mycobacterial antigen preparation

自己免疫性脳脊髄炎における マイコバクテリウム・パラ結核 による免疫原性増強

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.

著者スポットライト:実験的自己免疫性脳脊髄炎における自己抗原の免疫原性増強におけるパラ結核菌のアジュバント活性  

実験的自己免疫性脳脊髄炎における自己抗原の免疫原性増強における 抗酸菌パラ結核の アジュバント活性

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  

マイコバクテリア抗原調製

濃縮培地を含むT25フラスコでマイコバクテリウム・パラ結核K-10株を摂氏37度で2週間増殖させることから始めます。目視検査を通じてコロニーの成長を定期的に評価します。200ミリリットルの濃縮培養物を500ミリリットルの三角フラスコで1週間振とうインキュベーター内で成長させる。細菌懸濁液を摂氏70度で5〜10分間置き、それらを不活性化し、3000Gで10分間遠心分離します。ペレットを20ミリリットルのPBSで2回洗浄し、懸濁液を遠心分離します。ペレットを滅菌容器に移し、液体窒素で凍結します。凍結ペレットを直ちに凍結乾燥機チャンバーに移します。凍結乾燥後、バイオクリーニングベンチに移します。ステンレス製のヘラを用いて、容器内壁に付着した乾燥したMAP塊を取り除き、微粉末に粉砕します。粉砕した細胞を秤量し、手動で無菌10ミリリットルのバイアルに入れます。

Research

JoVE Journal

Immunology and Infection

Mycobacterial Antigen Preparation

265 ビュー.  Juntendo University. 濃縮培地を含むT25フラスコでマイコバクテリウム・パラ結核K-10株を摂氏37度で2週間増殖させることから始めます。目視検査を通じてコロニーの成長を定期的に評価します。200ミリリットルの濃縮培養物を500ミリリットルの三角フラスコで1週間振とうインキュベーター内で成長させる。細菌懸濁液を摂氏70度で5〜10分間置き、それらを不活性化し、3000Gで10分間遠心?...

ビデオ: 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 complete Freund&#39;s adjuvant (CFA) containing inactivated Mycobacterium tuberculosis. The antigenic components of the mycobacterium activate dendritic cells to stimulate T-cells to produce cytokines that promote the Th1 response via toll-like receptors. Therefore, the amount and species of mycobacteria present during the antigenic challenge are directly related to the development of EAE. This methods paper presents an alternative protocol to induce EAE in C57BL/6 mice using a modified incomplete Freund&#39;s adjuvant containing the heat-killed Mycobacterium avium subspecies paratuberculosis strain K-10.
M. paratuberculosis, a member of the Mycobacterium avium complex, is the causative agent of Johne&#39;s disease in ruminants and has been identified as a risk factor for several human T-cell-mediated disorders, including multiple sclerosis. Overall, mice immunized with Mycobacterium paratuberculosis showed earlier onset and greater disease severity than mice immunized with CFA containing the strain of M. tuberculosis H37Ra at the same doses of 4 mg/mL. The antigenic determinants of Mycobacterium avium subspecies paratuberculosis (MAP)&#160;strain K-10 were able to induce a strong Th1 cellular response during the effector phase, characterized by significantly higher numbers of T-lymphocytes (CD4+ CD27+), dendritic cells (CD11c+ I-A/I-E+), and monocytes (CD11b+ CD115+) in the spleen compared to mice immunized with CFA. Furthermore, the proliferative T-cell response to the MOG peptide appeared to be highest in M. paratuberculosis-immunized mice. The use of an encephalitogen (e.g., MOG35-55) emulsified in an adjuvant containing M. paratuberculosis in the formulation may be an alternative and validated method to activate dendritic cells for priming myelin epitope-specific CD4+ T-cells during the induction phase of EAE.

Here, we present an alternative protocol to actively induce experimental autoimmune encephalomyelitis in C57BL/6 mice, using the immunogenic epitope myelin oligodendrocyte glycoprotein (MOG)35-55 suspended in incomplete Freund's adjuvant containing the heat-killed Mycobacterium avium subspecies paratuberculosis.

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

免疫・感染学

Begin by growing mycobacterium paratuberculosis K-10 strain in T25 flasks containing enriched medium for two weeks at 37 degrees Celsius. Assess the colony growth regularly through visual inspection. Grow 200 milliliters of the enrichment culture in a 500 milliliter erlenmeyer flask in a shaking incubator for one week.Place the bacterial suspensions at 70 degrees Celsius for five to 10 minutes to inactivate them and centrifuge them at 3000G for 10 minutes. Wash the pellets in 20 milliliters of PBS twice and centrifuge the suspension. Transfer the pellet to a sterile container and freeze it with liquid nitrogen.Transfer the frozen pellet to a lyophilizer chamber immediately. After lyophilization transfer it to a bio cleaning bench. Using a stainless steel spatula dislodge the dried MAP lumps adhering to the inner walls of the container and pulverize them to a fine powder.Weigh the pulverized cells and manually place them in an aseptic 10 milliliter vial.

Preparation and Injection of Myelin Oligodendrocyte Glycoprotein (MOG35-55) Emulsion

Preparation and Injection of Myelin Oligodendrocyte Glycoprotein (MOG35-55) Emulsion  

Begin by grinding 10 milligrams of freeze-dried Mycobacterium paratuberculosis to a fine powder using a mortar and pestle. Add five milliliters of Incomplete Freund's adjuvant to obtain a 10 milligram per milliliter stock solution. Store the stock at minus four degrees Celsius.Before immunization, dilute the stock solution. Then, dilute the MOG 35-55 peptide to two milligrams per milliliter and store it at minus 20 degrees Celsius. Mix equal volumes of the MOG 35-55 with the adjuvant in a five milliliter tube with a homogenizer until a thick emulsion is formed.After every 10 seconds of mixing, place the solution on ice for 20 seconds and spin the tube to recover all the solution. Transfer the emulsion into a one milliliter syringe, ensuring it is free of air bubbles. Then, fit it with a 27-gauge needle.Subcutaneously, inject 200 microliters of the emulsion into the lower back of an anesthetized mouse. Then, intraperitoneally inject a dose of pertussis toxin on day zero and two after immunization. All mice manifested an acute monophasic disease, characterized by a single peak of disability observed at 14 to 17 days, followed by a partial recovery of symptoms over the next 10 days.Mice immunized with M.paratuberculosis showed an earlier onset after immunization and greater severity in the acute phase. Both groups demonstrated similar body weights. Proliferation assay with titrated thymidine incorporation performed on the EAE mice spleen cells showed a strong proliferative response to the peptide MOG 35-55, but not to ovalbumin.Cytofluorometric analysis showed increased T-lymphocytes, dendritic cells, and monocytes in the spleen of M.paratuberculosis-immunized EAE mice compared to CFA-immunized mice. Hematoxylin and and eosin tissue analysis revealed typical paravascular and meningeal mononuclear inflammatory infiltration in the brain and spinal cord.