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Acidentes por Quedas , Dor nas Costas , Rigidez Muscular Espasmódica , Adulto , Feminino , Humanos , Dor nas Costas/etiologia , Diagnóstico Diferencial , Perna (Membro) , Rigidez Muscular Espasmódica/complicações , Rigidez Muscular Espasmódica/diagnóstico , Rigidez Muscular Espasmódica/imunologia , Rigidez Muscular Espasmódica/terapia , Espasmo/etiologiaAssuntos
Autoanticorpos/sangue , Doenças Autoimunes/diagnóstico , Moléculas de Adesão Celular Neuronais/antagonistas & inibidores , Imunoterapia , Doenças Neurodegenerativas/etiologia , Idoso , Doenças Autoimunes/terapia , Diagnóstico Diferencial , Transtornos Neurológicos da Marcha/etiologia , Humanos , Imunoglobulinas Intravenosas/uso terapêutico , Masculino , Doença dos Neurônios Motores/etiologia , Transtornos do Sono-Vigília/etiologiaAssuntos
Encefalite/diagnóstico , Doença de Hashimoto/diagnóstico , Hiponatremia/etiologia , Idoso , Braço/fisiopatologia , Análise Química do Sangue , Diagnóstico Diferencial , Discinesias/etiologia , Encefalite/complicações , Encefalite/tratamento farmacológico , Face/fisiopatologia , Doenças Genéticas Ligadas ao Cromossomo X/diagnóstico , Glucocorticoides/uso terapêutico , Doença de Hashimoto/complicações , Doença de Hashimoto/tratamento farmacológico , Humanos , Síndrome de Secreção Inadequada de HAD/diagnóstico , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Metilprednisolona/uso terapêutico , Concentração Osmolar , Sódio/sangueRESUMO
OBJECTIVE: A novel autoimmune central nervous system (CNS) disorder with glial fibrillary acidic protein (GFAP)-IgG as biomarker was recently characterized. Here, 102 patients with GFAP-IgG positivity are described. METHODS: The 102 included patients had: (1) serum, cerebrospinal fluid (CSF), or both that yielded a characteristic astrocytic pattern of mouse tissue immunostaining; (2) confirmation of IgG reactive with specific GFAP isoforms (α, É, or κ) by cell-based assays; and (3) clinical data available. Control specimens (n = 865) were evaluated by tissue (n = 542) and cell-based (n = 323) assays. RESULTS: Median symptom onset age was 44 years (range = 8-103), and 54% were women. The predominant phenotype (83 patients; 81%) was inflammation of meninges, brain, spinal cord, or all 3 (meningoencephalomyelitis). Among patients, highest specificity for those phenotypes was observed for CSF testing (94%), and highest sensitivity was for the GFAPα isoform (100%). Rare GFAP-IgG positivity was encountered in serum controls by tissue-based assay (0.5%) or cell-based assay (1.5%), and in CSF controls by cell-based assay (0.9%). Among patients, striking perivascular radial enhancement was found on brain magnetic resonance imaging in 53%. Although cases frequently mimicked vasculitis, angiography was uniformly negative, and spinal imaging frequently demonstrated longitudinally extensive myelitic lesions. Diverse neoplasms encountered were found prospectively in 22%. Ovarian teratoma was most common and was predicted best when both N-methyl-D-aspartate receptor-IgG and aquaporin-4-IgG coexisted (71%). Six patients with prolonged follow-up had brisk corticosteroid response, but required additional immunosuppression to overcome steroid dependency. INTERPRETATION: GFAPα-IgG, when detected in CSF, is highly specific for an immunotherapy-responsive autoimmune CNS disorder, sometimes with paraneoplastic cause. Ann Neurol 2017;81:298-309.
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Astrócitos/patologia , Autoanticorpos/líquido cefalorraquidiano , Doenças Autoimunes do Sistema Nervoso/líquido cefalorraquidiano , Proteína Glial Fibrilar Ácida/imunologia , Síndromes Paraneoplásicas do Sistema Nervoso/líquido cefalorraquidiano , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Biomarcadores/líquido cefalorraquidiano , Criança , Feminino , Humanos , Imunoglobulina G , Imageamento por Ressonância Magnética , Masculino , Camundongos , Pessoa de Meia-Idade , Adulto JovemRESUMO
Autoimmune disorders are becoming increasingly recognized within the broader field of neurology. The discovery of multiple, novel, neutrally targeted autoantibodies over the past decade and their translation into commercially available testing, in particular, has aided in the more rapid diagnosis of these disorders. When considering imaging in autoimmune neurologic disorders, it is important, when possible, to visualize the autoimmune process itself, as well as to make sure that the patient does not have an associated malignancy driving the overall process. Positron emission tomographic scans can aid in the detection of small tumors with limited spread, as well as in the visualization of autoimmune processes affecting the brain and/or spinal cord. In autoimmune disorders of the central nervous system, imaging abnormalities can appear within the limbic system, extralimbic areas, and spinal cord. Such imaging abnormalities can serve as objective markers to follow over time to assess patients' responses to treatment. It is important to recognize that overlapping syndromes (for instance, both demyelinating and autoimmune or both infectious and autoimmune) exist and that inflammatory disorders can leave behind sequelae that can be recognized on subsequent imaging. Work is currently underway to develop more specific imaging techniques for autoimmune neurologic disorders.
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Autoanticorpos/imunologia , Doenças Autoimunes do Sistema Nervoso/diagnóstico por imagem , Doenças Autoimunes/diagnóstico por imagem , Sistema Nervoso Central/diagnóstico por imagem , Doenças Desmielinizantes/imunologia , Doenças Autoimunes do Sistema Nervoso/imunologia , Sistema Nervoso Central/imunologia , Doenças Desmielinizantes/patologia , Humanos , Miosite/imunologia , Miosite/terapiaRESUMO
The understanding of the manifestations, mechanisms, and management of autoimmune encephalitis has expanded dramatically in recent decades. Immune-mediated encephalitides are comparable in incidence and prevalence to infectious etiologies, and are associated with significant morbidity, especially when there is a delay in recognition and treatment. As such, clinicians from many specialties must develop a functional understanding of these disorders. Herein we provide an overview of the autoimmune and paraneoplastic encephalitides, including those associated with either intracellular or cell surface/synaptic neuronal autoantibodies. After briefly reviewing the current understanding of the pathobiology of autoimmune encephalitis, we combine a neuroanatomical approach with specific antibody syndromes to provide the reader with a clinically relevant review of these disorders. The clinical manifestations, diagnosis, and management of autoimmune encephalitis are reviewed, with an emphasis on clinical relevance. We also introduce updates in the field, including autoimmune encephalitis associated with novel cancer immunotherapies, infectious triggers of autoimmune encephalitis, and autoimmune encephalitis with demyelinating overlap syndromes.
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Autoanticorpos/imunologia , Encefalite/terapia , Doença de Hashimoto/terapia , Imunoterapia , Síndromes Paraneoplásicas/terapia , Doenças Autoimunes Desmielinizantes do Sistema Nervoso Central/imunologia , Doenças Autoimunes Desmielinizantes do Sistema Nervoso Central/terapia , Encefalite/imunologia , Doença de Hashimoto/imunologia , Humanos , Neurônios , Síndromes Paraneoplásicas/imunologiaRESUMO
In the past few years, many autoimmune encephalitides have been identified, with specific clinical syndromes and associated antibodies against neuronal surface antigens. There is compelling evidence that many of these antibodies are pathogenic and most of these encephalitides are highly responsive to immunotherapies. The clinical spectra of some of these antibody-mediated syndromes, especially those reported in only a few patients, are evolving. Others, such as anti-N-methyl-D-aspartate (NMDA) receptor encephalitis, are well characterized. Diagnosis involves recognizing the specific syndromes and identifying the antibody in a patient's cerebrospinal fluid (CSF) and/or serum. These syndromes are associated with variable abnormalities in CSF, magnetic resonance imaging, and electroencephalography. Treatment is often multidisciplinary and should be focused upon neutralizing the effects of antibodies and eliminating their source. Overlapping disorders have been noted, with some patients having more than one neurologic autoimmune disease. In other patients, viral infections such as herpes simplex virus encephalitis trigger robust antineuronal autoimmune responses.
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Autoanticorpos , Doenças Autoimunes do Sistema Nervoso/imunologia , Encefalopatias/imunologia , Encefalite/imunologia , Doença de Hashimoto/imunologia , Humanos , Neurônios/imunologiaRESUMO
Although they are relatively rare, the diagnosis of paraneoplastic neurologic syndromes (PNS) can be aided by the identification of neural autoantibodies in patients' serum and cerebrospinal fluid (CSF). They often clinically manifest as characteristic syndromes, including limbic encephalitis, opsoclonus-myoclonus syndrome, paraneoplastic cerebellar degeneration, and paraneoplastic encephalomyelitis. The antibodies are directed either toward intracellular targets, or epitopes on the cell surface. As compared to cell surface antibodies, intracellular paraneoplastic autoantibodies are more classically associated with cancer, most often lung, breast, thymoma, gynecologic, testicular, and/or neuroendocrine cancers. The malignancies themselves tend to be small and regionally contained, attesting to the strength of the immune system in cancer immunosurveillance. Typically, the intracellular antibodies are not directly pathogenic and tend to be associated with PNS that are poorly responsive to treatment. With some notable exceptions, including patients with PNS associated with testicular cancer, patients with intracellular antibodies are typically older individuals, in their 7th decade of life and beyond. Many of them are current or former smokers. Treatment strategies include tumor removal as well as immunotherapy to treat the concomitant PNS. Newer technologies and the ever-broadening use of cancer immunotherapies are contributing to the continued identification of novel intracellularly targeted autoantibodies.
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Encefalite Límbica , Síndromes Paraneoplásicas do Sistema Nervoso , Neoplasias Testiculares , Masculino , Humanos , Feminino , Síndromes Paraneoplásicas do Sistema Nervoso/terapia , Autoanticorpos , ImunoterapiaRESUMO
BACKGROUND: Host immune response is a critical component in tumorigenesis and immune escape. Radiation is widely used for glioblastoma (GBM) and can induce marked tissue inflammation and substantially alter host immune response. However, the role of myeloperoxidase (MPO), a key enzyme in inflammation and host immune response, in tumorigenesis after radiotherapy is unclear. In this study, we aimed to determine how post-radiation MPO activity influences GBM and outcome. METHODS: We injected C57BL/6J or MPO-knockout mice with 005 mouse GBM stem cells intracranially. To observe MPO's effects on post-radiation tumor progression, we then irradiated the head with 10 Gy unfractionated and treated the mice with a specific MPO inhibitor, 4-aminobenzoic acid hydrazide (ABAH), or vehicle as control. We performed semi-quantitative longitudinal molecular MRI, enzymatic assays and flow cytometry to assess changes in inflammatory response and tumor size, and tracked survival. We also performed cell culture experiments in murine and human GBM cells to determine the effect of MPO on these cells. RESULTS: Brain irradiation increased the number of monocytes/macrophages and neutrophils, and boosted MPO activity by ten-fold in the glioma microenvironment. However, MPO inhibition dampened radiation-induced inflammation, demonstrating decreased MPO-specific signal on molecular MRI and attenuated neutrophil and inflammatory monocyte/macrophage recruitment to the glioma. Compared to saline-treated mice, both ABAH-treated and MPO-knockout mice had accelerated tumor growth and reduced survival. We further confirmed that MPO decreased tumor cell viability and proliferation in cell cultures. CONCLUSION: Local radiation to the brain initiated an acute systemic inflammatory response with increased MPO-carrying cells both in the periphery and the GBM, resulting in increased MPO activity in the tumor microenvironment. Inhibition or absence of MPO activity increased tumor growth and decreased host survival, revealing that elevated MPO activity after radiation has an anti-tumor role.
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Glioblastoma , Peroxidase , Animais , Encéfalo , Glioblastoma/genética , Glioblastoma/radioterapia , Imageamento por Ressonância Magnética , Camundongos , Camundongos Endogâmicos C57BL , Peroxidase/metabolismo , Microambiente TumoralRESUMO
OBJECTIVE: Autoimmune encephalitis (AE) is a highly treatable neurologic condition that can cause psychosis. Screening for AE is not currently recommended in routine workup for first-episode psychosis (FEP), owing partly to the high cost of testing for AE-associated neuronal autoantibodies. METHODS: This study used a decision-analytic model to estimate the cost-effectiveness of routine serum screening for AE compared with clinically targeted screening in patients with FEP. Model parameters drawn from prior published literature included the prevalence of neuronal autoantibodies in FEP (4.5%), serum autoantibody panel cost (US $291), remission probability with antipsychotics (0.58), and remission probability with immunotherapy for patients diagnosed with AE (0.85). Outcomes included quality-adjusted life-years (QALYs), costs, and incremental cost-effectiveness ratios (ICERs), assessed over a 5-year horizon from the US health care sector and societal perspectives. ICER thresholds of $50,000/QALY to $150,000/QALY were used to define cost-effectiveness. The analysis was conducted between June 2018 and January 2020. RESULTS: Routine screening led to mean QALY gains of 0.008 among all patients and 0.174 among the subgroup of patients with neuronal autoantibodies. Mean costs increased by $780 from a societal perspective and $1,150 from a health care sector perspective, resulting in ICERs of $99,330/QALY and $147,460/QALY, respectively. Incorporating joint input data uncertainty, the likelihood routine screening has an ICER ≤ $150,000/QALY was 55% from a societal perspective and 37% from a health care sector perspective. The model parameter with the greatest contribution to overall uncertainty was the effectiveness of immunotherapy relative to antipsychotics. CONCLUSIONS: Routine screening for AE in patients with FEP may be cost-effective in the United States. As further immunotherapy effectiveness data become available, a more definitive recommendation to perform routine screening could be warranted.
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Autoanticorpos/sangue , Análise Custo-Benefício , Encefalite/diagnóstico , Doença de Hashimoto/diagnóstico , Custos de Cuidados de Saúde/estatística & dados numéricos , Transtornos Psicóticos/etiologia , Biomarcadores/sangue , Técnicas de Apoio para a Decisão , Encefalite/sangue , Encefalite/complicações , Encefalite/economia , Doença de Hashimoto/sangue , Doença de Hashimoto/complicações , Doença de Hashimoto/economia , Humanos , Modelos Econômicos , Transtornos Psicóticos/economia , Transtornos Psicóticos/terapia , Anos de Vida Ajustados por Qualidade de Vida , Estados UnidosRESUMO
Merkel cell carcinoma (MCC) is a rare and aggressive cutaneous neuroendocrine cancer with a high risk of recurrence and metastasis. MCC is generally associated with advanced age, fair skin, sun exposure, immunosuppression, and in the majority of cases, the Merkel cell polyomavirus. Neuroendocrine malignancies are associated with a variety of paraneoplastic neurological syndromes (PNS), characterized as autoimmune responses to malignancy-associated expression of neural antigens. Our literature review underscores previous case reports of MCC-associated PNS with voltage-gated calcium channel (VGCC) and anti-Hu (or ANNA-1) autoantibodies. We present the case of a 59-year-old male with regionally metastatic Merkel cell carcinoma complicated by the paraneoplastic manifestation of anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis. His primary lower neck subcutaneous MCC and metastasis were initially treated with surgery. Additional recurrent lymph node metastases were successfully treated with definitive intensity-modulated radiation therapy. His PNS improved with rituximab therapy. Although rare, this case highlights that in the setting of seizures and prominent psychiatric symptoms accompanying an MCC diagnosis, evaluation for autoimmune paraneoplastic encephalitis is warranted. Awareness and detection of preexisting PNS are crucial in the era of immune checkpoint inhibitors (ICI) for advanced MCC, where treatment with ICI has the potential to exacerbate preexisting autoimmune PNS and lead to worsened or even lethal neurologic immune-related adverse events (nirAEs).
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Importance: Recognizing the presenting and immunopathological features of Kelch-like protein-11 immunoglobulin G seropositive (KLHL11 IgG+) patients may aid in early diagnosis and management. Objective: To describe expanding neurologic phenotype, cancer associations, outcomes, and immunopathologic features of KLHL11 encephalitis. Design, Setting, and Participants: This retrospective tertiary care center study, conducted from October 15, 1998, to November 1, 2019, prospectively identified 31 KLHL11 IgG+ cases in the neuroimmunology laboratory. Eight were identified by retrospective testing of patients with rhomboencephalitis (confirmed by tissue-based-immunofluorescence and transfected-cell-based assays). Main Outcomes and Measures: Outcome variables included modified Rankin score and gait aid use. Results: All 39 KLHL11 IgG+ patients were men (median age, 46 years; range, 28-73 years). Initial clinical presentations were ataxia (n = 32; 82%), diplopia (n = 22; 56%), vertigo (n = 21; 54%), hearing loss (n = 15; 39%), tinnitus (n = 14; 36%), dysarthria (n = 11; 28%), and seizures (n = 9; 23%). Atypical neurologic presentations included neuropsychiatric dysfunction, myeloneuropathy, and cervical amyotrophy. Hearing loss or tinnitus preceded other neurologic deficits by 1 to 8 months in 10 patients (26%). Among patients screened for malignancy (n = 36), testicular germ-cell tumors (n = 23; 64%) or testicular microlithiasis and fibrosis concerning for regressed germ cell tumor (n = 7; 19%) were found in 83% of the patients (n = 30). In 2 patients, lymph node biopsy diagnosed metastatic lung adenocarcinoma in one and chronic lymphocytic leukemia in the other. Initial brain magnetic resonance imaging revealed T2 hyperintensities in the temporal lobe (n = 12), cerebellum (n = 9), brainstem (n = 3), or diencephalon (n = 3). Among KLHL11 IgG+ patients who underwent HLA class I and class II genotyping (n = 10), most were found to have HLA-DQB1*02:01 (n = 7; 70%) and HLA-DRB1*03:01 (n = 6; 60%) associations. A biopsied gadolinium-enhancing temporal lobe lesion demonstrated T cell-predominant inflammation and nonnecrotizing granulomas. Cerebellar biopsy (patient with chronic ataxia) and 2 autopsied brains demonstrated Purkinje neuronal loss and Bergmann gliosis, supporting early active inflammation and later extensive neuronal loss. Compared with nonautoimmune control peripheral blood mononuclear cells, cluster of differentiation (CD) 8+ and CD4+ T cells were significantly activated when patient peripheral blood mononuclear cells were cultured with KLHL11 protein. Most patients (58%) benefitted from immunotherapy and/or cancer treatment (neurological disability stabilized [n = 10] or improved [n = 9]). Kaplan-Meier curve demonstrated significantly higher probability of wheelchair dependence among patients without detectable testicular cancer. Long-term outcomes in KLHL11-IgG+ patients were similar to Ma2 encephalitis. Conclusions and Relevance: Kelch-like protein-11 IgG is a biomarker of testicular germ-cell tumor and paraneoplastic neurologic syndrome, often refractory to treatment. Described expanded neurologic phenotype and paraclinical findings may aid in its early diagnosis and treatment.
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Autoanticorpos/sangue , Proteínas de Transporte/sangue , Encefalite/sangue , Síndromes Paraneoplásicas do Sistema Nervoso/sangue , Fenótipo , Adulto , Idoso , Autoanticorpos/imunologia , Biomarcadores/sangue , Proteínas de Transporte/imunologia , Encefalite/diagnóstico , Encefalite/imunologia , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Síndromes Paraneoplásicas do Sistema Nervoso/diagnóstico , Síndromes Paraneoplásicas do Sistema Nervoso/imunologia , Estudos Retrospectivos , Linfócitos T/imunologia , Linfócitos T/metabolismoAssuntos
Doenças Autoimunes , Humanos , Imunofenotipagem , Doenças Autoimunes/diagnóstico , AutoimunidadeRESUMO
OBJECTIVE: To report the frequency of coexisting herpes viruses (herpes simplex virus 1 [HSV-1] or HSV-2, varicella zoster virus, Epstein-Barr virus [EBV], cytomegalovirus, or human herpes virus 6 [HHV-6]) and autoantibodies in patients with encephalitis (herpes or autoimmune) in clinical laboratory service. METHODS: Three groups were evaluated for herpes viruses and antibodies: group 1-patients whose CSF was positive for a herpes virus by real-time PCR over a period of 6 months; group 2-patients whose CSF was positive for an autoimmune encephalitis-associated antibody over 5 years (e.g., NMDA receptor [NMDA-R] antibody), and the same number of controls without autoimmune/infectious disease; and group 3-incidental autoimmune parainfectious encephalitis cases encountered over 1 year. RESULTS: In group 1, antibodies were detected in 27 of 100 herpes PCR-positive CSF specimens (CSFs), either unclassified neural or nonneural in all but one patient with NMDA-R antibody detected after EBV infection. Antibodies were also detected in 3 of 7 CSFs submitted for repeat PCR testing (unclassified, 2; AMPA receptor, 1). In group 2, herpes viruses were detected in 1 of 77 controls (HHV-6) and 4 of 77 patients with autoimmune encephalitis (EBV, 2; HHV-6, 2); autoantibodies targeted NMDA-R in 3/4 and GABAB-R in 1/4. In group 3, NMDA-R antibody was detected in 7 patients post-HSV-1 encephalitis. Of the remaining 3 patients, 2 had unclassified neural antibodies detected, and one had GABAB-R autoimmunity. Concomitant neoplasms were discovered in 2 patients each from groups 2 and 3. CONCLUSIONS: Autoantibodies and herpes virus DNA frequently coexist in encephalitic CSF. Some patients develop parainfectious autoimmunity following viral CNS infection (usually HSV-1 encephalitis). The significance of detecting herpes nucleic acids in others remains unclear.
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AIMS: Myeloperoxidase (MPO), a highly oxidative enzyme secreted by leukocytes has been implicated in human and experimental nonalcoholic steatohepatitis (NASH), but the underlying mechanisms remain unknown. In this study, we investigated how MPO contributes to progression from steatosis to NASH. RESULTS: In C57Bl/6J mice fed a diet deficient in methionine and choline to induce NASH, neutrophils and to a lesser extent inflammatory monocytes are markedly increased compared with sham mice and secrete abundant amounts of MPO. Through generation of HOCl, MPO directly causes hepatocyte death in vivo. In vitro experiments demonstrate mitochondrial permeability transition pore induction via activation of SAPK/JNK and PARP. MPO also contributes to activation of hepatic stellate cells (HSCs), the most important source of collagen in the liver. In vitro MPO-activated HSCs have an activation signature (MAPK and PI3K-AKT phosphorylation) and upregulate COL1A1, α-SMA, and CXCL1. MPO-derived oxidative stress also activates transforming growth factor ß (TGF-ß) in vitro, and TGF-ß signaling inhibition with SB-431542 decreased steatosis and fibrosis in vivo. Conversely, congenital absence of MPO results in reduced hepatocyte injury, decreased levels of TGF-ß, fewer activated HSCs, and less severe fibrosis in vivo. INNOVATION AND CONCLUSION: Cumulatively, these findings demonstrate important cross talk between inflammatory myeloid cells, hepatocytes, and HSCs via MPO and establish MPO as part of a proapoptotic and profibrotic pathway of progression in NASH, as well as a potential therapeutic target to ameliorate this disease.
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Comunicação Celular , Células Estreladas do Fígado/metabolismo , Hepatócitos/metabolismo , Cirrose Hepática/etiologia , Hepatopatia Gordurosa não Alcoólica/complicações , Hepatopatia Gordurosa não Alcoólica/metabolismo , Peroxidase/metabolismo , Transdução de Sinais , Animais , Apoptose/genética , Permeabilidade da Membrana Celular , Citocinas/biossíntese , Modelos Animais de Doenças , Ativação Enzimática , Expressão Gênica , Hepatócitos/patologia , Membranas Intracelulares/metabolismo , Cirrose Hepática/patologia , Camundongos , Camundongos Knockout , Mitocôndrias Hepáticas/metabolismo , Modelos Biológicos , Células Mieloides/imunologia , Células Mieloides/metabolismo , Neutrófilos/metabolismo , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/patologia , Estresse Oxidativo , Peroxidase/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
BACKGROUND: Enzymatic activity measurements of the highly oxidative enzyme myeloperoxidase (MPO), which is implicated in many diseases, are widely used in the literature, but often suffer from nonspecificity and lack of uniformity. Thus, validation and standardization are needed to establish a robust method that is highly specific, sensitive, and reproducible for assaying MPO activity in biological samples. PRINCIPAL FINDINGS: We found conflicting results between in vivo molecular MR imaging of MPO, which measures extracellular activity, and commonly used in vitro MPO activity assays. Thus, we established and validated a protocol to obtain extra- and intracellular MPO from murine organs. To validate the MPO activity assays, three different classes of MPO activity assays were used in spike and recovery experiments. However, these assay methods yielded inconsistent results, likely because of interfering substances and other peroxidases present in tissue extracts. To circumvent this, we first captured MPO with an antibody. The MPO activity of the resultant samples was assessed by ADHP and validated against samples from MPO-knockout mice in murine disease models of multiple sclerosis, steatohepatitis, and myocardial infarction. We found the measurements performed using this protocol to be highly specific and reproducible, and when performed using ADHP, to be highly sensitive over a broad range. In addition, we found that intracellular MPO activity correlated well with tissue neutrophil content, and can be used as a marker to assess neutrophil infiltration in the tissue. CONCLUSION: We validated a highly specific and sensitive assay protocol that should be used as the standard method for all MPO activity assays in biological samples. We also established a method to obtain extra- and intracellular MPO from murine organs. Extracellular MPO activity gives an estimate of the oxidative stress in inflammatory diseases, while intracellular MPO activity correlates well with tissue neutrophil content. A detailed step-by-step protocol is provided.
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Ensaios Enzimáticos/métodos , Peroxidase/metabolismo , Animais , Ativação Enzimática , Ensaios Enzimáticos/normas , Espaço Extracelular/metabolismo , Feminino , Espaço Intracelular/metabolismo , Contagem de Leucócitos , Masculino , Camundongos , Camundongos Knockout , Imagem Molecular , Neutrófilos/enzimologia , Peroxidase/genética , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Angiotensin (Ang) II promotes the phosphorylation of extracellular regulated kinase (ERK); however, the mechanisms leading to Ang II-induced ERK phosphorylation are debated. The currently accepted theory involves transactivation of epidermal growth factor receptor (EGFR). We have shown that generation of phosphatidic acid (PA) is required for the recruitment of Raf to membranes and the activation of ERK by multiple agonists, including Ang II. In the present report, we confirm that phospholipase D-dependent generation of PA is required for Ang II-mediated phosphorylation of ERK in Wistar-Kyoto and spontaneously hypertensive rat preglomerular smooth muscle cells (PGSMCs). However, EGF stimulation does not activate phospholipase D or generate PA. These observations indicate that EGF recruits Raf to membranes via a mechanism that does not involve PA, and thus, Ang II-mediated phosphorylation of ERK is partially independent of EGFR-mediated signaling cascades. We hypothesized that phosphoinositide-3-kinase (PI3K) can also act to recruit Raf to membranes; therefore, inhibition of PI3K should inhibit EGF signaling to ERK. Wortmannin, a PI3K inhibitor, inhibited EGF-mediated phosphorylation of ERK (IC50, approximately 14 nmol/L). To examine the role of the EGFR in Ang II-mediated phosphorylation of ERK we utilized 100 nmol/L wortmannin to inhibit EGFR signaling to ERK and T19N RhoA to block Ang II-mediated ERK phosphorylation. Wortmannin treatment inhibited EGF-mediated but not Ang II-mediated phosphorylation of ERK. Furthermore, T19N RhoA inhibited Ang II-mediated ERK phosphorylation, whereas T19N RhoA had significantly less effect on EGF-mediated ERK phosphorylation. We conclude that transactivation of the EGFR is not primarily responsible for Ang II-mediated activation of ERK in PGSMCs.