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

자가면역뇌척수염에서 Mycobacterium paratuberculosis에 의한 면역원성 향상

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.

저자 스포트라이트: 실험적 자가면역 뇌척수염 중 자가항원의 면역원성 향상에 대한 Mycobacterium paratuberculosis의 보조 활성  

실험적 자가면역 뇌척수염 동안 자가항원의 면역원성을 향상시키는 Mycobacterium paratuberculosis 의 보조 활성

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  

마이코박테리아 항원 준비

섭씨 37도에서 2주 동안 농축 배지가 들어 있는 T25 플라스크에서 mycobacterium paratuberculosis K-10 균주를 성장시키는 것으로 시작합니다. 육안 검사를 통해 정기적으로 식민지 성장을 평가하십시오. 1주일 동안 진탕 인큐베이터에서 500밀리리터 삼각 플라스크에서 농축 배양액 200ml를 성장시킵니다.박테리아 현탁액을 섭씨 70도에서 5-10분 동안 두어 비활성화하고 3000G에서 10분 동안 원심분리합니다. 펠릿을 20 밀리리터의 PBS로 2회 세척하고 현탁액을 원심분리한다. 펠렛을 멸균 용기에 옮기고 액체 질소로 동결시킵니다.냉동된 펠릿을 즉시 동결건조기 챔버로 옮깁니다. 동결건조 후 바이오 클리닝 벤치로 옮긴다. 스테인리스 스틸 주걱을 사용하여 용기의 내벽에 부착된 건조된 MAP 덩어리를 제거하고 미세한 분말로 분쇄합니다.분쇄된 세포의 무게를 측정하고 수동으로 무균 10밀리리터 바이알에 넣습니다.

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 ...