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1.
medRxiv ; 2023 Mar 17.
Artículo en Inglés | MEDLINE | ID: mdl-36993739

RESUMEN

In autoimmune Type 1 diabetes (T1D), immune cells progressively infiltrate and destroy the islets of Langerhans - islands of endocrine tissue dispersed throughout the pancreas. However, it is unclear how this process, called 'insulitis', develops and progresses within this organ. Here, using highly multiplexed CO-Detection by indEXing (CODEX) tissue imaging and cadaveric pancreas samples from pre-T1D, T1D, and non-T1D donors, we examine pseudotemporal-spatial patterns of insulitis and exocrine inflammation within large pancreatic tissue sections. We identify four sub-states of insulitis characterized by CD8 + T cells at different stages of activation. We further find that exocrine compartments of pancreatic lobules affected by insulitis have distinct cellularity, suggesting that extra-islet factors may make particular lobules permissive to disease. Finally, we identify "staging areas" - immature tertiary lymphoid structures away from islets where CD8 + T cells appear to assemble before they navigate to islets. Together, these data implicate the extra-islet pancreas in autoimmune insulitis, greatly expanding the boundaries of T1D pathogenesis.

2.
J Autoimmun ; 2017 Apr 04.
Artículo en Inglés | MEDLINE | ID: mdl-28389038

RESUMEN

Systemic Lupus Erythematosus (SLE) is a heterogeneous autoimmune disease with heightened disease severity in children. The incomplete understanding of the precise cellular and molecular events that drive disease activity pose a significant hurdle to the development of targeted therapeutic agents. Here, we performed single-cell phenotypic and functional characterization of pediatric SLE patients and healthy controls blood via mass cytometry. We identified a distinct CD14hi monocyte cytokine signature, with increased levels of monocyte chemoattractant protein-1 (MCP1), macrophage inflammatory protein-1ß (Mip1ß), and interleukin-1 receptor antagonist (IL-1RA). This signature was shared by every clinically heterogeneous patient, and reproduced in healthy donors' blood upon ex-vivo exposure to plasma from clinically active patients only. This SLE-plasma induced signature was abrogated by JAK1/JAK2 selective inhibition. This study demonstrates the utility of mass cytometry to evaluate immune dysregulation in pediatric autoimmunity, by identification of a multi-parametric immune signature that can be further dissected to delineate the events that drive disease pathogenesis.

3.
Leukemia ; 31(11): 2365-2375, 2017 11.
Artículo en Inglés | MEDLINE | ID: mdl-28331226

RESUMEN

Leukemias bearing CRLF2 and JAK2 gene alterations are characterized by aberrant JAK/STAT signaling and poor prognosis. The HDAC inhibitor givinostat/ITF2357 has been shown to exert anti-neoplastic activity against both systemic juvenile idiopathic arthritis and myeloproliferative neoplasms through inhibition of the JAK/STAT pathway. These findings led us to hypothesize that givinostat might also act against CRLF2-rearranged BCP-ALL, which lack effective therapies. Here, we found that givinostat inhibited proliferation and induced apoptosis of BCP-ALL CRLF2-rearranged cell lines, positive for exon 16 JAK2 mutations. Likewise, givinostat killed primary cells, but not their normal hematopoietic counterparts, from patients carrying CRLF2 rearrangements. At low doses, givinostat downregulated the expression of genes belonging to the JAK/STAT pathway and inhibited STAT5 phosphorylation. In vivo, givinostat significantly reduced engraftment of human blasts in patient-derived xenograft models of CRLF2-positive BCP-ALL. Importantly, givinostat killed ruxolitinib-resistant cells and potentiated the effect of current chemotherapy. Thus, givinostat in combination with conventional chemotherapy may represent an effective therapeutic option for these difficult-to-treat subsets of ALL. Lastly, the selective killing of cancer cells by givinostat may allow the design of reduced intensity regimens in CRLF2-rearranged Down syndrome-associated BCP-ALL patients with an overall benefit in terms of both toxicity and related complications.


Asunto(s)
Carbamatos/farmacología , Inhibidores de Histona Desacetilasas/farmacología , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Receptores de Citocinas/genética , Adolescente , Animales , Línea Celular Tumoral , Preescolar , Femenino , Humanos , Masculino , Ratones , Nitrilos , Fosforilación , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Pirazoles/farmacología , Pirimidinas , Factor de Transcripción STAT5/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto
4.
Leukemia ; 31(9): 1962-1974, 2017 09.
Artículo en Inglés | MEDLINE | ID: mdl-28008177

RESUMEN

Myeloproliferative neoplasms (MPNs) feature a malignant clone containing the JAK2 V617F mutation, or another mutation causing dysregulated JAK2 kinase activity. The multiple disease phenotypes of MPNs, and their tendency to transform phenotypically, suggest pathophysiologic heterogeneities beyond a common phenomenon of JAK2 hyperactivation. JAK2 has the potential to activate multiple other signaling molecules, either directly through downstream effectors, or indirectly through induction of target gene expression. We have interrogated myeloproliferative signaling in myelofibrosis (MF) and secondary acute myeloid leukemia (sAML) patient samples using mass cytometry, which allows the quantitative measurement of multiple signaling molecules simultaneously at the single-cell level, in cell populations representing a nearly complete spectrum of hematopoiesis. MF and sAML malignant cells demonstrated a high prevalence of hyperactivation of the JAK-STAT, MAP kinase, PI3 kinase and NFκB signaling pathways. Constitutive NFκB signaling was evident across MF and sAML patients. A supporting gene set enrichment analysis (GSEA) of MF showed many NFκB target genes to be expressed above normal levels in MF patient CD34+ cells. NFκB inhibition suppressed colony formation from MF CD34+ cells. This study indicates that NFκB signaling contributes to human myeloproliferative disease and is abnormally activated in MF and sAML.


Asunto(s)
Leucemia Mieloide Aguda/metabolismo , FN-kappa B/metabolismo , Mielofibrosis Primaria/metabolismo , Transducción de Señal , Antígenos CD34 , Médula Ósea , Línea Celular , Citometría de Flujo/métodos , Humanos , Janus Quinasa 2/genética , Leucemia Mieloide Aguda/etiología , Leucemia Mieloide Aguda/patología , Mielofibrosis Primaria/patología
5.
Interface Focus ; 3(4): 20130019, 2013 Aug 06.
Artículo en Inglés | MEDLINE | ID: mdl-24511382

RESUMEN

Many interesting studies aimed at elucidating the connectivity structure of biomolecular pathways make use of abundance measurements, and employ statistical and information theoretic approaches to assess connectivities. These studies often do not address the effects of the dynamics of the underlying biological system, yet dynamics give rise to impactful issues such as timepoint selection and its effect on structure recovery. In this work, we study conditions for reliable retrieval of the connectivity structure of a dynamic system, and the impact of dynamics on structure-learning efforts. We encounter an unexpected problem not previously described in elucidating connectivity structure from dynamic systems, show how this confounds structure learning of the system and discuss possible approaches to overcome the confounding effect. Finally, we test our hypotheses on an accurate dynamic model of the IGF signalling pathway. We use two structure-learning methods at four time points to contrast the performance and robustness of those methods in terms of recovering correct connectivity.

6.
Pac Symp Biocomput ; : 63-74, 2009.
Artículo en Inglés | MEDLINE | ID: mdl-19209696

RESUMEN

Bayesian network structure learning is a useful tool for elucidation of regulatory structures of biomolecular pathways. The approach however is limited by its acyclicity constraint, a problematic one in the cycle-containing biological domain. Here, we introduce a novel method for modeling cyclic pathways in biology, by employing our newly introduced Generalized Bayesian Networks (GBNs). Our novel algorithm enables cyclic structure learning while employing biologically relevant data, as it extends our cycle-learning algorithm to permit learning with singly perturbed samples. We present theoretical arguments as well as structure learning results from realistic, simulated data of a biological system. We also present results from a real world dataset, involving signaling pathways in T-cells.


Asunto(s)
Algoritmos , Modelos Biológicos , Transducción de Señal/fisiología , Inteligencia Artificial , Teorema de Bayes , Biometría , Bases de Datos Factuales , Somatomedinas/metabolismo , Linfocitos T/metabolismo
7.
Gene Ther ; 12(6): 467-76, 2005 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-15703764

RESUMEN

Acquired immunodeficiency syndrome (AIDS), caused by human immunodeficiency virus (HIV), kills millions worldwide every year. Vaccines against HIV still seem a distant promise. Pharmaceutical treatments exist, but these are not always effective, and there is increasing prevalence of viral strains with multidrug resistance. Highly active antiretroviral therapy (HAART) consists of inhibitors of viral enzymes (reverse transcriptase (RT) and protease). Gene therapy, first introduced as intracellular immunization, may offer hopes for new treatments to be used alone, or in conjunction with, conventional small molecule drugs. Gene therapy approaches against HIV-1, including suicide genes, RNA-based technology, dominant negative viral proteins, intracellular antibodies, intrakines, and peptides, are the subject of this review.


Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/terapia , Terapia Genética/métodos , VIH-1 , Síndrome de Inmunodeficiencia Adquirida/inmunología , Predicción , Técnicas de Transferencia de Gen , Ingeniería Genética , Vectores Genéticos/administración & dosificación , VIH-1/inmunología , Humanos , Proteínas Recombinantes/genética , Linfocitos T/inmunología , Replicación Viral
8.
Nucleic Acids Res ; 33(4): e39, 2005 Feb 24.
Artículo en Inglés | MEDLINE | ID: mdl-15731332

RESUMEN

We report a novel approach that allows for the rapid identification of proteins mediating phosphorylation in signaling cascades after specific stimulation. As a proof of concept, we used the interferon- gamma (IFN-gamma)-induced phosphorylation of signal transducer and activator of transcription-1 (Stat1) in a human promonocytic cell line, which was previously shown to be deficient in this signaling pathway. By using retroviral cDNA expression libraries, transduced selector cells expressing single cDNAs were stimulated with IFN-gamma, then fixed, permeabilized and stained intracellularly for phospho-Stat1 levels. Cells responding to the stimulation, which showed increased levels of phosphorylated Stat1, were enriched using fluorescence activated cell sorting (FACS). Genomic DNA was isolated from the enriched cell population and served as a template for cDNA amplification using PCR. After only one round of selection, a cDNA encoding the beta-chain of the IFN-gamma receptor (IFNGR2) was obtained and demonstrated to restore the selected phenotype. The approach now allows one to use phospho-events as reporters, alone or in tandem, for screening of signaling network states, overcoming a prior need to rely on the reporter genes that are often only indirect measures of phenotypes desired in a screen.


Asunto(s)
Biblioteca de Genes , Fosfoproteínas/análisis , Retroviridae/genética , Transducción de Señal , ADN Complementario/análisis , Proteínas de Unión al ADN/metabolismo , Fijadores , Citometría de Flujo , Formaldehído/química , Humanos , Interferón gamma/farmacología , Metanol/química , Fosforilación , Reacción en Cadena de la Polimerasa , Receptores de Interferón/genética , Factor de Transcripción STAT1 , Transactivadores/metabolismo , Células U937
9.
Nucleic Acids Res ; 32(2): e22, 2004 Jan 29.
Artículo en Inglés | MEDLINE | ID: mdl-14752044

RESUMEN

We present an approach that generates an oligomer-based library with minimal need for restriction site modification of sequences in the target vector. The technique has the advantage that it can be applied for generating peptide aptamer libraries at sites within proteins without the need for introducing flanking enzyme sites. As an example we present a phagemid retroviral shuttle vector that can be used to achieve stable expression of the library in mammalian cells for the purpose of screening for peptides with desired biological activity.


Asunto(s)
ADN de Cadena Simple/genética , Biblioteca de Genes , Oligonucleótidos/genética , Secuencia de Bases , Sitios de Unión/genética , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Clonación Molecular/métodos , ADN/genética , ADN/metabolismo , ADN de Cadena Simple/metabolismo , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Escherichia coli/genética , Vectores Genéticos/genética , Humanos , Datos de Secuencia Molecular , Pirrolidinonas/farmacología , Reproducibilidad de los Resultados , Retroviridae/genética , Moldes Genéticos , Transfección
10.
Gene Ther ; 10(15): 1248-57, 2003 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-12858190

RESUMEN

Although systemic administration of neutralizing anti-TNF antibodies has been used successfully in treating rheumatoid arthritis, there is a potential for side effects. We transduced a collagen reactive T-cell hybridoma with tissue-specific homing properties to assess therapeutic effects of local delivery to inflamed joints of anti-TNF single-chain antibodies (scFv) by adoptive cellular gene therapy. Cell culture medium conditioned with 1 x 10(6) scFv producer cells/ml had TNF neutralizing capacity in vitro equivalent to 50 ng/ml anti-TNF monoclonal antibody. Adding a kappa chain constant domain to the basic scFv (construct TN3-Ckappa) gave increased in vitro stability and in vivo therapeutic effect. TN3-Ckappa blocked development of collagen-induced arthritis in DBA/1LacJ mice for >60 days. Transgene expression was detected in the paws but not the spleen of treated animals for up to 55 days postinjection. No significant variations in cell proliferation or cytokine secretion were found in splenocytes or peripheral lymphocytes. IL-6 expression was blocked in the diseased paws of mice in the scFv treatment groups compared to controls. In conclusion, we have shown that local expression of an anti-inflammatory agent blocks disease development without causing demonstrable systemic immune function changes. This is encouraging for the potential development of safe adoptive cellular therapies to treat autoimmunity.


Asunto(s)
Artritis Experimental/prevención & control , Terapia Genética/métodos , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Artritis Experimental/inmunología , Células Cultivadas , Citocinas/biosíntesis , Expresión Génica , Vectores Genéticos , Ganglios Linfáticos/inmunología , Masculino , Ratones , Ratones Endogámicos DBA , Ratones Transgénicos , Retroviridae/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Bazo/inmunología , Factor de Necrosis Tumoral alfa/inmunología
11.
Anticancer Res ; 23(2B): 1165-72, 2003.
Artículo en Inglés | MEDLINE | ID: mdl-12820367

RESUMEN

Tumor endothelium could represent a novel target for active and passive immunotherapies of cancer. Here, we show that endothelial cells can be used as a vaccine in mice. In this study, three endothelial cell vaccine preparations from syngeneic (SVR), allogeneic (ISOS-1) and xenogeneic (ISO-HAS) sources were used to vaccinate mice. All mice developed humoral immune responses to endothelial cells and showed lower basal serum VEGF levels (37-45% lower) compared with unvaccinated control mice. Mice receiving the syngeneic SVR vaccine showed substantial inhibition of tumor growth after B16F10 melanoma challenge (50% of the mice in this group were tumor-free). The tumors that developed in the few mice in the syngeneic group had lower microvessel density counts (4-5 fold) compared with the other groups. The data suggests an in vivo antiangiogenic effect as the potential mechanism for the anti-cancer effect. In summary, further studies using other tumor models to demonstrate broad protection of this novel type of antiangiogenic vaccine are warranted.


Asunto(s)
Vacunas contra el Cáncer/inmunología , Endotelio Vascular/inmunología , Hemangioendotelioma/patología , Melanoma Experimental/prevención & control , Neovascularización Patológica/prevención & control , Animales , Anticuerpos Antineoplásicos/biosíntesis , Antígenos de Neoplasias/inmunología , Antígenos de Superficie/inmunología , Biomarcadores , Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/uso terapéutico , Línea Celular Transformada , Factores de Crecimiento Endotelial/sangre , Endotelio Vascular/citología , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunización Pasiva , Péptidos y Proteínas de Señalización Intercelular/sangre , Linfocinas/sangre , Melanoma Experimental/irrigación sanguínea , Melanoma Experimental/inmunología , Melanoma Experimental/patología , Melanoma Experimental/terapia , Ratones , Ratones Endogámicos C57BL , Neovascularización Patológica/tratamiento farmacológico , Páncreas/citología , Células Tumorales Cultivadas/trasplante , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular
12.
Leukemia ; 16(8): 1507-18, 2002 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-12145692

RESUMEN

The balance between hematopoietic cell viability and apoptosis is regulated by exogenous growth factors, however, the molecular mechanisms by which these trophic factors exert their effects remain obscure. A functional retroviral cDNA library-based screen was employed to identify genes that prevent growth factor withdrawal-mediated apoptosis in the myeloid progenitor cell 32Dcl3. This approach identified three classes of genes: those with known roles in apoptosis (bcl-X(L) and ornithine decarboxylase); genes previously identified but not linked directly to apoptotic signaling (O-linked N-acetylglucosamine transferase); and a previously uncharacterized gene we termed SPIN-2. In 32Dcl3 cells, expression of exogenous SPIN-2 provides 25% protection from apoptosis following growth factor withdrawal compared to controls which show approximately 1-2% survival. SPIN-2 overexpression slows cell growth rates and increases the percentage of cells in G(2)/M (32% vs control cells at 12%). Immunolocalization studies indicate that myc-epitope tagged SPIN-2 proteins, which retain their anti-apoptotic function, reside in the nucleus, whereas a C-terminal deletion mutant that loses its anti-apoptotic activity is located in the cytoplasm. These studies suggest that SPIN-2 is a novel nuclear protein that functions to regulate cell cycle progression and this activity is related to the inhibition of apoptosis following the removal of essential growth factors.


Asunto(s)
Apoptosis/genética , Proteínas de Ciclo Celular/aislamiento & purificación , Ciclo Celular/genética , Proteínas Nucleares/aislamiento & purificación , Células 3T3 , Secuencia de Aminoácidos , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiología , Línea Celular/citología , Línea Celular/metabolismo , Clonación Molecular , ADN Complementario/genética , Perfilación de la Expresión Génica , Biblioteca de Genes , Vectores Genéticos/genética , Células HL-60/química , Humanos , Interleucina-3/farmacología , Células Jurkat/citología , Ratones , Datos de Secuencia Molecular , Células Mieloides/citología , Células Mieloides/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/fisiología , ARN Mensajero/genética , ARN Neoplásico/genética , Proteínas Recombinantes de Fusión/fisiología , Retroviridae/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad , Cromosoma X/genética
14.
Immunity ; 15(5): 687-90, 2001 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-11728331

RESUMEN

HIV-1 budding appears to require Vps4 and Tsg101-two proteins that have links to endosomal sorting machinery. A picture emerges wherein divergent viruses recruit endosomal proteins like Tsg101 to gain access to ubiquitin processes that play a crucial role during viral budding.


Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/virología , Adenosina Trifosfatasas , Proteínas Portadoras/fisiología , VIH-1/fisiología , Proteínas de Saccharomyces cerevisiae , Proteínas de Unión al ADN/fisiología , Complejos de Clasificación Endosomal Requeridos para el Transporte , Endosomas/fisiología , Proteínas Fúngicas/fisiología , Humanos , Factores de Transcripción/fisiología , Replicación Viral
16.
Oncogene ; 20(30): 4019-28, 2001 Jul 05.
Artículo en Inglés | MEDLINE | ID: mdl-11494130

RESUMEN

To gain better understanding of the molecular alterations responsible for the aggressive growth potential of epidermal growth factor receptor (EGFR)-positive breast cancers, we utilized an expression cloning strategy to seek gene products that mediate the EGF-independent growth of human breast cancer cells. A retroviral cDNA expression library was constructed from the EGFR-positive SUM-149PT cell line, and transduced into growth factor-dependent human mammary epithelial (HME) cells. Recipient cells were functionally selected for their ability to proliferate in serum-free, EGF-free medium. Library cDNAs were recovered from EGF-independent colonies by PCR amplification or by biological rescue. Clone H55a#1 contained a library insert encoding amphiregulin. This EGFR ligand was able to confer EGF independence when transduced into HME cells. SUM-149PT and H55a#1 cells overexpressed amphiregulin transcripts, and secreted moderate EGF-like activity in conditioned media, indicating a possible autocrine loop. EGFR membrane levels and constitutive phosphorylation were consistent with this hypothesis, as well as the sensitivity of the cells to an ErbB-specific kinase inhibitor. Expression of the WT1 Wilms' tumor suppressor gene, a transcriptional activator of amphiregulin, did not parallel amphiregulin transcript levels, suggesting that another factor regulates amphiregulin in SUM-149PT. Our data confirm the importance of amphiregulin in the etiology of breast cancer.


Asunto(s)
Neoplasias de la Mama/patología , Mama/citología , Factor de Crecimiento Epidérmico/farmacología , Técnicas Genéticas , Glicoproteínas/fisiología , Sustancias de Crecimiento/fisiología , Péptidos y Proteínas de Señalización Intercelular , Anfirregulina , División Celular/efectos de los fármacos , Transformación Celular Neoplásica/genética , Células Cultivadas/efectos de los fármacos , Células Clonales/citología , Células Clonales/efectos de los fármacos , Medios de Cultivo Condicionados/farmacología , Medio de Cultivo Libre de Suero , ADN Complementario/genética , Familia de Proteínas EGF , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Receptores ErbB/genética , Receptores ErbB/fisiología , Femenino , Biblioteca de Genes , Genes del Tumor de Wilms , Prueba de Complementación Genética , Vectores Genéticos/genética , Glicoproteínas/genética , Sustancias de Crecimiento/genética , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiología , Células Madre Neoplásicas/citología , Células Madre Neoplásicas/efectos de los fármacos , Fenotipo , Retroviridae/genética , Transfección , Células Tumorales Cultivadas/citología , Células Tumorales Cultivadas/efectos de los fármacos
17.
Eur J Immunol ; 31(1): 285-93, 2001 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-11265645

RESUMEN

T cell factor / lymphocyte enhancer factor (Tcf/Lef) transcription factors complex with the transcriptional co-activator beta-catenin to transduce Wnt signals in a variety of developmental systems. The prototypic family member Tcf-1 is highly expressed in T lineage cells. Tcf1-/- mice are defective in cell cycling of early thymocyte stages. Here, we show that the interaction of beta-catenin with Tcf-1 is required for full thymocyte development. This interaction may be established by signals mediated by Wnt1 and Wnt4, leading to increased Tcf-dependent transcriptional activity in thymocytes, as demonstrated in Tcf-LacZ reporter mice. Transduction of fetal thymocytes with Wnt1 and Wnt4 results in increased survival in an in vitro cell culture system. Retroviral expression of soluble Wnt receptor mutants that block Wnt signaling inhibits thymocyte development. These results imply an important role for the Wnt cascade in thymocyte development.


Asunto(s)
Proteínas de Unión al ADN/fisiología , Proteínas Proto-Oncogénicas/fisiología , Transducción de Señal , Linfocitos T/fisiología , Transactivadores , Factores de Transcripción/fisiología , Activación Transcripcional , Proteínas de Pez Cebra , Animales , Proteínas del Citoesqueleto/fisiología , Factor Nuclear 1-alfa del Hepatocito , Factor de Unión 1 al Potenciador Linfoide , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 1 de Transcripción de Linfocitos T , Proteínas Wnt , Proteína Wnt1 , beta Catenina
18.
Mol Ther ; 3(2): 186-96, 2001 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-11237675

RESUMEN

Angiostatin and endostatin are potent endothelial cell growth inhibitors that have been shown to inhibit angiogenesis in vivo and tumor growth in mice. However, tumor shrinkage requires chronic delivery of large doses of these proteins. Here we report synergistic antitumor activity and survival of animals when these factors are delivered in combination to tumors by retroviral gene transfer. We have demonstrated this efficacy in both murine leukemia and melanoma models. Complete loss of tumorigenicity was seen in 40% of the animals receiving tumors transduced by the combination of angiostatin and endostatin in the leukemia model. The synergy was also demonstrated in vitro on human umbilical vein endothelial cell differentiation and this antiangiogenic activity may suggest a mechanism for the antitumor activity in vivo. These findings imply separate pathways by which angiostatin and endostatin mediate their antiangiogenic effects. Together, these data suggest that a combination of antiangiogenic factors delivered by retroviral gene transfer may produce synergistic antitumor effects in both leukemia and solid tumors, thus avoiding long-term administration of recombinant proteins. The data also suggest that novel combinations of antiangiogenic factors delivered into tumors require further investigation as therapeutic modalities.


Asunto(s)
Inhibidores de la Angiogénesis/uso terapéutico , Antineoplásicos/uso terapéutico , Colágeno/uso terapéutico , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Leucemia/terapia , Melanoma/terapia , Neovascularización Patológica , Fragmentos de Péptidos/uso terapéutico , Plasminógeno/uso terapéutico , Inhibidores de la Angiogénesis/administración & dosificación , Angiostatinas , Animales , Antineoplásicos/administración & dosificación , Western Blotting , División Celular/efectos de los fármacos , Separación Celular , Supervivencia Celular , Colágeno/administración & dosificación , Colágeno/metabolismo , Regulación hacia Abajo , Combinación de Medicamentos , Endostatinas , Femenino , Citometría de Flujo , Vectores Genéticos , Proteínas Fluorescentes Verdes , Humanos , Inmunohistoquímica , Laminina/metabolismo , Proteínas Luminiscentes/metabolismo , Melanoma Experimental/terapia , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Modelos Genéticos , Virus de la Leucemia Murina de Moloney/genética , Fragmentos de Péptidos/administración & dosificación , Plasminógeno/administración & dosificación , Pruebas de Precipitina , Proteoglicanos/metabolismo , Retroviridae/genética , Factores de Tiempo , Transducción Genética , Transfección
19.
Clin Immunol ; 98(2): 220-8, 2001 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-11161978

RESUMEN

The Type II EBV malignancies nasopharyngeal carcinoma and EBV(+) Hodgkin's disease express three subdominant antigens, latency membrane protein (LMP) 1, LMP2, and EBNA-1. While adoptive immunotherapy with T cell lines for Type III EBV malignancy (such as posttransplant lymphoma, PTLD, which expresses the immunodominant EBNA-3 antigens) has been used to prevent and treat PTLD, the generation of class I MHC-restricted CTL suitable for the immunotherapy of Type II EBV malignancy is difficult. This is primarily due to the lack of anti-LMP or EBNA-1 CTL activity in many healthy volunteers. We have engineered, by retroviral transduction of the TCR, CTL that have the potential to recognize subdominant EBV latency antigens. Using the SAMEN retroviral vector we demonstrate the ability to transfer CTL activity from a LMP2 peptide-specific CTL clone to a stimulated PBMC population. TCR-transduced PBMC also secrete IFN-gamma upon coculture with LMP2 targets and maintain expression of the transduced TCR during subsequent mitogenic expansion.


Asunto(s)
Vectores Genéticos/genética , Herpesvirus Humano 4/inmunología , Virus de la Leucemia Murina/genética , Leucocitos Mononucleares/inmunología , Fragmentos de Péptidos/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Linfocitos T Citotóxicos/inmunología , Proteínas de la Matriz Viral/inmunología , Secuencia de Aminoácidos , Presentación de Antígeno , Epítopos/genética , Epítopos/inmunología , Antígeno HLA-A2/inmunología , Herpesvirus Humano 4/genética , Humanos , Interferón gamma/metabolismo , Activación de Linfocitos , Fragmentos de Péptidos/genética , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Linfocitos T Citotóxicos/metabolismo , Transfección , Proteínas de la Matriz Viral/genética
20.
Nat Genet ; 27(1): 23-9, 2001 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-11137994

RESUMEN

We have expressed libraries of peptides in mammalian cells to select for trans-dominant effects on intracellular signaling systems. As an example-and to reveal pharmacologically relevant points in pathways that lead to Taxol resistance-we selected for peptide motifs that confer resistance to Taxol-induced cell death. Of several peptides selected, one, termed RGP8.5, was linked to upregulation of expression of the gene ABCB1 (also known as MDR1, for multiple drug resistance) in HeLa cells. Our data indicate that trans-dominant effector peptides can point to potential mechanisms by which signaling systems operate. Such tools may be useful in functional genomic analysis of signaling pathways in mammalian disease processes.


Asunto(s)
Genes MDR/genética , Paclitaxel/farmacología , Péptidos/farmacología , Transducción de Señal/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cisteína Endopeptidasas/metabolismo , Resistencia a Antineoplásicos , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes Dominantes/genética , Células HeLa , Humanos , Datos de Secuencia Molecular , Complejos Multienzimáticos/antagonistas & inhibidores , Complejos Multienzimáticos/metabolismo , Biblioteca de Péptidos , Péptidos/química , Péptidos/genética , Péptidos/metabolismo , Complejo de la Endopetidasa Proteasomal , Unión Proteica , Subunidades de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , Alineación de Secuencia , Técnicas del Sistema de Dos Híbridos
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