RESUMEN
Sialic acid acetylesterase (SIAE) is an enzyme that negatively regulates B lymphocyte antigen receptor signalling and is required for the maintenance of immunological tolerance in mice. Heterozygous loss-of-function germline rare variants and a homozygous defective polymorphic variant of SIAE were identified in 24/923 subjects of European origin with relatively common autoimmune disorders and in 2/648 controls of European origin. All heterozygous loss-of-function SIAE mutations tested were capable of functioning in a dominant negative manner. A homozygous secretion-defective polymorphic variant of SIAE was catalytically active, lacked the ability to function in a dominant negative manner, and was seen in eight autoimmune subjects but in no control subjects. The odds ratio for inheriting defective SIAE alleles was 8.6 in all autoimmune subjects, 8.3 in subjects with rheumatoid arthritis, and 7.9 in subjects with type I diabetes. Functionally defective SIAE rare and polymorphic variants represent a strong genetic link to susceptibility in relatively common human autoimmune disorders.
Asunto(s)
Acetilesterasa/genética , Enfermedades Autoinmunes/enzimología , Enfermedades Autoinmunes/genética , Autoinmunidad/genética , Hidrolasas de Éster Carboxílico/genética , Predisposición Genética a la Enfermedad/genética , Mutación de Línea Germinal/genética , Ácido N-Acetilneuramínico/metabolismo , Acetilación , Acetilesterasa/metabolismo , Alelos , Animales , Anticuerpos Antinucleares/sangre , Artritis Reumatoide/enzimología , Artritis Reumatoide/genética , Linfocitos B/metabolismo , Biocatálisis , Hidrolasas de Éster Carboxílico/metabolismo , Estudios de Casos y Controles , Línea Celular , Diabetes Mellitus Tipo 1/enzimología , Diabetes Mellitus Tipo 1/genética , Europa (Continente)/etnología , Exones/genética , Humanos , Ratones , Oportunidad Relativa , Polimorfismo de Nucleótido Simple/genética , Tamaño de la Muestra , Análisis de Secuencia de ADNRESUMEN
Facilitating cells (FC) are bone marrow-derived cells that facilitate allogeneic hematopoietic stem cell (SC) engraftment and induce transplantation tolerance without causing graft vs. host disease. Although there is evidence for FC directing the development of FoxP3+CD4+CD25+ regulatory T cells, the specific FC subsets that control regulatory T cell development have not been defined. The current study investigates the role of FC-CD3epsilon+ and FC-CD3epsilon- subpopulations in the development of FoxP3+CD4+CD25+ regulatory T cells. Here, we demonstrate that the induction of FoxP3+CD4+CD25+ regulatory T cells in coculture is mediated by not only the FC-CD3epsilon- subset but also the FC-CD3epsilon+ subset, which is distinct from plasmacytoid precursor dendritic cells (p-preDC). The identification of cell populations distinct from p-preDC that efficiently induce the generation of FoxP3+CD4+CD25+ regulatory T cells may prove useful for future therapeutic applications for the induction of tolerance following allogeneic SC transplantation.
Asunto(s)
Células de la Médula Ósea/inmunología , Células Dendríticas/inmunología , Factores de Transcripción Forkhead/biosíntesis , Subunidad alfa del Receptor de Interleucina-2/biosíntesis , Linfocitos T Reguladores/inmunología , Animales , Células de la Médula Ósea/citología , Antígenos CD4/biosíntesis , Diferenciación Celular/inmunología , Células Cultivadas , Técnicas de Cocultivo , Citocinas/biosíntesis , Citocinas/genética , Femenino , Expresión Génica/inmunología , Genes Codificadores de la Cadena beta de los Receptores de Linfocito T , Interferón gamma/biosíntesis , Interferón gamma/genética , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/biosíntesis , Subgrupos de Linfocitos T/inmunología , Linfocitos T Reguladores/citología , Receptor Toll-Like 9/biosíntesis , Receptor Toll-Like 9/genética , Factor de Crecimiento Transformador beta/biosíntesis , Factor de Crecimiento Transformador beta/genéticaRESUMEN
Catalytically defective rare variants of Sialic acid Acetyl Esterase (SIAE) have previously been linked to autoimmunity. Studies presented here confirm that the M89V SIAE protein and all other products of common variant alleles of SIAE are catalytically normal. Although overexpressing transfected non-lymphoid cells secrete small amounts of SIAE that can associate with the cell surface, normal human lymphocytes do not exhibit cell surface SIAE, supporting genetic evidence in mice that indicates that this protein functions in a lymphocyte intrinsic manner. Analyses of the plasma proteome also indicate that SIAE is not secreted in vivo. A re-analysis exclusively of catalytically defective rare variant alleles of SIAE in subjects in which this gene was completely sequenced confirmed an association of SIAE with autoimmunity. A subset of catalytically defective rare variant SIAE alleles has previously been typed in a large genotyping study comparing a diverse group of disease subjects and controls; our re-analysis of this data shows that catalytically defective alleles are enriched in disease subjects. These data suggest that SIAE may be associated with autoimmunity and that further study of catalytically defective rare variant SIAE alleles in terms of autoimmune disease susceptibility is strongly warranted.
Asunto(s)
Acetilesterasa/genética , Enfermedades Autoinmunes/genética , Autoinmunidad , Mutación , Polimorfismo de Nucleótido Simple , Acetilesterasa/metabolismo , Alelos , Animales , Enfermedades Autoinmunes/enzimología , Biocatálisis , Pruebas de Enzimas , Expresión Génica , Frecuencia de los Genes , Predisposición Genética a la Enfermedad , Genotipo , Células HEK293 , Humanos , RatonesRESUMEN
OBJECTIVES: Lung cancer is associated with a significant rate of locoregional recurrence after surgical resection, particularly when nonanatomic wedge resections are performed. The primary aim of this study was to assess the feasibility of a microsphere drug delivery system to locally deliver chemotherapy and prevent the establishment and growth of lung cancer cells and establish proof of concept for a potential future approach to target occult microscopic disease remaining at the surgical resection margin. METHODS: Poly-(D,L-lactic-co-glycolic acid) (PLGA) microspheres loaded with the antineoplastic agent paclitaxel were prepared and tested for antitumor efficacy in an in vitro cell proliferation assay for tumor inhibition and induction of apoptosis. The in vivo prevention of Lewis lung carcinoma cell establishment and growth in subcutaneous tissues of mice was also assessed by comparing 4 treatment groups: Lewis lung carcinoma cells alone, Lewis lung carcinoma cells combined with 100 x 10(6) unloaded (carrier alone) PLGA microspheres, and Lewis lung carcinoma cells combined with 50 x 10(6) or 100 x 10(6) paclitaxel-loaded PLGA microspheres. After the coinjection of Lewis lung carcinoma cells with or without microspheres, in vivo tumor growth was monitored, and tumor weight was recorded on death. RESULTS: Paclitaxel-loaded PLGA microspheres were found to effectively prevent growth of tumor cells in culture through the induction of apoptosis. Similarly, paclitaxel-loaded PLGA microspheres significantly inhibited tumor growth in vivo at both the 50 x 10(6) and 100 x 10(6) microsphere dose (0.497 +/- 0.183 and 0.187 +/- 0.083 g total tumor weight, respectively) compared with 2.91 +/- 0.411 g for Lewis lung carcinoma cells with unloaded microspheres and 3.37 +/- 0.433 g for untreated tumor (P < .001). Toxicity was not clinically apparent in any animal treated with paclitaxel-loaded PLGA microspheres. CONCLUSIONS: Paclitaxel-loaded PLGA microspheres induce tumor apoptosis and inhibit the establishment and growth of lung cancer cells both in vitro and in vivo without obvious systemic toxicity. By using models consistent with localized microscopic tumor burdens, these results suggest that local delivery of paclitaxel through a microsphere system might lead to an effective future method of decreasing local tumor recurrence in non-small cell lung cancer when applied to the surgical margins at risk for microscopic tumor foci. Such an approach might be particularly efficacious after wedge resection in the setting of poor pulmonary reserve or significant comorbidity, where local recurrence rates are increased and acceptable alternative treatment options are limited.
Asunto(s)
Antineoplásicos Fitogénicos/administración & dosificación , Neoplasias Pulmonares/prevención & control , Recurrencia Local de Neoplasia/prevención & control , Paclitaxel/administración & dosificación , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Estudios de Factibilidad , Femenino , Ratones , Ratones Endogámicos C57BL , MicroesferasRESUMEN
The establishment of donor cell lineages following allogeneic bone marrow transplantation is frequently associated with the development of graft-vs-host disease (GVHD). The identification of cell populations that are capable of supporting allogeneic stem cell (SC) engraftment and the induction of tolerance without inducing GVHD could expand the use of this therapy. CD8(+)TCR(-) facilitating cells (FC) have been shown to promote allogeneic SC engraftment with resulting transplantation tolerance across complete MHC barriers without inducing GVHD. Although donor reconstitution in SC plus FC recipients is associated with the induction of regulatory T cell-associated factors, it is not known whether an induction of regulatory T cells and subsequent tolerance is a direct effect of the FC. The current study demonstrates that 1) SC plus FC transplantation results in the induction of donor CD4(+)25(+) regulatory T cells and that FC are present in the spleen of recipients before the induction of these cells, 2) activation of FC with CpG-oligodeoxynucleotide promotes CD4(+)25(-) T cell differentiation into CD4(+)25(+) regulatory T cells in vitro, as demonstrated by cytokine and forkhead/winged helix transcription factor (FoxP3) gene and protein expression, and 3) direct contact between FC and CD4(+)25(-) T cells is required for FoxP3(+)CD4(+)25(+) regulatory T cell induction and is dependent on CD86 expression on FC. This is the first report to demonstrate a mechanism for FC in the induction of regulatory T cells following allogeneic SC plus FC transplantation. The transplantation of donor FC may provide an alternative approach to permit clinical SC engraftment and induction of transplantation tolerance in the future.
Asunto(s)
Células de la Médula Ósea/inmunología , Factores de Transcripción Forkhead/inmunología , Supervivencia de Injerto/inmunología , Trasplante de Células Madre Hematopoyéticas , Linfocitos T Reguladores/inmunología , Tolerancia al Trasplante/inmunología , Animales , Antígeno B7-2/inmunología , Antígeno B7-2/metabolismo , Células de la Médula Ósea/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/inmunología , Citocinas/inmunología , Citocinas/metabolismo , Femenino , Factores de Transcripción Forkhead/biosíntesis , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Enfermedad Injerto contra Huésped/inmunología , Enfermedad Injerto contra Huésped/metabolismo , Ratones , Oligonucleótidos/farmacología , Linfocitos T Reguladores/metabolismo , Tolerancia al Trasplante/efectos de los fármacos , Trasplante HomólogoRESUMEN
Transplantation of purified allogeneic hemopoietic stem cells (SC) alone is characterized by a decreased risk of graft-vs-host disease but increased incidence of engraftment failure. It has been established that the facilitating cell (FC) promotes allogeneic SC reconstitution and results in donor-specific transplantation tolerance across MHC disparities, without graft-vs-host disease. Although the requirements for this facilitating function are not well-characterized, it is known that facilitation is dependent on FC expression of a unique heterodimer consisting of the TCR beta-chain (TCRbeta) and a 33-kDa protein, FCp33. The current study confirms that CD3epsilon and TCRbeta expression are present on the FC at the time of transplantation and demonstrates that the majority of cells in the FC population express the TCR signaling molecule, FcRgamma, rather than the more conventional CD3zeta receptor. Of particular significance, we have now demonstrated that FC-mediated allogeneic SC reconstitution is critically dependent on FcRgamma expression and that FcRgamma coprecipitates with the TCRbeta-FCp33 heterodimer. The mandatory requirement of TCRbeta and FcRgamma for FC function provides the first evidence of a previously undescribed role for FcRgamma in the facilitation of allogeneic SC reconstitution and establishes that FcRgamma is part of the TCRbeta-FCp33 complex uniquely expressed on FC.
Asunto(s)
Trasplante de Médula Ósea/inmunología , Proteínas Portadoras/fisiología , Trasplante de Células Madre Hematopoyéticas , Isoantígenos/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/fisiología , Receptores de IgG/fisiología , Animales , Complejo CD3/biosíntesis , Complejo CD3/genética , Complejo CD3/fisiología , Dimerización , Femenino , Células Madre Hematopoyéticas/inmunología , Células Madre Hematopoyéticas/metabolismo , Inmunofenotipificación , Isoantígenos/biosíntesis , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Antígenos de Linfocitos T alfa-beta/biosíntesis , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Receptores de IgG/biosíntesis , Receptores de IgG/metabolismo , Transcripción GenéticaRESUMEN
Graft-versus-host disease (GVHD) and failure of engraftment limit clinical bone marrow transplantation (BMT) to patients with closely matched donors. Engraftment failure of purified allogeneic hematopoietic stem cells (HSCs) has been decreased in various BMT models by including donor BM-derived CD8(+)/alphabetagammadeltaTCR(-) facilitating cells (FCs) or CD8(+)/alphabetaTCR(+) T cells in the BM inoculum. To aggressively investigate the GVHD potential of these donor CD8(+) populations, a purified cell model of lethal GVHD was established in a murine semiallogeneic parent --> F(1) combination. Lethally irradiated recipients were reconstituted with purified donor HSCs alone or in combination with splenic T cells (T(SP)), BM-derived T cells (T(BM)), or the FC population. In marked contrast to the lethal GVHD present in recipients of HSCs plus T(SP) or CD8(+) T(BM), recipients of donor HSC+FC inocula did not exhibit significant clinical or histologic evidence of GVHD. Instead, HSC+FC recipients were characterized by increased splenocyte expression of transforming growth factor-beta (TGF-beta) and the induction of the regulatory T-cell genes CTLA4, GITR, and FoxP3. These findings suggest that the FCs, which express a unique FCp33-TCRbeta heterodimer in place of alphabetaTCR, permits HSC alloengraftment and prevents GVHD through the novel approach of regulatory T-cell induction in vivo.