RESUMEN

Some of the early complications of hematopoietic stem cell transplantation (HSCT) concerning the small vessels can be summarized as transplant-associated systemic vasculopathies (TASV). One enzyme known to play a major role in inflammation, tissue remodeling, and repair processes as well as tumor metastasis is heparanase (HPSE). HPSE genetic variants have recently been associated with significant influence on the risk of developing certain TASV such as a sinusoidal obstruction syndrome. This study aimed to validate the two known HPSE single nucleotide polymorphisms (SNPs)-rs4693608 and rs4364254-as a genetic predictor of TASV in a cohort of 494 patients and were correlated retrospectively with the clinical course post-HSCT. Significant association was revealed for rs4364254, showing that the incidence of TASV (38.0% vs. 57.8%, p = .009) and in particular of acute graft-versus-host disease (aGvHD) (36.3% vs. 54.0%, p = .0138) was lower in wildtype CC carriers than in TC/TT carriers. Moreover, compared with all other genotypes, the allelic combination GG-CC had the lowest incidence of TASV (34.9% vs. 57.4%, p = .0109) and aGvHD in particular (34.9% vs. 53.5%, p = .0315). A competing risk regression analysis confirmed a significantly reduced risk for a TASV in patients with GG (subhazard ratio [SHR] = 0.670, p = .043) and CC (SHR = 0.598, p = .041) compared with the corresponding homozygote SNP as well as for allelic combinations correlated with low HPSE gene expression (SHR = 0.630, p = .016) and in correlation with clinical risk factors. In summary, our study emphasizes an association of HPSE gene SNPs with TASV, in particular with aGvHD, which could be implementable as pre-transplant risk stratification if validated prospectively.

Asunto(s)

Enfermedad Injerto contra Huésped , Trasplante de Células Madre Hematopoyéticas , Humanos , Estudios Retrospectivos , Incidencia , Genotipo , Polimorfismo de Nucleótido Simple , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Enfermedad Injerto contra Huésped/genética

RESUMEN

A dendritic cell sarcoma cell line, U-DCS, was established from a dendritic cell sarcoma in a 53-year-old Caucasian male patient. Since its establishment, U-DCS has maintained stable phenotypic characteristics in vitro and has a doubling time of approximately 2 days under standard culture conditions. U-DCS is growing with typical dendritic cell morphology in tissue and expresses the dendritic cell sarcoma immunophenotypic markers S100 protein, MHCI, MHCII, and vimentin. Expression analysis revealed transcripts for the toll-like receptors TLR3, -4, -9 and DDX58 (RIG-I), but not for TLR2. U-DCS shows functional features of dendritic cells with the ability of phagocytosis and antigen-specific T cell stimulation. Karyotype-, CGH-, and mFISH analysis point to a chromosomal instability and a hypotetraploid karyotype with approximately 130 chromosomes. U-DCS is the first immortalized human dendritic cell sarcoma cell line and has some morphological and functional features of dendritic cells without dependency on growth factors.

Asunto(s)

Técnicas de Cultivo de Célula/métodos , Células Dendríticas/citología , Sarcoma/metabolismo , Línea Celular Tumoral , Inestabilidad Cromosómica , Proteína 58 DEAD Box/genética , Proteína 58 DEAD Box/metabolismo , Células Dendríticas/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Inmunofenotipificación , Hibridación Fluorescente in Situ , Cariotipo , Masculino , Persona de Mediana Edad , Fagocitosis , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Proteínas S100/metabolismo , Sarcoma/genética , Receptor Toll-Like 3/genética , Receptor Toll-Like 3/metabolismo , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismo , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/metabolismo , Vimentina/metabolismo

RESUMEN

We studied the developmental and functional mechanisms behind NK cell-mediated antitumor responses against metastatic colorectal carcinoma (CRC) in mice. In particular, we focused on investigating the significance of T-box transcription factors and the immunotherapeutic relevance of IL-15 in the development and function of tumor-reactive NK cells. Pulmonary CRC metastases were experimentally seeded via an adoptive i.v. transfer of luciferase-expressing CT26 CRC cells that form viewable masses via an in vivo imaging device; genetically deficient mice were used to dissect the antitumor effects of developmentally different NK cell subsets. IL-15 precomplexed to IL-15 receptor-α was used in immunotherapy experiments. We found that mice deficient for the T-box transcription factor T-bet lack terminally differentiated antitumor CD27(low)KLRG1(+) NK cells, leading to a terminal course of rapid-onset pulmonary CRC metastases. The importance of this NK cell subset for effective antitumor immunity was shown by adoptively transferring purified CD27(low)KLRG1(+) NK cells into T-bet-deficient mice and, thereby, restoring immunity against lung metastasis formation. Importantly, immunity to metastasis formation could also be restored in T-bet-deficient recipients by treating mice with IL-15 precomplexed to IL-15 receptor-α, which induced the development of eomesodermin(+)KLRG1(+) NK cells from existing NK cell populations. Thus, contingent upon their T-bet-dependent development and activation status, NK cells can control metastatic CRC in mice, which is highly relevant for the development of immunotherapeutic approaches in the clinic.

Asunto(s)

Neoplasias Colorrectales/patología , Células Asesinas Naturales/inmunología , Neoplasias Pulmonares/secundario , Receptores Inmunológicos/metabolismo , Proteínas de Dominio T Box/genética , Traslado Adoptivo , Animales , Diferenciación Celular/inmunología , Células Cultivadas , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/terapia , Proteínas de Homeodominio/genética , Inmunoterapia , Interferón gamma/genética , Interleucina-15/metabolismo , Células Asesinas Naturales/citología , Lectinas Tipo C , Neoplasias Pulmonares/prevención & control , Neoplasias Pulmonares/terapia , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Perforina , Proteínas Citotóxicas Formadoras de Poros/genética , Receptores de Interleucina-15/metabolismo , Proteínas Recombinantes de Fusión/uso terapéutico , Proteínas de Dominio T Box/deficiencia , Proteínas de Dominio T Box/metabolismo , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismo

RESUMEN

Although Th1, Th2, and Th17 cells are thought to be major effector cells in adaptive alloimmune responses, their respective contribution to allograft rejection remains unclear. To precisely address this, we used mice genetically modified for the Th1 and Th17 hallmark transcription factors T-bet and RORγt, respectively, which allowed us to study the alloreactive role of each subset in an experimental transplant setting. We found that in a fully mismatched heterotopic mouse heart transplantation model, T cells deficient for T-bet (prone to Th17 differentiation) versus RORγt (prone to Th1 differentiation) rejected allografts at a more accelerated rate, indicating a predominance of Th17- over Th1-driven alloimmunity. Importantly, T cells doubly deficient for both T-bet and RORγt differentiated into alloreactive GATA-3-expressing Th2 cells, which promptly induced allograft rejection characterized by a Th2-type intragraft expression profile and eosinophilic infiltration. Mechanistically, Th2-mediated allograft rejection was contingent on IL-4, as its neutralization significantly prolonged allograft survival by reducing intragraft expression of Th2 effector molecules and eosinophilic allograft infiltration. Moreover, under IL-4 neutralizing conditions, alloreactive double-deficient T cells upregulated Eomesodermin (Eomes) and IFN-γ, but not GATA-3. Thus, in the absence of T-bet and RORγt, Eomes may salvage Th1-mediated alloimmunity that underlies IL-4 neutralization-resistant allograft rejection. We summarize that, whereas Th17 cells predictably promote allograft rejection, IL-4-producing GATA-3(+) Th2 cells, which are generally thought to protect allogeneic transplants, may actually be potent facilitators of organ transplant rejection in the absence of T-bet and RORγt. Moreover, Eomes may rescue Th1-mediated allograft rejection in the absence of IL-4, T-bet, and RORγt.

Asunto(s)

Aloinjertos/inmunología , Rechazo de Injerto/inmunología , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Proteínas de Dominio T Box/metabolismo , Células Th2/inmunología , Traslado Adoptivo , Animales , Diferenciación Celular , Eosinófilos/inmunología , Factor de Transcripción GATA3/biosíntesis , Trasplante de Corazón/efectos adversos , Interferón gamma/biosíntesis , Interleucina-4/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/deficiencia , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Proteínas de Dominio T Box/biosíntesis , Proteínas de Dominio T Box/deficiencia , Proteínas de Dominio T Box/genética

RESUMEN

c-Kit is a receptor tyrosine kinase (RTK) with a pivotal role in melanogenesis, gametogenesis, and hematopoiesis. Aberrantly activated RTK and related downstream signaling partners were identified as key elements in the molecular pathogenesis of several malignancies. This finding culminated in a two-class model integrating constitutive activating and maturation arrest-inducing mutations as key elements for the pathogenesis of acute myelogenous leukemia (AML). c-Kit is expressed by myeloblasts in about 60% to 80% of patients, and the most frequently observed activating RTK mutations in AML (next to FLT3) are mutations or internal tandem duplications in c-Kit, with an overall incidence of 17%. The identification of small-molecule tyrosine kinase inhibitors capable of blocking key kinase switches introduced a paradigm change in the treatment of diseases like gastrointestinal stromal tumors and chronic myelogenous leukemia. Despite encouraging preclinical data, it appears that a complex clonal disease like AML will probably benefit from a synergistic approach of targeted drugs used (at least for now) in combination with conventional chemotherapy.

Asunto(s)

Leucemia Mieloide/tratamiento farmacológico , Leucemia Mieloide/genética , Mutación , Proteínas Proto-Oncogénicas c-kit/genética , Enfermedad Aguda , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Regulación Leucémica de la Expresión Génica , Humanos , Leucemia Mieloide/patología , Pronóstico , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/uso terapéutico , Resultado del Tratamiento

RESUMEN

The cellular prion protein (PrP(c)) plays a crucial role in the pathogenesis of prion diseases, but its physiological function is far from understood. Several candidate functions have been proposed including binding and internalization of metal ions, a superoxide dismutase-like activity, regulation of cellular antioxidant activities, and signal transduction. The transmembrane (TM1) region of PrP(c) (residues 110-135) is particularly interesting because of its very high evolutionary conservation. We investigated a possible role of TM1 in the antioxidant defense, by assessing the impact of overexpressing wt-PrP or deletion mutants in N(2)A mouse neuroblastoma cells on intracellular reactive oxygen species (ROS) levels. Under conditions of oxidative stress, intracellular ROS levels were significantly lowered in cells overexpressing either wild-type PrP(c) (wt-PrP) or a deletion mutant affecting TM1 (Delta8TM1-PrP), but, as expected, not in cultures overexpressing a deletion mutant lacking the octapeptide region (Deltaocta-PrP). Overexpression of wt-PrP, Delta8TM1-PrP, or Deltaocta-PrP did not affect basal ROS levels. Interestingly, the mitochondrial membrane potential was significantly lowered in Deltaocta-PrP-transfected cultures in the absence of oxidative stress. We conclude that the protective effect of PrP(c) against oxidative stress involves the octarepeat region but not the TM1 domain nor the high-affinity copper binding site described for human residues His96/His111.

Asunto(s)

Antioxidantes/farmacología , Neuroblastoma/metabolismo , Priones/metabolismo , Animales , Cobre/metabolismo , Células HeLa , Humanos , Peróxido de Hidrógeno/farmacología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Neuroblastoma/patología , Oxidantes/farmacología , Estrés Oxidativo/efectos de los fármacos , Estructura Terciaria de Proteína , Especies Reactivas de Oxígeno/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos , Eliminación de Secuencia

RESUMEN

BACKGROUND: Poly(ADP-ribosyl)ation is a posttranslational modification of nuclear proteins catalysed by poly(ADP-ribose) polymerases (PARPs), using NAD+ as a substrate. Activation of PARP-1 is in immediate response to DNA damage generated by endogenous and exogenous damaging agents. It has been implicated in several crucial cellular processes including DNA repair and maintenance of genomic stability, which are both intimately linked with the ageing process. The measurement of cellular poly(ADP-ribosyl)ation capacity, defined as the amount of poly(ADP-ribose) produced under maximal stimulation, is therefore relevant for research on ageing, as well as for a variety of other scientific questions. RESULTS: This paper reports a new, robust protocol for the measurement of cellular poly(ADP-ribosyl)ation capacity in PBMC or Jurkat T-cells using flow cytometry, based on a previously established immuno-dot-blot assay. In order to validate the new assay, we determined the dose-response curve of 3-aminobenzamide, a well-known competitive PARP inhibitor, and we derived an IC50 that is very close to the published value. When testing a set of PBMC samples taken from fifteen healthy young human donors, we could confirm the presence of a substantial interindividual variation, as previously observed using a radiometric assay. CONCLUSION: The methodology described in this paper should be generally useful for the determination of cellular poly(ADP-ribosyl)ation capacity in a wide variety of settings, especially for the comparison of large sets of samples, such as population studies. In contrast to previously published radiometric or immuno-dot-blot assays, the new FACS-based method allows (i) selective analysis of mononuclear cells by gating and (ii) detection of a possible heterogeneity in poly(ADP-ribosyl)ation capacity between cells of the same type.