RESUMEN
BACKGROUND: Mismatched family donor and unrelated donor BM transplants are associated with a high risk of acute GvHD. White T-cell depletion is the best method to reduce risk of acute GvHD, there was a reluctance to use T-cell depletion in the mismatched setting because of increased risk of rejection and relapse. Partial T-cell depletion, by the panning of CDS and CD8 positive T cells may reduce complications related to GvHD without compromising outcomes. METHOD: In a long-term follow-up of a Phase II study of partial T-cell depletion by panning for BM transplant, 32 recipients received transplants from a single-Ag (HLA A, B, or DR) mismatched family donor; or an HLA serologically-matched unrelated donor. Patients were studied for engraftment, GHD, relapse and survival. RESULTS: 30 (94%) of the patients marrow engrafted. The cumulative risk of Grade 2-4 acute GvHD was 62 - 9%; of Grade 3-4 GvHD, 11 - 6%. The 4-year cumulative risk of relapse was 18 - 8% and actuarial survival was 44 - 9%. DISCUSSION: Partial T-cell depletion had a low rate of severe acute GvHD without compromising engrafment or relapse risk.
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Trasplante de Médula Ósea/métodos , Antígenos CD5/biosíntesis , Linfocitos T CD8-positivos/metabolismo , Adolescente , Adulto , Células de la Médula Ósea/citología , Niño , Femenino , Estudios de Seguimiento , Enfermedad Injerto contra Huésped/inmunología , Enfermedad Injerto contra Huésped/prevención & control , Histocompatibilidad , Humanos , Leucemia/terapia , Linfoma/terapia , Masculino , Recurrencia , Resultado del TratamientoRESUMEN
Immunization with specific tumor-associated antigen (Ag) (TAA)-pulsed dendritic cells (DC) has proven to be efficacious in a variety of animal models and is being investigated for the treatment of cancer patients. Use of DC pulsed with specific peptides or transfected with TAA genes has been a focused area of investigation for the induction of potent tumor and viral immune responses. In this study we demonstrate transgene expression, including expression of the MART-1 gene, in DC transfected with plasmid DNA and cationic liposome complexes. These transiently transfected DC, derived from healthy donor monocytes cultured with granulocyte macrophage colony-stimulating factor and interleukin-4, express the transgene and can stimulate naive CD8+ T cells to elicit an antitumor immune response. These cytotoxic T lymphocytes (CTL) were capable of recognizing both known and unknown TAA epitopes and were able to exhibit cytolytic activity against human histocompatibility leukocyte Ag-matched tumor cells expressing the Ag. In addition to their cytolytic function, the CTL displayed an oligoclonal T-cell receptor repertoire, indicating that the presented Ag induced alterations in the T-cell population. The ability to induce tumor-specific CTL in vitro using gene-modified DC transiently expressing TAAs demonstrates the potential use of these Ag-presenting cells to generate future in vivo cancer vaccine strategies.
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Antígeno Carcinoembrionario/genética , Células Dendríticas/fisiología , Proteínas de Neoplasias/genética , Linfocitos T Citotóxicos/inmunología , Antígenos de Neoplasias , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Antígeno Carcinoembrionario/metabolismo , Células Cultivadas , Cloranfenicol O-Acetiltransferasa/genética , Cloranfenicol O-Acetiltransferasa/metabolismo , Células Dendríticas/citología , Células Dendríticas/efectos de los fármacos , Dependovirus/genética , Genes MHC Clase I , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Humanos , Interleucina-4/farmacología , Liposomas , Antígeno MART-1 , Complejo Mayor de Histocompatibilidad , Monocitos , Proteínas de Neoplasias/metabolismo , Receptores de Antígenos de Linfocitos T/efectos de los fármacos , Receptores de Antígenos de Linfocitos T/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfección , TransgenesRESUMEN
The isolation and culture of human CD34+ cells could have broad clinical application for hematologic support following high-dose chemotherapy or bone marrow transplantation. The need for reproducible, animal product-free conditions for the culture of progenitors is crucial to the widespread clinical implementation of ex vivo cell therapies. In these studies, we explored the use of animal serum-free (ASF) medium for the culture of isolated human bone marrow and peripheral blood CD34+ cells. In this ASF system, isolated CD34+ cells were cultured using a variety of different growth factor combinations. Such ASF culture conditions yielded equivalent to superior cell and progenitor growth when directly compared with culture containing 10% fetal calf serum (FCS). In cultures containing IL-1, IL-3, and stem cell factor, total cell numbers increased, on average, 33-fold over the first 2 weeks. On phenotypic analysis, the ASF cultures demonstrated sustained proliferation of CD33+ myeloid cells throughout the culture period. CD34+ cell numbers increased during the first 7-10 days of culture, with a mean 3.4-fold expansion. Concomitant with the CD34+ cell expansion was an average 8.2-fold expansion of colony-forming unit-granulocyte-macrophage (CFU-GM) and a 102.0-fold increase in burst-forming units-erythrocytes (BFU-E). Likewise, a mean 4929-fold expansion of CD41a+ megakaryocyte progenitors was observed in these CD34+ cultures. Different combinations of growth factors affected the fold increase in cell and progenitor number. When CD34+ cell cultures from normal healthy volunteers mobilized with either G-CSF or GM-CSF were compared, similar expansions of total cell and progenitor cells resulted. However, CD41+ cells expansions were greater in those samples from G-CSF-mobilized volunteers in every case tested. These studies established the feasibility of this ASF CD34+ cell culture system to generate a population of maturing progenitors for potential use in transfusion support during cytopenic periods following high-dose chemotherapy or bone marrow transplantation.
Asunto(s)
Antígenos CD34/sangre , Factor Estimulante de Colonias de Granulocitos/farmacología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Movilización de Célula Madre Hematopoyética/métodos , Células Madre Hematopoyéticas/efectos de los fármacos , Animales , Linaje de la Célula , Separación Celular , Células Cultivadas , Medio de Cultivo Libre de Suero , Células Precursoras Eritroides/efectos de los fármacos , Células Madre Hematopoyéticas/inmunología , Humanos , Inmunofenotipificación , Megacariocitos/efectos de los fármacos , Valores de Referencia , Reproducibilidad de los ResultadosRESUMEN
We have established rapid procedures that negatively deplete and positively select for specific murine cell populations. By using polystyrene tissue culture flasks containing a covalently bound mouse anti-rat antibody and specific anti-mouse, cell-surface antigen antibodies, we easily and efficiently depleted greater than 90% of the mature lineage cells from murine bone marrow. This selection procedure resulted in an enrichment of progenitor colonies (CFU-Cs) in murine bone marrow. Using the same polystyrene tissue culture devices, we can directly isolate CD117+ (c-kit+) murine hematopoietic cells. As few as 2000 of these CD117+ cells rescued and reconstituted lethally irradiated recipients in a murine bone marrow transplant model.
Asunto(s)
Separación Celular/métodos , Técnicas de Cultivo/métodos , Células Madre Hematopoyéticas , Animales , Trasplante de Médula Ósea , Linaje de la Célula , Técnicas de Cultivo/instrumentación , Citometría de Flujo/métodos , Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Proteínas Proto-Oncogénicas c-kit/inmunologíaRESUMEN
The development of serum-free systems for the maintenance and expansion of both primitive and committed hematopoietic progenitors has numerous applications in both basic and clinical research. Many different media have been tested and refined over the years, and current formulations now yield results similar to those observed with fetal bovine serum-based medias. Using these serum-free culture systems, the impact of the cell microenvironment and individual growth factors on primitive and maturing stem cells have both been studied. The utility of progenitor populations expanded ex vivo under serum-free conditions is under investigation.
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Células Madre Hematopoyéticas/fisiología , Animales , Bovinos , Células Cultivadas , Medio de Cultivo Libre de Suero , Humanos , SolubilidadRESUMEN
Human CD34+ cells were subfractionated into three size classes using counterflow centrifugal elutriation followed by immunoadsorption to polystyrene cell separation devices. The three CD34+ cell fractions (Fr), Fr 25/29, Fr 33/37, and Fr RO, had mean sizes of 8.5, 9.3 and 13.5 microns, respectively. The majority of cells in the large Fr RO CD34+ cell population expressed the committed stage antigens CD33, CD19, CD38, or HLA-DR and contained the majority of granulocyte-macrophage colony-forming units (CFU-GM), burst-forming units-erythroid (BFU-E), and CFU-mixed lineage (GEMM). In contrast, the small Fr 25/29 CD34+ cells were devoid of committed cell surface antigens and lacked colony-forming activity. When seeded to allogeneic stroma, Fr RO CD34+ cells produced few CFU-GM at week 5, whereas cells from the Fr 25/29 CD34+ cell population showed a 30- to 55-fold expansion of myeloid progenitors at this same time point. Furthermore, CD34+ cells from each size fraction supported ontogeny of T cells in human thymus/liver grafts in severe combined immunodeficient (SCID) mice. Upon cell cycle analyses, greater than 97% of the Fr 25/29 CD34+ cells were in G0/G1 phase, whereas greater proportions of the two larger CD34+ cell fractions were in active cell cycle. Binding of the cytokines interleukin (IL)-1 alpha, IL-3, IL-6, stem cell factor (SCF), macrophage inhibitory protein (MIP)-1 alpha, granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage (GM)-CSF to these CD34+ cell populations was also analyzed by flow cytometry. As compared with the larger CD34+ cell fractions, cells in the small Fr 25/29 CD34+ cell population possessed the highest numbers of receptors for SCF, MIP1 alpha, and IL-1 alpha. Collectively, these results indicate that the Fr 25/29 CD34+ cell is a very primitive, quiescent progenitor cell population possessing a high number of receptors for SCF and MIP1 alpha and capable of yielding both myeloid and lymphoid lineages when placed in appropriate in vitro or in vivo culture conditions.
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Antígenos de Superficie/análisis , Células de la Médula Ósea , Separación Celular/métodos , Factores de Crecimiento de Célula Hematopoyética/metabolismo , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas , Receptores de Citocinas/análisis , Animales , Antígenos CD/análisis , Antígenos CD34 , Ciclo Celular , Centrifugación por Gradiente de Densidad , Citometría de Flujo , Factores de Crecimiento de Célula Hematopoyética/farmacología , Células Madre Hematopoyéticas/química , Células Madre Hematopoyéticas/citología , Humanos , Inmunofenotipificación , Técnicas de Inmunoadsorción , Ratones , Ratones SCID , Trasplante HeterólogoRESUMEN
We investigated the in vitro drug adsorption of PQ 10150 sodium silicate gel (AIS, Santa Clara, CA) with particle size of 230 microns and surface area of 400 m2/g. We observed 99% to 88% adsorption of gentamicin; a mean 91% of disopyramide; a mean 89% of quinidine at low concentration, falling to 75% at higher concentration. Insulin was 88% adsorbed at low concentrations but less so (65%) at higher concentrations. We observed a mean 83% adsorption of procainamide, a mean 84% of N-acetyl procainamide, 74% of lidocaine, 73% of amitriptyline, and 44% of desipramine. We found an average of 14% reduction of total digoxin concentration when serum containing digoxin (2 to 33 ng/mL) was exposed to sodium silicate, while the reduction in free digoxin concentration was 16%. Five percent ethosuximide was also removed. The adsorption of theophylline, phenobarbital, acetaminophen, phenytoin, ethylene glycol, methotrexate, salicylate, thiocyanate and diazepam was minimal and not significant. We conclude that significant amounts of charged, non-albumin bound drugs can be removed by PQ 10150 sodium silicate gel.
Asunto(s)
Preparaciones Farmacéuticas/química , Silicatos/química , Adsorción , Cromatografía Líquida de Alta Presión , Electroquímica , Polarización de Fluorescencia , Geles , Hemoperfusión , RadioinmunoensayoRESUMEN
In this report, we evaluated the short-term expansion of murine bone marrow mononuclear cells (BMMNC) and enriched stem cell populations to determine the capacity of these cells for long-term rescue and engraftment to lethally irradiated recipients. In our study, nonadherent bone marrow mononuclear cell (NBM-MNC) and Thy1+Lin- stem cells populations were cultured with interleukin-3 (IL-3) or IL-3 plus stem cell factor (SCF) for periods up to 6 days. By day 6 of culture, the mononuclear cells (MNC) decreased to 6% of input cell number, whereas Thy1+Lin- cells increased by 2310%. Doses of 95,000; 100,000; 50,000; and 250,000 NBM-MNCs at 0, 1, 2, and 6 days of culture, respectively, rescued 50% of lethally irradiated mice. When 250,000 MNCs were cultured for 0, 1, 2, and 6 days, 71, 61, 100, and 50% of the animals survived lethal irradiation for greater than 24 weeks. In contrast, doses of 8,000 and 21,000 Thy1+Lin- cells cultured 0 and 1 day, respectively, yielded 50% survival rates. These same cells cultured for 6 days failed to rescue recipients even at high doses. Twenty thousand Thy1+Lin- cells cultured for 0, 1, 2, and 6 days, even in the presence of SCF, produced decreasing survival rates of 86, 43, 26, and 0%, respectively. The proliferative responses of these different populations in combination with their long-term rescue abilities indicated that the absolute number of long-term rescue units (LD50, 24 weeks) in the cultured Thy1+Lin- population decreased faster than in similarly cultured NBM-MNCs. Studies evaluating donor cell engraftment demonstrated that animals rescued with cultured Thy1+Lin- and NBM-MNCs maintained high levels of donor reconstitution [7]. The percent donor T cell engraftment did not significantly change between 2 and 17 months post-bone marrow transplantation (post-BMT). Therefore, those animals who received sufficient cells to survive lethal irradiation generally established and maintained high levels of donor engraftment. The data suggest a role for accessory cells and/or factors in the preservation of stem cell activity.
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Células de la Médula Ósea , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/citología , Animales , Separación Celular , Supervivencia Celular , Células Cultivadas , Femenino , Técnicas In Vitro , Ratones , Ratones Endogámicos C57BL , Quimera por Radiación , Linfocitos T/citología , Antígenos Thy-1/análisis , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
Realization of the full potential of human umbilical cord blood (HUCB) as a source of transplantable hematopoietic progenitor cells (HPC) will be contingent on the development of a reliable method for ex vivo cell processing and stem cell enrichment. A first step in stem cell enrichment protocols involves depletion of the red blood cells (RBC) in the sample. We have compared several RBC depletion methods on the basis of recovery of total nucleated cells. It was found that 3% gelatin was effective at depleting RBC with a nucleated cell recovery of 72.4 +/- 7.3% (mean +/- standard deviation [SD]) in the HUCB samples. Several lectins were also used for processing HUCB and compared for their ability to remove RBC. Of these, soybean agglutinin (SBA) was found to remove RBC without substantial loss of HPC. Moreover, sequential treatment of HUCB with 3% gelatin and the AIS MicroCELLector SBA resulted in a product with < 1% hematocrit, 57% reduction in T cells, 35% nucleated cell recovery, and relatively high yields of burst-forming units-erythroid (BFU-E) (61%) and colony-forming units-granulocyte/macrophage (CFU-GM) (58%) and -mixed (CFU-GEMM) (66%). In a separate series of studies, enrichment of the CD34+ subset in HUCB was accomplished by processing HUCB with Ficoll followed by sequential treatment with the AIS MicroCELLector SBA and AIS MicroCELLector CD34. The CD34+ fraction was enriched for BFU-E (14-fold), CFU-GM (nine-fold), and CFU-GEMM (five-fold) with an overall purity ¿ > or = 92% CD34+ by flow cytometry. This report demonstrates that 3% gelatin and the AIS MicroCELLector SBA can be combined as an ex vivo processing technique to reduce the volume of the HUCB product by depleting RBC and T cells while still maintaining a high recovery of HPC. Moreover, separation of highly enriched CD34+ cells from HUCB is achievable and opens up the possibility of using CD34+ cells from HUCB for ex vivo progenitor cell expansion for transplantation, transfusion, and gene therapy.
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Antígenos CD/análisis , Separación Celular/métodos , Eritrocitos , Sangre Fetal/citología , Células Madre Hematopoyéticas/citología , Lectinas , Lectinas de Plantas , Proteínas de Soja , Antígenos CD34 , Centrifugación por Gradiente de Densidad , Ensayo de Unidades Formadoras de Colonias , Femenino , Citometría de Flujo , Gelatina , Células Madre Hematopoyéticas/inmunología , Humanos , Inmunofenotipificación , Lectinas/metabolismo , EmbarazoRESUMEN
Septic shock syndrome is a potentially fatal medical condition that is associated with elevated blood levels of low molecular weight proteins known as cytokines. Adsorption was investigated as a potential method for removing cytokines from blood. Saline with 50 mg/mL human serum albumin (HAS) spiked with pathological concentrations (ng-pg/mL) of radiolabeled cytokine was used to study cytokine adsorption. Adsorption isotherms were linear in the pathological concentration range, with adsorption constants ranging from 33.0 mL/g to 173 mL/g for tumor necrosis factor (TNF-alpha), interleukin-8 (IL-8),interleukin-6 (IL-6), and C3a. Adsorption constants were also determined for interleukin-1alpha (IL-1alpha), IL-1beta, and interferon-gamma (IFN-gamma). The adsorption of cytokines by several different silica adsorbents was investigated. Increased concentrations of NaCl reduced cytokine adsorption, but did not completely eliminate adsorption even at high concentrations, suggesting that adsorption wads not entirely electrostatic in nature. Possible mechanisms of cytokine adsorption are discussed. Data for batch adsorption for TNF-alpha was used to estimate the minimum amount of silica required to treat septic shock. It was concluded that a silica adsorbent has a sufficiently high capacity to be used for hemoperfusion. Adsorption of myoglobin and cytochrome c was also investigated as possible marker proteins for future dynamic adsorption studies in hemoperfusion devices.
RESUMEN
Murine systems have demonstrated improved anti-tumor efficacy when tumor-infiltrating lymphocytes (TIL) are combined with interleukin-2 (IL-2). One goal of human adoptive immunotherapy is to identify and expand TIL with specific activity against autologous tumor. In this study we attempted to isolate and characterize such cells by phenotype and cytokine expression pattern. Fourteen unselected (bulk) TIL and 5 CD8+ selected cultures were established from primary renal cell carcinoma. All cultures were grown in the presence of IL-2; triplicate cultures of each TIL culture were grown in IL-2 alone or were intermittently cocultured with irradiated allogeneic or autologous tumor. The in vitro cytotoxicity, phenotype, and cytokine expression pattern as defined by the secretion of tumor necrosis factor-alpha (TNF-alpha), interferon-gamma, granulocyte-macrophage colony-stimulating factor, IL-4, IL-6, and IL-12 were evaluated for each culture. While there was significant heterogeneity among the cultures under differing culture conditions as characterized by cytotoxicity, phenotype, and cytokine secretion pattern, we identified 8 TIL cultures which demonstrated specific enhanced lysis of only autologous tumor upon exposure to irradiated autologous tumor in vitro. These cultures demonstrate a decreased proportion of cells expressing the phenotype CD11b+. More importantly, these cultures were defined by the cytokine secretion phenotype TNF(+)/IL-6(-) upon exposure to irradiated autologous tumor. When utilized in vivo in adoptive immunotherapy of metastatic renal cell carcinoma together with IL-2, complete resolution of all metastatic tumor has only been achieved in patients who received TIL with the cytokine profile TNF(+)/IL-6(-). These findings suggest that tumor-specific renal TIL with enhanced tumor-specific cytotoxicity can be generated in vitro and can be characterized by a specific pattern of cytokine secretion. In addition, patients who receive TIL characterized by the cytokine profile TNF(+)/IL-6(-) may have an improved outcome when receiving immunotherapy.
Asunto(s)
Antígenos CD8/inmunología , Carcinoma de Células Renales/inmunología , Carcinoma de Células Renales/terapia , Citocinas/metabolismo , Neoplasias Renales/inmunología , Neoplasias Renales/terapia , Linfocitos Infiltrantes de Tumor/metabolismo , Células Cultivadas , Citotoxicidad Inmunológica , Humanos , Inmunofenotipificación , Inmunoterapia Adoptiva , Interleucina-2/farmacología , Interleucina-6/metabolismo , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/inmunología , Antígeno de Macrófago-1/fisiología , Evaluación de Resultado en la Atención de Salud , Fenotipo , Subgrupos de Linfocitos T/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
OBJECTIVES: (1) To determine the safety and feasibility of repetitive reinfusions of activated autologous CD8 cells followed by low-dose continuous interleukin (IL)-2 infusion in patients with AIDS. (2) To study the relationships between clinical responses, surface marker phenotypic distributions and cytokine expression patterns of both cultured CD8 cells and lymphocytes in the peripheral blood compartment. DESIGN: Six adult patients with Centers for Disease Control and Prevention group IV HIV-1 disease ranging from mild to severe, were studied. All patients were receiving zidovudine prior to and during the study period, and had initial CD4 and CD8 cell counts > 50 and 200 x 10(6)/l, respectively. METHODS: Autologous CD8 T cells (10(8)-10(10)) were reinfused five times after ex vivo culture and stimulation with phytohemagglutinin and recombinant (r) IL-2. The fifth such infusion was followed by 5 days of rIL-2 infusion. Phenotypes and cytokine expression patterns of the expanded cells were determined as well as serum levels of immune mediators throughout the study. RESULTS: Patients showed stable CD4 and CD8 cell counts, p24 antigenemia, and minimal toxicity over the 24-week protocol study. Clinical improvement was observed in lymphadenopathy (six out of six), oral hairy leukoplakia (three out of four), and Kaposi's sarcoma (KS; two out of two) in the patients studied. In vivo induction of detectable levels of bioactive acid-stable interferon (IFN)-alpha, but not of other cytokines studied, upon activated CD8 cell reinfusion was associated consistently with improvement of oral hairy leukoplakia. However, partial regression of KS was observed after the CD8 cell infusion cycles and without IFN-alpha induction. In one of the two patients studied, KS regression was associated with decreased IL-1 alpha serum levels. In the other patient, who had failed previous IFN-alpha therapy, KS regression was observed after a decline in reinfused CD8 cell-associated gene expression of tumor necrosis factor (TNF)-beta. Both IL-1 alpha and TNF-beta are growth factors for KS cells. CONCLUSIONS: These observations demonstrate the feasibility and safety of ex vivo CD8 cell activation, expansion, and reinfusion, and rIL-2 infusion in AIDS patients. The findings in this Phase I trial suggest potential clinical efficacy and encourage Phase II trials. The correlations obtained between clinical and immunological states could contribute to an understanding of the relationship between CD8 T-cell function and HIV-1-associated disease progression.
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Síndrome de Inmunodeficiencia Adquirida/inmunología , Linfocitos T CD8-positivos/inmunología , Inmunoterapia Adoptiva , Interleucina-2/uso terapéutico , Síndrome de Inmunodeficiencia Adquirida/complicaciones , Síndrome de Inmunodeficiencia Adquirida/terapia , Antígenos CD/análisis , Transfusión de Sangre Autóloga , Citotoxicidad Inmunológica , Femenino , Antígenos HLA-DR/análisis , Humanos , Infusiones Intravenosas , Interleucina-2/administración & dosificación , Transfusión de Linfocitos , Linfotoxina-alfa/biosíntesis , Masculino , Sarcoma de Kaposi/complicaciones , Sarcoma de Kaposi/inmunología , Sarcoma de Kaposi/terapiaRESUMEN
Bovine conglutinin (BC), a member of the mammalian C-type collectin subfamily, is a serum protein synthesized in liver that is believed to play a role in natural host defense. Previously, we have characterized a full length BC cDNA and we now describe the partial characterization of a genomic clone that encodes for the BC gene (CGN1). BC is encoded by nine exons spanning > 11 kb and has been localized previously to band 18 of bovine (Bos taurus) chromosome 28. Genomic sequencing demonstrated that the signal peptide/amino-terminal domain, the carbohydrate recognition domain, and the linking peptide, a domain between the collagenous region and the carbohydrate recognition domain, are each encoded by a single exon. The collagenous domain is split into five exons, with the 5' most region being located within the exon that also encodes the signal peptide/amino terminus. The remaining four collagenous domain exons are tandemly arranged with lengths of 117, 108, 108, and 117 bp, respectively. Overall, the BC genomic organization is very similar to that of the human surfactant protein-D gene, SFTP4. On the basis of identical collagen domain structures, we suggest that conglutinin and bovine surfactant protein-D evolved from a gene duplication event occurring in Bovidae after divergence from other mammals.
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Colectinas , Glicoproteínas/genética , Surfactantes Pulmonares/genética , Seroglobulinas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Evolución Biológica , Bovinos , Cartilla de ADN/química , Exones , Genes , Datos de Secuencia Molecular , Polimorfismo Genético , Proteína D Asociada a Surfactante PulmonarRESUMEN
We have used cationic liposomes to facilitate adeno-associated virus (AAV) plasmid transfections of primary and cultured cell types. AAV plasmid DNA complexed with liposomes showed levels of expression several fold higher than those of complexes with standard plasmids. In addition, long-term expression (> 30 days) of the gene, unlike the transient expression demonstrated by typical liposome-mediated transfection with standard plasmids, was observed. Southern analysis of chromosomal DNA further substantiated the hypothesis that the long-term expression was due to the presence of the transgene in the AAV plasmid-transfected group and not in the standard plasmid-transfected group. AAV plasmid-liposome complexes induced levels of transgene expression comparable to those obtained by recombinant AAV transduction. Primary breast, ovarian, and lung tumor cells were transfectable with the AAV plasmid DNA-liposome complexes. Transfected primary and cultured tumor cells were able to express transgene product even after lethal irradiation. High-level gene expression was also observed in freshly isolated CD3+, CD4+, and CD8+ T cells from normal human peripheral blood. Transfection efficiency ranged from 10 to 50% as assessed by intracellular interleukin-2 levels in interleukin-2-transfected cells. The ability to express transgenes in primary tumor and lymphoid cells may be applied toward tumor vaccine studies and protocols which may eventually permit highly specific modulation of the cellular immune response in cancer and AIDS.
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Neoplasias de la Mama/metabolismo , ADN/genética , Dependovirus/genética , Expresión Génica , Interleucina-2/biosíntesis , Neoplasias Pulmonares/metabolismo , Neoplasias Ováricas/metabolismo , Plásmidos/administración & dosificación , Linfocitos T/metabolismo , Transfección/métodos , Células Tumorales Cultivadas/metabolismo , Síndrome de Inmunodeficiencia Adquirida/inmunología , Animales , Southern Blotting , Células Cultivadas , Cloranfenicol O-Acetiltransferasa/análisis , Cloranfenicol O-Acetiltransferasa/biosíntesis , ADN/administración & dosificación , Portadores de Fármacos , Femenino , Vectores Genéticos , Humanos , Interleucina-2/genética , Liposomas , Masculino , Neoplasias de la Próstata , Ratas , Subgrupos de Linfocitos T/metabolismo , Neoplasias de la Vejiga UrinariaRESUMEN
Bovine conglutinin (BC) is a C-type lectin isolated from bovine serum that appears to play a role in first-line host defense. The BC cDNA was cloned from a bovine liver library and the nucleotide (nt) sequence of 1519 bp was determined. The longest open reading frame encoded a 20-amino-acid (aa) signal sequence and a mature protein of 351 aa. Analysis of the nt and deduced aa sequences revealed 87 and 78% identity, respectively, with the sequences of another vertebrate lectin: bovine surfactant protein-D (SP-D). Of interest, the expression of the BC mRNA, as determined by RNase protection assay, is restricted to liver, unlike bovine SP-D, a lung-surfactant protein.
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Colectinas , Hígado/metabolismo , ARN Mensajero/biosíntesis , Seroglobulinas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , Glicoproteínas/genética , Datos de Secuencia Molecular , Especificidad de Órganos , Proteína D Asociada a Surfactante Pulmonar , Surfactantes Pulmonares/genética , Homología de Secuencia de Ácido NucleicoRESUMEN
The plant lectin, soybean agglutinin (SBA), has been widely used to separate heterogeneous populations of cells. In the field of bone marrow transplantation, SBA has been used for partial depletion of T cells from bone marrow allografts to reduce graft-vs.-host disease. SBA's high affinity for many different tumor cells has also indicated its use as a tumor purging agent for autologous bone marrow transplants. We have compared two methods of cell separation using either soluble SBA agglutination, or SBA covalently attached to an activated polystyrene surface. The nonbinding SBA-cell populations generated by these two procedures were very similar in terms of cell recovery, light scatter properties, and phenotypic profile. Notably, both SBA- fractions were enriched in cells with the known progenitor markers, CD34, CD33, and HLA-DR, and were relatively depleted of SBA binding cells. In addition, the activity of each SBA- cell population was measured in vitro in short-term progenitor assays. Here, both SBA- populations were significantly enriched for CFU-GM. When device-separated SBA- cell populations were seeded into long-term bone marrow culture, they produced both increased progenitor activity and cell proliferation compared to unseparated BMMCs. The polystyrene technology described here could reduce or eliminate many of the drawbacks of soluble SBA agglutination, making SBA cell separation a viable and convenient technique for clinical application.