RESUMO
Arginine is a semi-essential amino acid which serves as a substrate for nitric oxide (NO) production by nitric oxide synthase (NOS) and a precursor for various metabolites including ornithine, creatine, polyamines, and agmatine. Arginase competes with nitric oxide synthase for substrate arginine to produce orthinine and urea. There is contradictory evidence in the literature on the role of nitric oxide in the pathophysiology of traumatic brain injury (TBI). These contradictory perspectives are likely due to different NOS isoforms - endothelial (eNOS), inducible (iNOS) and neuronal (nNOS) which are expressed in the central nervous system. Of these, the role of nNOS in acute injury remains less clear. This study aimed to employ a genetic approach by overexpressing arginase isoforms specifically in neurons using a Thy-1 promoter to manipulate cell autonomous NO production in the context of TBI. The hypothesis was that increased arginase would divert arginine from pathological NO production. We generated 2 mouse lines that overexpress arginase I (a cytoplasmic enzyme) or arginase II (a mitochondrial enzyme) in neurons of FVB mice. We found that two-weeks after induction of controlled cortical injury, overexpressing arginase I but not arginase II in neurons significantly reduced contusion size and contusion index compared to wild-type (WT) mice. This study establishes enhanced neuronal arginase levels as a strategy to affect the course of TBI and provides support for the potential role of neuronal NO production in this condition.
Assuntos
Arginase/genética , Lesões Encefálicas Traumáticas/genética , Neurônios/enzimologia , Óxido Nítrico/genética , Animais , Arginina/metabolismo , Lesões Encefálicas Traumáticas/patologia , Linhagem Celular , Modelos Animais de Doenças , Regulação Enzimológica da Expressão Gênica , Humanos , Camundongos , Neurônios/patologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo I/genética , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo III/genética , Antígenos Thy-1/genéticaRESUMO
PURPOSE: Cerebrospinal cavernous malformations (CCMs) are vascular lesions characterized by dilated and leaky capillary caverns. CCMs can cause seizures, focal neurological deficits or acute intracranial hemorrhage; however, most patients are asymptomatic. CCMs occur either sporadically or as a familial autosomal-dominant disorder. We present a clinical and molecular study of a patient with distinctive cerebral and spinal cavernous malformations following radiochemotherapy for a malignant brain tumor. METHODS: The patient had multiple magnet resonance imaging (MRI) examinations of his brain and spine following radiochemotherapy for a primary intracranial germ cell tumor (GCT), as part of his oncologic follow-up. The MRI sequences included susceptibility-weighted imaging (SWI). The coding exons and their flanking intronic regions of KRIT1/CCM1 gene were analyzed for mutations by polymerase chain reaction (PCR) and direct sequencing. RESULTS: MRI revealed numerous cerebral and spinal microhemorrhages and pronounced cavernous malformations that progressed with subsequent follow-up imaging. Genetic analysis demonstrated a novel heterozygous KRIT1/CCM1 two base pair deletion (c.1535_1536delTG) in exon 14. This deletion leads to a frameshift with a premature stop codon at nucleotide position 1553 and a highly likely loss of function of the KRIT1 protein. CONCLUSION: We describe a patient with a novel heterozygous germ line loss of function mutation in KRIT1, which is associated with rapid-onset and highly progressive CCMs after radiochemotherapy for a malignant brain tumor.
Assuntos
Quimiorradioterapia/efeitos adversos , Hemangioma Cavernoso do Sistema Nervoso Central/etiologia , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Proteína KRIT1/genética , Mutação com Perda de Função/genética , Análise Mutacional de DNA , Seguimentos , Hemangioma Cavernoso do Sistema Nervoso Central/diagnóstico por imagem , Humanos , Processamento de Imagem Assistida por Computador , Imageamento por Ressonância Magnética , Masculino , Neoplasias Meníngeas/genética , Neoplasias Meníngeas/terapia , Neoplasias Embrionárias de Células Germinativas/genética , Neoplasias Embrionárias de Células Germinativas/terapia , Adulto JovemRESUMO
B-cell acute lymphoblastic leukemia (B-ALL) is the commonest childhood cancer and the prognosis of children with relapsed or therapy refractory disease remains a challenge. Treatment with chimeric antigen receptor-modified T cells targeting the CD19 antigen (CART-19 therapy) has been presented as a promising approach toward improving the outcome of relapsed or refractory disease. However, 10%-20% of the patients suffer another relapse. Epitope-loss under therapy pressure has been suggested as a mechanism of tumor cells to escape the recognition from CART-19 therapy. In this work, we analyzed the expression of CD19 isoforms in a cohort of 14 children with CD19 B-ALL and 6 nonleukemia donors. We showed that an alternatively spliced CD19 mRNA isoform lacking exon 2, and therefore the CART-19 epitope, but not isoforms lacking the transmembrane and cytosolic domains are expressed in leukemic blasts at diagnosis in children and in the bone marrow of nonleukemia donors. Furthermore, we clarified the sequence of a further isoform lacking the epitope recognized by CART-19 therapy and disclosed the presence of new isoforms. In comparison with the children, we showed that alternatively spliced CD19 mRNA isoforms affecting exon 2 are also expressed in 6 adult patients with CD19 B-ALL. On top of that, one of the adults expressed an isoform lacking the CD19 transmembrane and cytosolic domains. In conclusion, we proved that some of the CD19 isoforms contributing to CART-19 escape already preexist at diagnosis and could evolve as a dominant clone during CART-19 therapy suggesting the application of combined treatment approaches.
Assuntos
Antígenos CD19/metabolismo , Vacinas Anticâncer/imunologia , Epitopos de Linfócito T/metabolismo , Imunoterapia Adotiva/métodos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Isoformas de Proteínas/metabolismo , Linfócitos T/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos CD19/genética , Criança , Pré-Escolar , Estudos de Coortes , Epitopos de Linfócito T/genética , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Isoformas de Proteínas/genética , Receptores de Antígenos de Linfócitos T/genética , Proteínas Recombinantes de Fusão/genética , Especificidade do Receptor de Antígeno de Linfócitos T , Linfócitos T/transplante , Resultado do Tratamento , Evasão Tumoral , Adulto JovemRESUMO
BACKGROUND: Considering the improved outcome, a better understanding of the late effects in Wilms tumor survivors (WT-S) is needed. This study was aimed at evaluating renal function and determining the prevalence of clinical and subclinical renal dysfunction in a cohort of WT-S using a multimodal diagnostic approach. METHODS: Thirty-seven WT-S were included in this prospective cross-sectional single center study. To evaluate renal function, glomerular filtration rate (GFR) and urinary protein excretion were assessed. Additionally, kidney sonomorphology and blood pressure were analyzed. RESULTS: All examined WT-S (mean age 28.7 years, mean follow-up 24.8 years) had been treated with a combination of surgery and chemotherapy; 59.5% had received adjuvant radiotherapy. Impaired glomerular renal function was detected in a considerable proportion of WT-S, with age-adjusted cystatin-based GFR estimation below age norm in 55.9%. A lower cystatin-based estimated GFR (eGFR) correlated with longer follow-up time and higher irradiation dose. In 5 patients (13.5%) albuminuria was identified. Analysis of sonomorphology detected compensatory contralateral renal hypertrophy in 83.3% of WT-S. Chronic kidney disease (CKD) ≥ stage II was present in 55.9% of WT-S. Blood pressure measurements revealed arterial hypertension in 15 (40.5%) WT-S (newly diagnosed n=10). In 24.3% both CKD ≥ stage II and arterial hypertension were determined. CONCLUSION: Even though WT-S are believed to carry a low risk for end-stage renal disease, in this study, a remarkable number of WT-S presented with previously unidentified subclinical signs of renal function impairment and secondary morbidity. Therefore, it is important to continue regular follow-up, especially after transition into adulthood.
Assuntos
Sobreviventes de Câncer/estatística & dados numéricos , Neoplasias Renais/fisiopatologia , Rim/fisiopatologia , Insuficiência Renal Crônica/epidemiologia , Tumor de Wilms/fisiopatologia , Adolescente , Adulto , Antineoplásicos/efeitos adversos , Quimioterapia Adjuvante , Estudos Transversais , Feminino , Seguimentos , Taxa de Filtração Glomerular , Humanos , Rim/diagnóstico por imagem , Rim/efeitos dos fármacos , Rim/cirurgia , Neoplasias Renais/mortalidade , Neoplasias Renais/terapia , Masculino , Pessoa de Meia-Idade , Nefrectomia/efeitos adversos , Nefrectomia/métodos , Prevalência , Estudos Prospectivos , Proteinúria/epidemiologia , Proteinúria/fisiopatologia , Proteinúria/urina , Insuficiência Renal Crônica/fisiopatologia , Insuficiência Renal Crônica/urina , Ultrassonografia , Tumor de Wilms/mortalidade , Tumor de Wilms/terapia , Adulto JovemRESUMO
High grade neuroepithelial tumor of the central nervous system with BCOR alteration (CNS HGNET-BCOR) is a recently described new tumor entity with a dismal prognosis. The objective of this study was to identify and validate pathways deregulated in CNS HGNET-BCOR as basis for targeted therapy approaches.We characterized the BCOR alteration in a pediatric patient with CNS HGNET-BCOR diagnosis by Sanger sequencing and demonstrated an elevated BCOR expression by qRT-PCR and western blot. By whole transcriptome sequencing and Ingenuity Pathway Analysis, we identified the activation of the Sonic Hedgehog (SHH) and of the WNT signaling pathway in two different regions of the primary tumor and of one inoculation metastasis compared to normal brain. We validated the activation of the SHH and of the WNT pathway by qRT-PCR analysis of GLI1 and AXIN2 respectively. GLI1 and AXIN2 were upregulated in the primary tumor and in two inoculation metastases compared to normal brain. Mutational analysis of SMO, PTCH1 and SUFU, three key components of the SHH pathway, revealed a Single Nucleotide Polymorphism (SNP) in PTCH1 (rs357564). We tested the effect of the GLI-inhibitor arsenic trioxide (ATO) on a short-term cell culture isolated from the metastasis. ATO was able to reduce the viability of the cells with an IC50 of 1.3 µM.In summary, these results provide functional evidence of altered BCOR expression and homogeneous coactivation of both the SHH and WNT signaling pathways, building the basis for potential novel therapeutic approaches for patients with a CNS HGNET-BCOR diagnosis.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias Encefálicas/genética , Carcinoma Basocelular/genética , Neoplasias Neuroepiteliomatosas/genética , Medicina de Precisão , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/genética , Antineoplásicos/farmacologia , Trióxido de Arsênio , Arsenicais/farmacologia , Proteína Axina/genética , Biomarcadores Tumorais/metabolismo , Biópsia , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Carcinoma Basocelular/tratamento farmacológico , Carcinoma Basocelular/metabolismo , Carcinoma Basocelular/secundário , Sobrevivência Celular/efeitos dos fármacos , Criança , Análise Mutacional de DNA , Relação Dose-Resposta a Droga , Predisposição Genética para Doença , Proteínas Hedgehog/genética , Humanos , Concentração Inibidora 50 , Imageamento por Ressonância Magnética , Masculino , Mutação , Neoplasias Neuroepiteliomatosas/tratamento farmacológico , Neoplasias Neuroepiteliomatosas/metabolismo , Neoplasias Neuroepiteliomatosas/secundário , Óxidos/farmacologia , Receptor Patched-1/genética , Fenótipo , Polimorfismo de Nucleotídeo Único , Valor Preditivo dos Testes , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/metabolismo , Receptor Smoothened/genética , Células Tumorais Cultivadas , Regulação para Cima , Via de Sinalização Wnt/genética , Proteína GLI1 em Dedos de Zinco/antagonistas & inibidores , Proteína GLI1 em Dedos de Zinco/genética , Proteína GLI1 em Dedos de Zinco/metabolismoRESUMO
Although neuro- and nephroblastoma are common solid tumors in children, the simultaneous occurrence is very rare and is often associated with syndromes. Here, we present a unique case of synchronous occurrence of neuro- and nephroblastoma in an infant with no signs of congenital anomalies or a syndrome. We performed genetic testing for possible candidate genes as underlying mutation using the next-generation sequencing (NGS) approach to target 94 genes and 284 single-nucleotide polymorphisms (SNPs) involved in cancer. We uncovered a novel heterozygous germline missense mutation p.F58L (c.172TâC) in the anaplastic lymphoma kinase (ALK) gene and one novel heterozygous rearrangement Q418Hfs(*)11 (c.1254_1264delins TTACTTAGTACAAGAACTG) in the Fanconi anemia gene FANCD2 leading to a truncated protein. Besides, several SNPs associated with the occurrence of neuroblastoma and/or nephroblastoma or multiple primary tumors were identified. The next-generation sequencing approach might in the future be useful not only in understanding tumor etiology but also in recognizing new genetic markers and targets for future personalized therapy.