: 3.6 x 10(6) cells/kg; range 1.9-6.6 x 10(6) cells/kg) and improved the immune properties of the mobilized stem cells, including an increase in CD8+ T cells expressing an NK activating receptor called NKG2D (P less than 0.004), cells that are extremely potent at killing myeloma cells using non-MHC-I restricted and TCR-independent mechanisms. Novel mobilization techniques can improve the mobilized graft and may improve clinical outcomes in myeloma patients.
Asunto(s)
Linfocitos/inmunología , Mieloma Múltiple/inmunología , Línea Celular Tumoral , Citometría de Flujo , HumanosRESUMEN
Cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl(-) secretion across fluid-transporting epithelia is regulated, in part, by modulating the number of CFTR Cl(-) channels in the plasma membrane by adjusting CFTR endocytosis and recycling. However, the mechanisms that regulate CFTR recycling in airway epithelial cells remain unknown, at least in part, because the recycling itineraries of CFTR in these cells are incompletely understood. In a previous study, we demonstrated that CFTR undergoes trafficking in Rab11a-specific apical recycling endosomes in human airway epithelial cells. Myosin Vb is a plus-end-directed, actin-based mechanoenzyme that facilitates protein trafficking in Rab11a-specific recycling vesicles in several cell model systems. There are no published studies examining the role of myosin Vb in airway epithelial cells. Thus, the goal of this study was to determine whether myosin Vb facilitates CFTR recycling in polarized human airway epithelial cells. Endogenous CFTR formed a complex with endogenous myosin Vb and Rab11a. Silencing myosin Vb by RNA-mediated interference decreased the expression of wild-type CFTR and DeltaF508-CFTR in the apical membrane and decreased CFTR-mediated Cl(-) secretion across polarized human airway epithelial cells. A recombinant tail domain fragment of myosin Vb attenuated the plasma membrane expression of CFTR by arresting CFTR recycling. The dominant-negative effect was dependent on the ability of the myosin Vb tail fragment to interact with Rab11a. Taken together, these data indicate that myosin Vb is required for CFTR recycling in Rab11a-specific apical recycling endosomes in polarized human airway epithelial cells.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Endosomas/metabolismo , Células Epiteliales/citología , Regulación de la Expresión Génica , Cadenas Pesadas de Miosina/fisiología , Miosina Tipo V/fisiología , Proteínas de Unión al GTP rab/metabolismo , Secuencia de Aminoácidos , Línea Celular , Endocitosis , Silenciador del Gen , Humanos , Modelos Biológicos , Datos de Secuencia Molecular , Cadenas Pesadas de Miosina/química , Miosina Tipo V/química , Interferencia de ARN , TransfecciónRESUMEN
The family of core-binding factors includes the DNA-binding subunits Runx1-3 and their common non-DNA-binding partner CBFbeta. We examined the collective role of core-binding factors in hematopoiesis with a hypomorphic Cbfb allelic series. Reducing CBFbeta levels by 3- or 6-fold caused abnormalities in bone development, megakaryocytes, granulocytes, and T cells. T-cell development was very sensitive to an incremental reduction of CBFbeta levels: mature thymocytes were decreased in number upon a 3-fold reduction in CBFbeta levels, and were virtually absent when CBFbeta levels were 6-fold lower. Partially penetrant consecutive differentiation blocks were found among early T-lineage progenitors within the CD4- CD8- double-negative 1 and downstream double-negative 2 thymocyte subsets. Our data define a critical CBFbeta threshold for normal T-cell development, and situate an essential role for core-binding factors during the earliest stages of T-cell development.
Asunto(s)
Desarrollo Óseo/fisiología , Subunidad beta del Factor de Unión al Sitio Principal/fisiología , Granulocitos/patología , Hematopoyesis/fisiología , Megacariocitos/patología , Subgrupos de Linfocitos T/patología , Alelos , Animales , Linaje de la Célula , Ensayo de Unidades Formadoras de Colonias , Subunidades alfa del Factor de Unión al Sitio Principal/fisiología , Subunidad beta del Factor de Unión al Sitio Principal/deficiencia , Subunidad beta del Factor de Unión al Sitio Principal/genética , Hígado/embriología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Quimera por Radiación , Bazo/embriología , Timo/embriologíaRESUMEN
PDZ domains are ubiquitous peptide-binding modules that mediate protein-protein interactions in a wide variety of intracellular trafficking and localization processes. These include the pathways that regulate the membrane trafficking and endocytic recycling of the cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial chloride channel mutated in patients with cystic fibrosis. Correspondingly, a number of PDZ proteins have now been identified that directly or indirectly interact with the C terminus of CFTR. One of these is CAL, whose overexpression in heterologous cells directs the lysosomal degradation of WT-CFTR in a dose-dependent fashion and reduces the amount of CFTR found at the cell surface. Here, we show that RNA interference targeting endogenous CAL specifically increases cell-surface expression of the disease-associated DeltaF508-CFTR mutant and thus enhances transepithelial chloride currents in a polarized human patient bronchial epithelial cell line. We have reconstituted the CAL-CFTR interaction in vitro from purified components, demonstrating for the first time that the binding is direct and allowing us to characterize its components biochemically and biophysically. To test the hypothesis that inhibition of the binding site could also reverse CAL-mediated suppression of CFTR, a three-dimensional homology model of the CAL.CFTR complex was constructed and used to generate a CAL mutant whose binding pocket is correctly folded but has lost its ability to bind CFTR. Although produced at the same levels as wild-type protein, the mutant does not affect CFTR expression levels. Taken together, our data establish CAL as a candidate therapeutic target for correction of post-maturational trafficking defects in cystic fibrosis.
Asunto(s)
Proteínas Portadoras/fisiología , Membrana Celular/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/biosíntesis , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Proteínas de la Membrana/fisiología , Mutagénesis , Interferencia de ARN , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Animales , Células COS , Proteínas Portadoras/biosíntesis , Chlorocebus aethiops , Células Epiteliales/metabolismo , Proteínas de la Matriz de Golgi , Humanos , Proteínas de la Membrana/biosíntesis , Proteínas de Transporte de Membrana , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación Proteica , Homología de Secuencia de Aminoácido , Transactivadores/metabolismoRESUMEN
Hematopoietic stem cells (HSCs) are first found in the aorta-gonad-mesonephros region and vitelline and umbilical arteries of the midgestation mouse embryo. Runx1 (AML1), the DNA binding subunit of a core binding factor, is required for the emergence and/or subsequent function of HSCs. We show that all HSCs in the embryo express Runx1. Furthermore, HSCs in Runx1(+/-) embryos are heterogeneous and include CD45(+) cells, endothelial cells, and mesenchymal cells. Comparison with wild-type embryos showed that the distribution of HSCs among these various cell populations is sensitive to Runx1 dosage. These data provide the first morphological description of embryonic HSCs and contribute new insight into their cellular origin.