RESUMO
The spleen plays a role in innate and adaptive immunity, and autoimmune diseases like rheumatoid arthritis (RA). We investigated the effect of splenectomy in early and moderate stages of autoimmune arthritis in a mouse model. To induce recombinant human G1-induced arthritis (GIA), BALB/c mice were immunized intraperitoneally three times in 4-week intervals with the rhG1 antigen. Mice were splenectomized on day 7 (SPE1) or day 35 (SPE2) after the initiation of immunization; tested for clinical severity, joint radiological and histological changes, serum levels of inflammatory cytokines and autoantibodies, and rhG1-specific immune responses; and compared to those in control mice with spleen left intact. Circulating Tregs and T-helper subset ratios in the spleen and inguinal lymph nodes (LNs) were also examined using flow cytometry. The onset of severe inflammatory response was significantly delayed in SPE1 and SPE2 groups compared to control mice at early stages of GIA, which was associated with increased circulating Tregs. After the third immunization, as disease progressed, the severity scores were robustly increased in all mice. Nevertheless, in splenectomized mice, we observed reduced joint deterioration and cartilage damage, more Th2 cells in LNs, and reduced levels of pro-inflammatory cytokines and autoantibodies in their sera. Mesenteric LN cells of splenectomized mice exhibited weaker response in vitro against the rhG1 antigen compared to control mice spleen. In conclusion, splenectomy in the early stages of GIA delayed the inflammatory response, suggesting a protective effect against the development and progression of severe destructive arthritis.
Assuntos
Artrite Reumatoide , Autoanticorpos , Citocinas , Camundongos Endogâmicos BALB C , Esplenectomia , Linfócitos T Reguladores , Animais , Camundongos , Linfócitos T Reguladores/imunologia , Autoanticorpos/imunologia , Autoanticorpos/sangue , Humanos , Artrite Reumatoide/imunologia , Artrite Reumatoide/cirurgia , Baço/imunologia , Feminino , Artrite Experimental/imunologia , Linfonodos/imunologia , Modelos Animais de Doenças , Articulações/patologia , Articulações/imunologia , Articulações/cirurgia , Células Th2/imunologia , Inflamação/imunologia , Proteínas Recombinantes/imunologiaRESUMO
OMICS methods brought significant advancements to the understanding of tumor cell biology, which transformed the treatment and prognosis of several cancers. Clinical practice and outcomes, however, changed significantly less in the case of glioblastoma (GBM). In this study, we aimed to assess the utility of whole exome (WES) sequencing in the clinical setting. Ten pairs of formalin-fixed, paraffin-embedded (FFPE) GBM specimens were obtained at onset (GBM-P) and at recurrence (GBM-R). Histopathological and molecular features of all samples supported the diagnosis of GBM based on WHO CNS5. WES data were filtered, applying a strict and custom-made pipeline, and occurrence of oncogenic and likely oncogenic variants in GBM-P, GBM-R or both were identified by using the VarSeq program version 2.5.0 (Golden Helix, Inc.). Characteristics and recurrence of the variants were analyzed in our own cohort and were also compared to those available in the COSMIC database. The lists of oncogenic and likely oncogenic variants corresponded to those identified in other studies. The average number of these variants were 4 and 5 out of all detected 24 and 34 variants in GBM-P and GBM-R samples, respectively. On average, one shared oncogenic/likely oncogenic variant was found in the pairs. We assessed the identified variants' therapeutic significance, also taking into consideration the guidelines by the Association for Molecular Pathology (AMP). Our data support that a thorough WES analysis is suitable for identifying oncogenic and likely oncogenic variants in an individual clinical sample or a small cohort of FFPE glioma specimens, which concur with those of comprehensive research studies. Such analyses also allow us to monitor molecular dynamics of sequential GBM. In addition, careful evaluation of data according to the AMP guideline reveal that though therapeutic applicability of the variants is generally limited in the clinic, such information may be valuable in selected cases, and can support innovative preclinical and clinical trials.
Assuntos
Neoplasias Encefálicas , Sequenciamento do Exoma , Genômica , Glioblastoma , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Genômica/métodos , Masculino , Feminino , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Pessoa de Meia-Idade , Idoso , Adulto , Recidiva Local de Neoplasia/genética , MutaçãoRESUMO
Background and purpose:
Glioblastoma (GBM), a highly aggressive form of brain tumors, has been extensively studied using OMICS methods, and the most characteristic molecular determinants have been incorporated into the histopathological diagnosis. Research data, nevertheless, only partially have been adopted in clinical practice. Here we aimed to present results of our epigenomic GBM profiling to better understand early and late determinants of these tumors, and to share main elements of our findings with practicing professionals.
. Methods:GBM specimens were surgically obtained after first diagnosis (GBM1) and at recurrence (GBM2). DNA was extracted from 24 sequential pairs of formalin-fixed, paraffin-embedded tumor tissues. The Reduced Representation Bisulfite Sequencing kit was used for library preparation. Pooled libraries were sequenced on an Illumina NextSeq 550 instrument. Methylation controls (MC) were obtained from a publicly available database. Bioinformatic analyses were performed to identify differentially methylated pathways and their elements in cohorts of MC, GBM1 and GBM2.
. Results:Several differentially methylated pathways involved in basic intracellular and brain tissue developmental processes were identified in the GBM1 vs. MC and GBM2 vs. MC comparisons. Among differentially methylated pathways, those involved in immune regulation, neurotransmitter (particularly dopaminergic, noradrenergic and glutaminergic) responses and regulation of stem cell differentiation and proliferation stood out in the GBM2 vs. GBM1 comparisons.
. Conclusion:Our study revealed biological complexity of early and late gliomagenesis encompassing mechanisms from basic intracellular through distorted neurodevelopmental processes to more specific immune and highjacked neurotransmitter pathways in the tumor microenvironment. These findings may offer considerations for therapeutic approaches.
.Assuntos
Neoplasias Encefálicas , Glioblastoma , Humanos , Glioblastoma/patologia , Epigenômica , Metilação de DNA , Encéfalo/patologia , Neoplasias Encefálicas/patologia , Neurotransmissores , Regulação Neoplásica da Expressão Gênica , Microambiente TumoralRESUMO
Recent advances in molecular diagnostics are transforming the classification of malignant tumours, it has long played a major role in the field of CNS tumours. Examination of 1p/19q codeletion is indispensable in case of diffuse gliomas. Glioblastoma may be diagnosed even in the absence of characteristic morphological features, when EGFR amplification, TERT promoter mutation or +7/-10 copy number abnormalities are present. The number of entities defined by a genetic abnormality is growing. Comprehensive analysis of DNA methylation may be of considerable help in addition to histology and basic molecular studies, especially in case of small samples. Keeping up with the ever-expanding diagnostic repertoire is difficult, however, advantages and disadvantages of these methods and the context in which they may be useful should be understood by those who are involved in the diagnosis of CNS tumours. This summary provides a general overview of the main methods used in molecular diagnostics.