Name: Mycobacterial Antigen Preparation
Uploaded: 2023-05-12T14:55:00
Description: Watch this Scientific Journal Video about Mycobacterial Antigen Preparation at JoVE.com

mycobacterial antigen preparation

Aumento da imunogenicidade por Mycobacterium paratuberculosis na encefalomielite autoimune

Experimental autoimmune encephalomyelitis (EAE) is a common model for the study of human demyelinating disorders1. There are several models of EAE: active immunization using different myelin peptides in combination with potent adjuvants, passive immunization by in vitro transfer of myelin-specific CD4+ lymphocytes, and transgenic models of spontaneous EAE2. Each of these models has specific features that allow different aspects of EAE to be studied, such as the onset, effector phase, or chronic phase. The myelin oligodendrocyte glycoprotein (MOG) model of EAE is a good model to study the immune-mediated mechanisms of chronic neuroinflammation and demyelination, as it is characterized by mononuclear inflammatory infiltration, demyelination in peripheral white matter, and reduced recovery after the disease peak1.
MOG-EAE is induced by immunization of susceptible mice with the peptide MOG35-55 in complete Freund&#39;s adjuvant (CFA), followed by an intraperitoneal injection of pertussis toxin. This increases the permeability of the blood-brain barrier and allows myelin-specific T-cells activated in the periphery to reach the central nervous system (CNS), where they will be reactivated3. CFA plays a key role in the induction of EAE by enhancing the antigen uptake by antigen-presenting cells and the expression of cytokines related to humoral- and cell-mediated responses4. This mechanism is mainly due to the presence of killed Mycobacterium tuberculosis emulsified in oil, the components of which provide a strong stimulus for the immune system5. In fact, the induction of EAE is directly related to the amount of mycobacterium present during the antigenic challenge6.
The addition of other killed mycobacteria, such as Mycobacterium butyricum, to incomplete Freund&#39;s adjuvant7, as well as the effect of adjuvant combinations8, can modulate the clinical course of EAE and consequently influence the reproducibility of the results. Mycobacterium avium subspecies paratuberculosis (MAP), the etiologic agent of Johne&#39;s disease in ruminants, has been associated with inflammatory disorders of the human CNS9, as its antigenic components are capable of eliciting a strong humoral- and cell-mediated response in patients with multiple sclerosis and neuromyelitis optica spectrum disorder9. Therefore, in this protocol, we show an alternative and reproducible method to induce MOG-EAE by replacing M. tuberculosis in CFA with M. paratuberculosis.

Autor em destaque: Atividade adjuvante de Mycobacterium paratuberculosis no aumento da imunogenicidade de autoantígenos durante a encefalomielite autoimune experimental  

Atividade Adjuvante de Mycobacterium paratuberculosis no Aumento da Imunogenicidade de Autoantígenos Durante Encefalomielite Autoimune Experimental

We studied the pathogenesis of several neurological diseases with new techniques developed by our experts to improve treatment for patient with movement disorders who have interactive diseases. Part of our research focuses on the role of pathogens and the etiology of new inflammatory diseases such as multiple sclerosis or NMOSD, which are characterized by a complex interplay of genetic and environmental factors. Various pathogens have been associated with these diseases, although their role is unclear.We found that antigenic components of Mycobacterium avian cell species paratuberculosis can elicit a strong humoral and cell-mediated response in patient with multiple sclerosis. Furthermore, we demonstrated that Mycobacteria provide inset autogenic potential by acting on the gut immune brain axis in the EAE model. We identified Mycobacterium paratuberculosis as a potent adjuvant candidate in the induction of active EAE, which resulted in a more severe disease than that induced by inactivated Mycobacterium tuberculosis.This difference could be because Mycobacterium paratuberculosis produces a lipo pentapeptide antigen on the cell wall surface instead of glycopeptide lipids. One of the goals of our future research is to elucidate the crosstalk between the immune response of the peripheral and central nervous system, and to understand the mechanism of infection-mediated neuroinflammation. For this, we were developing unique genetics model of neuroinflammations.

Watch this Scientific Journal Video about Mycobacterial Antigen Preparation at JoVE.com

Mycobacterial Antigen Preparation  

Preparação de antígenos micobacterianos

Comece cultivando a cepa K-10 da mycobacterium paratuberculosis em frascos T25 contendo meio enriquecido por duas semanas a 37 graus Celsius. Avaliar o crescimento da colônia regularmente através de inspeção visual. Cultivar 200 mililitros da cultura de enriquecimento em um frasco erlenmeyer de 500 mililitros em uma incubadora de agitação por uma semana.Coloque as suspensões bacterianas a 70 graus Celsius por cinco a 10 minutos para inativá-las e centrifugue-as a 3000G por 10 minutos. Lave os pellets em 20 mililitros de PBS duas vezes e centrifugue a suspensão. Transfira o pellet para um recipiente estéril e congele-o com nitrogênio líquido.Transfira o pellet congelado para uma câmara liofilizadora imediatamente. Após a liofilização, transfira-o para uma bancada de biolimpeza. Usando uma espátula de aço inoxidável, desaloje os grumos MAP secos aderidos às paredes internas do recipiente e pulverize-os em um pó fino.Pese as células pulverizadas e coloque-as manualmente em um frasco asséptico de 10 mililitros.

Research

JoVE Journal

Immunology and Infection

Mycobacterial Antigen Preparation

Adjuvant Activity of Mycobacterium paratuberculosis in Enhancing the Immunogenicity of Autoantigens During Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG) requires immunization by a MOG peptide emulsified in ...