RESUMO
We recently identified Galectin-1 (Gal-1), a ß-galactoside-binding lectin, as a novel immune regulator in neuroblastoma (NB). Here, we characterized the tolerogenic function of Gal-1 within the CD8+ T cell compartment and further evaluated its relevance as an antigen for effective DNA vaccination against NB in a mouse model. NB cells with Gal-1 knockdown (NXS-2L) exhibited significantly reduced tumor growth compared to NXS-2 NB cells. Administration of anti-CD8 antibodies prevented this antitumor effect, with primary tumor growth comparable to that from Gal-1 (G1)-sufficient NB cells. Peptide epitope screening with online databases and in silico docking experiments predicted the sequences "FDQADLTI" (#1), "GDFKIKCV" (#2), and "AHGDANTI" (#3) to have superior H2-KK binding affinities and "KFPNRLNM" (#4), "DGDFKIKCV" (#5), and "LGKDSNNL" (#6) to have superior H2-DD binding affinities. Minigenes encoding G1-KK (#1-#2-#3), G1-DD (#4-#5-#6) and the triplet with the highest affinity, G1-H (#1-#2-#4), were generated and cloned into a ubiquitin-containing plasmid (pU). Mice receiving pU-G1-KK or pU-G-1H presented a reduction in the s.c. tumor volume and weight of up to 80% compared to control mice; this reduction was associated with increased cytotoxicity of isolated splenocytes from vaccinated animals. Vaccination with pUG1-DD showed a lower capability to suppress primary tumor progression. In conclusion, Gal-1 expression by NB negatively regulates CD8+ T cells. Vaccination with DNA plasmids encoding Gal-1 epitopes overcomes immune escape, enhances CD8+ T cell-dependent immunity and displays effective antitumor activity against NB.
Assuntos
Vacinas Anticâncer/farmacologia , Galectina 1/imunologia , Epitopos Imunodominantes , Neuroblastoma/tratamento farmacológico , Animais , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/genética , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral , Citotoxicidade Imunológica/efeitos dos fármacos , Mapeamento de Epitopos , Feminino , Galectina 1/genética , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/imunologia , Camundongos , Neuroblastoma/genética , Neuroblastoma/imunologia , Carga Tumoral/efeitos dos fármacos , Evasão Tumoral/efeitos dos fármacos , Microambiente Tumoral , Vacinação , Vacinas de DNA/farmacologiaRESUMO
Congenital plasminogen (Plg) deficiency leads to the development of ligneous membranes on mucosal surfaces. Here, we report our experience with local and intravenous fresh frozen plasma (FFP). We retrospectively reviewed medical files of 17 patients and their eight first-degree relatives. Conjunctivitis was the main complaint. Thirteen patients were treated both with intravenous and conjunctival FFP. Venous thrombosis did not develop in any. Genetic evaluation revealed heterogeneous mutations as well as polymorphisms. Diagnosis and treatment of Plg deficiency is challenging; topical and intravenous FFP may be an alternative treatment.
Assuntos
Transfusão de Componentes Sanguíneos , Conjuntivite/terapia , Doenças Genéticas Inatas/terapia , Plasma , Plasminogênio/deficiência , Pré-Escolar , Conjuntivite/diagnóstico , Conjuntivite/genética , Feminino , Doenças Genéticas Inatas/diagnóstico , Doenças Genéticas Inatas/genética , Humanos , Lactente , Recém-Nascido , Masculino , Mutação , Polimorfismo GenéticoRESUMO
Heme oxygenase (HO)-1 catalyzes the degradation of cytotoxic heme into biliverdin and blocks antitumor immune responses, thus protecting cancer against host defense. Whether this scenario also applies to neuroblastoma (NB), the most common extracranial solid childhood tumor, is not known. Here, we demonstrate for the first time a prognostic relevance of HO-1 expression in samples from NB patients and show that targeting of HO-1 prevents both cancer resistance against cellular stress and immune escape in the syngeneic NXS2 A/J mouse model of NB. High HO-1 RNA expression in NB tissues emerged as unfavorable prognostic marker, in particular for patients older than 18 months as indicated by univariate as well as multivariate survival probability analyses including disease stage and MYCN status. On the basis of this observation we aimed to target HO-1 by systemic as well as tumor-specific zinc protoporphyrin-mediated HO-1 suppression in a syngeneic immunocompetent NB mouse model. This resulted in 50% reduction of primary tumor growth and a suppression of spontaneous liver metastases. Importantly, HO-1 inhibition abrogated immune cell paralysis affecting CD4 and CD8 T-effector cells. This in turn reverted HO-1-dependent immune escape mechanisms in NB by increasing NB apoptosis and improved DC maturation. In summary, HO-1 emerges as a novel immune regulator in NB and emerges as a promising target for the development of therapeutic approaches.
Assuntos
Biomarcadores Tumorais/análise , Heme Oxigenase-1/imunologia , Neuroblastoma/imunologia , Evasão Tumoral/imunologia , Animais , Western Blotting , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Modelos Animais de Doenças , Feminino , Heme Oxigenase-1/metabolismo , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Camundongos , Neuroblastoma/enzimologia , Neuroblastoma/patologia , Prognóstico , Modelos de Riscos Proporcionais , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Reliable diagnostic strategies for individuals with cancer demand practical methods for highly sensitive and specific detection of tumor cells. Amplification of genomic regions that include putative oncogenes is common in tumor cells of various types. Genomic array platforms offer the opportunity to identify and precisely map amplified genomic regions (ampGRs). The stable existence of these tumor cellspecific genomic aberrations during and after therapy, in theory, make ampGRs optimal targets for cancer diagnostics. In this study, we mapped ampGRs around the proto-oncogene MYCN of human neuroblastomas using a high-resolution tiling array (HR-TA). Based on the HR-TA data, we were able to precisely describe the telomeric and centromeric borders of the ampGRs and deduce virtual fusion sites of the joined ampGRs (amplicon fusion sites [AFSs]). These AFSs served as blueprints for the subsequent design of AFS bridging PCR assays (AFS-PCRs). Strikingly, these assays were absolutely tumor cell specific and capable of detecting 1 tumor cell in 1 × 10(6) to 8 × 10(6) control cells. We successfully proved the in vivo practicability of AFS-PCR by detecting and quantifying the specific AFS DNA of human MYCN-amplified neuroblastomas in the patients' corresponding peripheral blood and bone marrow samples. Thus, we believe AFS-PCR could become a powerful and nevertheless feasible personalized diagnostic tool applicable to a large number of cancer patients, including children with MYCN-amplified neuroblastomas.
Assuntos
Neuroblastoma/diagnóstico , Neuroblastoma/genética , Reação em Cadeia da Polimerase/métodos , Sequência de Bases , Linhagem Celular Tumoral , Criança , Humanos , Dados de Sequência Molecular , Neuroblastoma/patologia , Oncogenes , Proto-Oncogene MasRESUMO
PURPOSE: We compared the expression of the insulin receptor-related receptor (IRR) in primary human neuroblastomas with other biological and clinical parameters and the impact of its expression on prognostic outcome. EXPERIMENTAL DESIGN: We studied 49 neuroblastomas of different clinical stages and histological subtypes for (a) IRR, insulin-like growth factor 1 receptor (IGF-1R), TrkA, p75 neurotrophin receptor, and MYCN mRNA expression by reverse transcription-PCR; (b) MYCN gene amplification by Southern blot analyses; (c) cyclin A protein expression by Western blot analyses indicating proliferation rate; and (d) apoptotic index (AI) by terminal deoxynucleotidyl transferase (Tdt)-mediated dUTP nick end-labeling assay. RESULTS: IRR mRNA expression was found in 25 (51%) neuroblastomas and correlated with stages 1, 2, 3, and 4S disease and with age =12 months at diagnosis. IRR was expressed predominantly in neuroblastomas without MYCN gene amplification and coexpressed with IGF-1R, TrkA, and p75 neurotrophin receptor. IRR mRNA expression also correlated with an undifferentiated histology but not with the proliferation rate. In coexpression with IGF-1R, the IRR was associated with enhanced AI. IRR expression was significantly correlated with a good prognosis in all 49 neuroblastomas (6-year survival probability, 91.8% versus 49.7% for IRR nonexpression; P = 0.003). CONCLUSION: IRR expression is a new marker for a favorable prognosis in neuroblastoma that is independent of MYCN amplification and age at diagnosis. Our data suggest an influence of IRR on IGF signaling via IGF-1R because coexpression of these two receptor tyrosine kinases was significantly correlated with an undifferentiated histology, a high AI, and an advanced survival probability.