RESUMO
Correctly diagnosing a rare childhood cancer such as sarcoma can be critical to assigning the correct treatment regimen. With a finite number of pathologists worldwide specializing in pediatric/young adult sarcoma histopathology, access to expert differential diagnosis early in case assessment is limited for many global regions. The lack of highly-trained sarcoma pathologists is especially pronounced in low to middle-income countries, where pathology expertise may be limited despite a similar rate of sarcoma incidence. To address this issue in part, we developed a deep learning convolutional neural network (CNN)-based differential diagnosis system to act as a pre-pathologist screening tool that quantifies diagnosis likelihood amongst trained soft-tissue sarcoma subtypes based on whole histopathology tissue slides. The CNN model is trained on a cohort of 424 centrally-reviewed histopathology tissue slides of alveolar rhabdomyosarcoma, embryonal rhabdomyosarcoma and clear-cell sarcoma tumors, all initially diagnosed at the originating institution and subsequently validated by central review. This CNN model was able to accurately classify the withheld testing cohort with resulting receiver operating characteristic (ROC) area under curve (AUC) values above 0.889 for all tested sarcoma subtypes. We subsequently used the CNN model to classify an externally-sourced cohort of human alveolar and embryonal rhabdomyosarcoma samples and a cohort of 318 histopathology tissue sections from genetically engineered mouse models of rhabdomyosarcoma. Finally, we investigated the overall robustness of the trained CNN model with respect to histopathological variations such as anaplasia, and classification outcomes on histopathology slides from untrained disease models. Overall positive results from our validation studies coupled with the limited worldwide availability of sarcoma pathology expertise suggests the potential of machine learning to assist local pathologists in quickly narrowing the differential diagnosis of sarcoma subtype in children, adolescents, and young adults.
Assuntos
Rabdomiossarcoma Embrionário , Rabdomiossarcoma , Adolescente , Animais , Criança , Humanos , Aprendizado de Máquina , Camundongos , Redes Neurais de Computação , Patologistas , Rabdomiossarcoma/diagnóstico , Rabdomiossarcoma Embrionário/patologia , Adulto JovemRESUMO
The anti-oxidant lipoic acid (LA) is beneficial in murine models of multiple sclerosis (MS) and has recently been shown to slow brain atrophy in secondary progressive MS. The mechanism of these effects by LA is incompletely understood but may involve effects on microglia. The objective of this study is to understand how LA affects microglial cells. We cultured primary microglial cells from C57BL/6 adult mice brains and stimulated the cells with lipopolysaccharide (LPS) and interferon gamma (IFN-γ) in the presence or absence of LA. We demonstrate the inhibition of phagocytosis, rearrangement of actin, and formation of membrane blebs in stimulated microglia in the presence of LA. These experiments suggest that LA causes changes in microglial actin, which may lead to alterations in phagocytosis, mobility, and migration.
Assuntos
Antioxidantes/farmacologia , Movimento Celular/fisiologia , Microglia/fisiologia , Fagocitose/fisiologia , Ácido Tióctico/farmacologia , Animais , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Feminino , Interferon gama/toxicidade , Lipopolissacarídeos/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Fagocitose/efeitos dos fármacosRESUMO
Oligodendrocyte processes wrap axons to form neuroprotective myelin sheaths, and damage to myelin in disorders, such as multiple sclerosis (MS), leads to neurodegeneration and disability. There are currently no approved treatments for MS that stimulate myelin repair. During development, thyroid hormone (TH) promotes myelination through enhancing oligodendrocyte differentiation; however, TH itself is unsuitable as a remyelination therapy due to adverse systemic effects. This problem is overcome with selective TH agonists, sobetirome and a CNS-selective prodrug of sobetirome called Sob-AM2. We show here that TH and sobetirome stimulated remyelination in standard gliotoxin models of demyelination. We then utilized a genetic mouse model of demyelination and remyelination, in which we employed motor function tests, histology, and MRI to demonstrate that chronic treatment with sobetirome or Sob-AM2 leads to significant improvement in both clinical signs and remyelination. In contrast, chronic treatment with TH in this model inhibited the endogenous myelin repair and exacerbated disease. These results support the clinical investigation of selective CNS-penetrating TH agonists, but not TH, for myelin repair.
Assuntos
Acetatos/farmacologia , Esclerose Múltipla/tratamento farmacológico , Bainha de Mielina/efeitos dos fármacos , Fenóis/farmacologia , Hormônios Tireóideos/agonistas , Substância Branca/efeitos dos fármacos , Acetatos/uso terapêutico , Animais , Axônios/efeitos dos fármacos , Axônios/patologia , Diferenciação Celular/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Técnicas de Silenciamento de Genes , Gliotoxina/toxicidade , Humanos , Imageamento por Ressonância Magnética , Masculino , Camundongos , Camundongos Transgênicos , Esclerose Múltipla/etiologia , Esclerose Múltipla/patologia , Bainha de Mielina/patologia , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/patologia , Fenóis/uso terapêutico , Pró-Fármacos/farmacologia , Pró-Fármacos/uso terapêutico , Remielinização/efeitos dos fármacos , Remielinização/genética , Hormônios Tireóideos/administração & dosagem , Fatores de Transcrição/genética , Substância Branca/citologia , Substância Branca/diagnóstico por imagem , Substância Branca/patologiaRESUMO
Cortical lesions are a crucial part of MS pathology and it is critical to determine that new MS therapies have the ability to alter cortical inflammatory lesions given the differences between white and gray matter lesions. We tested lipoic acid (LA) in a mouse focal cortical EAE model. Brain sections were stained with antibodies against CD4, CD11b and galectin-3. Compared with vehicle, treatment with LA significantly decreased CD4+ and galectin-3+ immune cells in the brain. LA treated mice had fewer galectin-3+ cells with no projections indicating decrease in the number of infiltrating monocytes. LA significantly reduces inflammation in a focal cortical model of MS.
Assuntos
Antioxidantes/uso terapêutico , Córtex Cerebral/patologia , Encefalite/tratamento farmacológico , Encefalite/etiologia , Encefalomielite Autoimune Experimental/complicações , Encefalomielite Autoimune Experimental/patologia , Ácido Tióctico/uso terapêutico , Animais , Antígeno CD11b/metabolismo , Córtex Cerebral/efeitos dos fármacos , Citocinas/metabolismo , Citocinas/farmacologia , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/induzido quimicamente , Feminino , Galectina 3/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Glicoproteína Mielina-Oligodendrócito/imunologia , Fragmentos de Peptídeos/imunologia , Estatísticas não Paramétricas , Fatores de TempoRESUMO
Increased remissions in multiple sclerosis (MS) during late pregnancy may result from high levels of sex steroids such as estrogen and estriol. Estrogen (E2=17ß-estradiol) protects against experimental autoimmune encephalomyelitis (EAE), but the cellular basis for E2-induced protection remains unclear. Treatment with relatively low doses of E2 can protect against clinical and histological signs of MOG-35-55 induced EAE through mechanisms involving the PD-1 coinhibitory pathway and B-cells. The current study evaluated the contribution of PD-1 ligands, PD-L1 and PD-L2, on B-cells in E2-mediated protection against EAE in WT, PD-L1-/- and PD-L2-/- mice. Unlike PD-L2-/- mice that were fully protected against EAE after E2 treatment, E2-implanted PD-L1-/- mice were fully susceptible to EAE, with increased numbers of proliferating Th1/Th17 cells in the periphery and severe cellular infiltration and demyelination in the CNS. Moreover, transfer of B-cells from MOG-immunized PD-L1-/- or PD-L2-/- donors into E2-preconditioned B-cell deficient µMT-/- recipient mice revealed significantly reduced E2-mediated protection against EAE in recipients of PD-L1-/- B-cells, but near-complete protection in recipients of PD-L2-/- B-cells. We conclude that PD-1 interaction with PD-L1 but not PD-L2 on B-cells is crucial for E2-mediated protection in EAE and that strategies that enhance PD-1/PD-L1 interactions might potentiate E2 treatment effects in MS.
RESUMO
Although multiple sclerosis (MS) has traditionally been considered to be an inflammatory disease, recent evidence has brought neurodegeneration into the spotlight, suggesting that accumulated damage and loss of axons is critical to disease progression and the associated irreversible disability. Proposed mechanisms of axonal degeneration in MS posit cytosolic and subsequent mitochondrial Ca(2+) overload, accumulation of pathologic reactive oxygen species (ROS), and mitochondrial dysfunction leading to cell death. In this context, the role of the p66 isoform of ShcA protein (p66) may be significant. The ShcA isoform is uniquely targeted to the mitochondrial intermembrane space in response to elevated oxidative stress, and serves as a redox enzyme amplifying ROS generation in a positive feedforward loop that eventually mediates cell death by activation of the mitochondrial permeability transition pore. Consequently, we tested the hypothesis that genetic inactivation of p66 would reduce axonal injury in a murine model of MS, experimental autoimmune encephalomyelitis (EAE). As predicted, the p66-knockout (p66-KO) mice developed typical signs of EAE, but had less severe clinical impairment and paralysis than wild-type (WT) mice. Histologic examination of spinal cords and optic nerves showed significant axonal protection in the p66-KO tissue, despite similar levels of inflammation. Furthermore, cultured p66-KO neurons treated with agents implicated in MS neurodegenerative pathways showed greater viability than WT neurons. These results confirm the critical role of ROS-mediated mitochondrial dysfunction in the axonal loss that accompanies EAE, and identify p66 as a new pharmacologic target for MS neuroprotective therapeutics.
Assuntos
Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/prevenção & controle , Proteínas Adaptadoras da Sinalização Shc/deficiência , Animais , Axônios/patologia , Axônios/ultraestrutura , Proliferação de Células , Células Cultivadas , Córtex Cerebral/citologia , Peptidil-Prolil Isomerase F , Ciclofilinas/deficiência , Citocinas/metabolismo , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/induzido quimicamente , Encefalomielite Autoimune Experimental/patologia , Adjuvante de Freund/efeitos adversos , Glicoproteínas/efeitos adversos , Peróxido de Hidrogênio/farmacologia , Infiltração Leucêmica/tratamento farmacológico , Infiltração Leucêmica/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Glicoproteína Mielina-Oligodendrócito , Fibras Nervosas Mielinizadas/patologia , Neurônios/metabolismo , Neurônios/ultraestrutura , Nervo Óptico/imunologia , Nervo Óptico/metabolismo , Nervo Óptico/patologia , Nervo Óptico/ultraestrutura , Fragmentos de Peptídeos/efeitos adversos , Proteínas Adaptadoras da Sinalização Shc/metabolismo , Medula Espinal/imunologia , Medula Espinal/metabolismo , Medula Espinal/patologia , Medula Espinal/ultraestrutura , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologiaRESUMO
Lipoic acid (LA) is an antioxidant that is effective in treating experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis (MS). C57BL/6 mice with EAE develop experimental autoimmune optic neuritis (EAON), which models acute optic neuritis in humans. Here we determined whether LA is therapeutically effective in EAON. We immunized C57BL/6 mice with MOG 35-55 peptide. Mice received either daily subcutaneous injections of LA (100mg/kg) or saline in early or late suppression paradigms. In the early suppression paradigm, optic nerve cross-sections showed 14.9±3.8% (mean±SEM) damage in mice receiving saline (n=7) and 2.0±0.4% damage in mice given LA (n=7, p=0.001). In the late suppression paradigm, optic nerve sections showed 24.6±3.5% damage in mice treated with saline (n=7) and 8.4±2.5% in mice treated with LA (n=7, p=0.004). Thus a dramatic reduction in axonal injury was seen after LA administration in both experimental paradigms. Compared with saline treated mice with EAON, optic nerves from mice receiving LA had significantly fewer CD4+ and CD11b+ cells in both paradigms. This study provides a rationale for investigating the therapeutic efficacy of LA in acute optic neuritis in humans.