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

We studied the pathogenesis of several neurological diseases with new techniques developed by our experts to improve treatment for patients with movement disorders who are interactive diseases. Part of our research focuses on the role of pathogens in 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 avion cell species paratuberculosis can elicit a strong humeral and cell-mediated response in patient with multiple scoliosis. Furthermore, we demonstrated that mycobacteria provide encephalitogenic 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 microbacterium tuberculosis.

This difference could be because mycobacterium paratuberculosis produces a lipopentapeptide antigen on the cell wall's face instead of glycopeptide lipids. Well, the goal of our future research, it is 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 neuroinflammation.

Begin by growing mycobacterium paratuberculosis K10 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 3, 000G 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 a 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. 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 eosin tissue analysis revealed typical paravascular and meningeal mononuclear inflammatory infiltration in the brain and spinal cord.