RESUMEN
BACKGROUND: The introduction of pegylated interferon (PEG-IFN)-α in the treatment of chronic hepatitis C has led to an increase in sustained virological response. Despite reduced immunogenicity of the pegylated form in comparison with native interferon (IFN)-α, a high frequency of adverse cutaneous reactions has been reported with pegylated IFN-α. Here, we aimed to investigate the immunological mechanisms underlying pegylated IFN-α-induced drug eruptions. METHODS: Hepatitis C patients suffering from drug eruptions in association with administration of pegylated interferons were enrolled in the study (n = 22). Subjects were tested for sensitivity to pegylated IFN-α2a , pegylated IFN-α2b , or ribavirin using intradermal, scratch, and/or patch tests, as well as lymphocyte activation tests (LATs). Skin biopsies obtained from pegylated IFN-α-associated exanthemas, as well as from localized inflammatory skin reactions at pegylated IFN-α injection sites, were analyzed for the expression of relevant chemokines by quantitative real-time PCR and immunohistochemistry. RESULTS: A subset of patients suffering from pegylated IFN-α-associated exanthemas displayed positive intradermal tests to PEG-IFNs but not to conventional IFN (11/22). In selected patients, this observation correlated with the presence of pegylated IFN-specific T cells (3/11). Chemokine profiles of inflammatory skin reactions at the injection sites reflected an IFN-α-signature, whereas lesional skin of exanthemas showed induction of TH2-associated chemokines. CONCLUSIONS: Our results indicate that specific sensitizations are one cause of exanthemas under therapy with PEG-IFNs. Clinical proof-of-concept analyses demonstrate that affected patients may benefit from a switch to conventional, nonpegylated drugs, enabling IFN-α therapy continuation without drug-associated skin eruptions.
Asunto(s)
Antivirales/efectos adversos , Erupciones por Medicamentos/etiología , Interferón-alfa/efectos adversos , Polietilenglicoles/efectos adversos , Antivirales/uso terapéutico , Citocinas/genética , Citocinas/metabolismo , Erupciones por Medicamentos/diagnóstico , Expresión Génica , Hepatitis C Crónica/complicaciones , Hepatitis C Crónica/tratamiento farmacológico , Humanos , Factores Reguladores del Interferón/genética , Factores Reguladores del Interferón/metabolismo , Interferón alfa-2 , Interferón-alfa/uso terapéutico , Activación de Linfocitos , Polietilenglicoles/uso terapéutico , Proteínas Recombinantes/efectos adversos , Proteínas Recombinantes/uso terapéutico , Piel/patología , Pruebas Cutáneas , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismoRESUMEN
Collagen-induced arthritis (CIA) is an experimental arthritis model used to study the inflammatory processes in this disease and test potential therapeutics. In order to better characterize this model, we conducted the first comprehensive gene expression analysis of rat CIA. To evaluate how closely the rat model reflects human rheumatoid arthritis (RA), we also analysed gene expression in human RA, using genome-wide Affymetrix gene arrays. By applying multiple strategies, including comparison of the highest induced genes, expression of immunological-associated genes as well as Ingenuity Pathway Analysis (IPA), we were able to compare the two expression profiles. Among the highest induced genes in RA were several B-cell-associated genes, including immunoglobulins, B-cell markers such as CD20, and cytokines and chemokines that act on B cells such as TNFSF13b/BLyS and CXCL13, none of which was upregulated in CIA. The latter was instead characterized by the upregulation of genes expressed primarily in macrophages and dendritic cells. Of the 22 pathways identified as significant in both diseases by IPA, only three (IL6, chemokine signalling and antigen presentation) were present in both settings. We conclude that there are significant differences in the inflammatory mechanisms between human RA and rat CIA, and that genome-wide comparative gene expression analyses are useful tools to evaluate the relevance of animal models to human disease.
Asunto(s)
Artritis Experimental/genética , Artritis Reumatoide/genética , Expresión Génica , Animales , Perfilación de la Expresión Génica , Humanos , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , RatasRESUMEN
Adenomyosis is a common gynaecological disorder characterized by the abnormal growth of endometrium into the myometrium and myometrial hypertrophy/hyperplasia. Uterine fibroids are benign neoplasms of the myometrium, and they represent a diagnostic pitfall for adenomyosis. In this study, we have used the genome-wide Affymetrix U133 Plus 2.0 microarray platform to compare the gene expression patterns of adenomyosis, uterine fibroids, normal endometrium and myometrium. Unsupervised principal component analysis (PCA) revealed that these four tissue types could be segregated from one another solely based on their gene expression profiles. Analysis of variance (ANOVA), followed by Tukey means separation test, significance analysis of microarrays (SAM) and 2-fold change threshold, identified 7415 probe sets as differentially expressed among the four groups of samples. Supervised cluster analysis based on these probe sets clustered adenomyosis most closely with endometrium and uterine fibroids with myometrium, consistent with the anatomic origin of these two diseases. The Tukey means separation post hoc testing found 2073 probe sets altered between adenomyosis and normal endometrium or myometrium, and 2327 probe sets altered in expression when comparing uterine fibroids with myometrium. Using Ingenuity Pathways Analysis (IPA), we found 9 highly significant functional networks in adenomyosis and 10 in uterine fibroids. Notably, the top network in both cases was associated with functions implicated in cancer and cell death. Finally, we compared the gene expression profiles of adenomyosis and uterine fibroids and identified 471 differentially expressed probe sets that may represent potential biomarkers for the differential diagnosis of these diseases.
Asunto(s)
Endometriosis/genética , Perfilación de la Expresión Génica , Análisis de Varianza , Biomarcadores , Biomarcadores de Tumor , Diagnóstico Diferencial , Endometriosis/diagnóstico , Endometriosis/metabolismo , Endometriosis/patología , Endometrio/metabolismo , Femenino , Humanos , Leiomioma/diagnóstico , Leiomioma/genética , Leiomioma/metabolismo , Miometrio/metabolismo , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Análisis de Componente Principal , Neoplasias Uterinas/diagnóstico , Neoplasias Uterinas/genética , Neoplasias Uterinas/metabolismoRESUMEN
CC chemokine receptor 7 (CCR7) is involved in the initiation of immune responses by mediating the migration of naïve T cells and mature dendritic cells to T-cell-rich zones of secondary lymphoid organs where antigen presentation occurs. To address whether CCR7 plays a role in the development of autoimmunity, we induced experimental autoimmune encephalomyelitis in CCR7-deficient mice on a C57BL/6 background (CCR7(-/-)) using the neuroantigen, myelin oligodendrocyte glycoprotein 35-55 amino acid peptide (MOG((35-55))) and Bordetella pertussis toxin (PTX). CCR7(-/-) mice acquired disease with an intensity similar to wild-type littermates. MOG((35-55))-specific lymphocyte responses were dominant in the spleen of CCR7(-/-) mice, rather than in lymph nodes as observed in wild-type mice. These results indicate that effective immune responses (with altered kinetics) can develop in the absence of CCR7 but develop in the spleen rather than lymph nodes as CCR7 is necessary for T and dendritic cells to enter lymph nodes.
Asunto(s)
Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/inmunología , Receptores de Quimiocina/deficiencia , Receptores de Quimiocina/genética , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Secuencia de Aminoácidos , Animales , Movimiento Celular/genética , Movimiento Celular/inmunología , Células Cultivadas , Quimiocinas CC/metabolismo , Enfermedad Crónica , Células Dendríticas/citología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Encefalomielitis Autoinmune Experimental/patología , Femenino , Inmunofenotipificación , Ganglios Linfáticos/citología , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Receptores CCR7 , Receptores de Quimiocina/fisiología , Bazo/inmunología , Bazo/metabolismo , Bazo/patología , Subgrupos de Linfocitos T/patologíaRESUMEN
Genomics-based approaches are increasingly being used to identify disease-associated genes that represent potential new drug targets. As a first step in the validation of genes of unknown function, we describe a method for rapidly determining the subcellular localization of the gene product. If an immunotherapeutic approach is being considered, it is of particular interest to identify targets that are either on the cell-surface or secreted. Transient expression in COS cells combined with immunofluorescent staining provides a semi-high throughput method for determining the subcellular localization of multiple targets in parallel. COS cells are ideal for this purpose since: (i) they transfect easily; (ii) the high levels of expression that can be achieved transiently allow detection after 24 h; and (iii) the relatively large size and spread morphology of these cells allows the subcellular organelles to be easily visualized. To evaluate the system, we show prototype staining patterns for known cytoplasmic,secreted, Golgi-associated, endoplasmic reticulum-associated, and plasma membrane proteins, as well as data for novel targets. The localization of novel secretory and cell-surface proteins as determined by immunofluorescent staining, was confirmed by independent methods.
RESUMEN
The v-abl and v-src oncogenes encode activated cytoplasmic tyrosine kinases with considerable sequence similarity. The v-abl oncogene of the Abelson murine leukemia virus exhibits a narrow tissue tropism for transformation, almost exclusively forming pre-B-cell tumors, while the v-src oncogene can induce a variety of sarcomas and other tumors. To localize the determinants of the narrow tropism of the v-abl gene, we generated a series of hybrid retroviral genomes containing portions of the v-abl and v-src oncogenes in a Moloney murine leukemia virus backbone. Each virus was tested for transforming activity in NIH3T3 cells; for transforming activity on bone marrow cultures; and for pathogenicity in newborn mice. Many of the hybrid oncogenes carried by these viruses exhibited transforming activity, and demonstrate that the SH2 domain of each oncogene can be utilized by the kinase domain of the other oncogene for that activity. The results further suggest that a portion of the C-terminal region of v-abl is necessary for the pre-B-cell specificity of the oncogene.
Asunto(s)
Transformación Celular Viral , Genes abl , Genes src , Células 3T3 , Animales , Animales Recién Nacidos , Células de la Médula Ósea , Células Cultivadas , ADN Recombinante , Reordenamiento Génico de Cadena Pesada de Linfocito B , Genes de Inmunoglobulinas , Técnicas In Vitro , Ratones , Ratones Endogámicos BALB C , Neoplasias Experimentales/genética , Proteínas Recombinantes de Fusión/genética , Retroviridae/genéticaRESUMEN
Two sets of mutants of the Abelson murine leukemia virus, generated by linker insertion mutagenesis of a cloned proviral DNA, were tested for their ability to transform bone marrow cultures in vitro. All the viruses retained an intact tyrosine kinase domain and were competent for transformation of NIH3T3 fibroblasts in culture. One series contained 12-bp linker insertions in the regions flanking the kinase domain, and the other contained frameshift mutations that truncated the gene product downstream of the kinase domain. The majority of the 12-bp insertion mutants retained full bone marrow-transforming activity; only one insertion in the SH2 domain showed reduced activity. This mutant suggests that some aspect of the SH2 domain may be more important in transformation of lymphocytes than fibroblasts. In contrast to the first set of mutants, the bone marrow-transforming activity of the majority of the truncation mutants was significantly reduced or completely lost. We conclude that there is a broad requirement for an intact C-terminal domain of the v-abl protein for the transformation of pre-B cells, but that no single part of this domain is critical.
Asunto(s)
Virus de la Leucemia Murina de Abelson/genética , Linfocitos B/patología , Transformación Celular Neoplásica , Células Madre Hematopoyéticas/patología , Mutagénesis Insercional , Células 3T3 , Animales , Médula Ósea/patología , Células Cultivadas , ADN Viral/análisis , Ratones , Mutación , Proteínas Oncogénicas v-abl/análisis , Proteínas Oncogénicas v-abl/toxicidad , Relación Estructura-ActividadRESUMEN
A series of recombinant retroviral genomes was generated by cotransformation of NIH 3T3 cells with a mixture of cloned DNAs: a proviral copy of the wild-type Moloney murine leukemia virus, and Moloney-based vectors containing defective copies of the chicken v-src and the murine v-abl oncogenes. Morphologically transformed foci, appearing at low frequencies in these cultures, released high titers of transforming viruses. Analysis of one group of these viruses showed that the genomes were recombinants containing portions of the viral gag gene juxtaposed to the v-src oncogene. Biologically active cloned DNAs of two of these viruses were obtained and mapped in detail. One of these viruses did not cause disease after inoculation into newborn mice, but the other induced rapidly fatal hemangiosarcomas located exclusively in the brain.