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

ऑटोइम्यून एन्सेफेलोमाइलाइटिस में माइकोबैक्टीरियम पैराट्यूबरकुलोसिस द्वारा इम्युनोजेनेसिटी एन्हांसमेंट

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.

लेखक स्पॉटलाइट: प्रायोगिक ऑटोइम्यून एन्सेफैलोमाइलाइटिस के दौरान ऑटोएंटिजेन्स की इम्युनोजेनेसिटी को बढ़ाने में माइकोबैक्टीरियम पैराट्यूबरकुलोसिस की सहायक गतिविधि  

प्रायोगिक ऑटोइम्यून एन्सेफैलोमाइलाइटिस के दौरान ऑटोएंटीजन की इम्युनोजेनेसिटी को बढ़ाने में माइकोबैक्टीरियम पैराट्यूबरकुलोसिस की सहायक गतिविधि

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  

माइकोबैक्टीरियल एंटीजन तैयारी

37 डिग्री सेल्सियस पर दो सप्ताह के लिए समृद्ध माध्यम वाले टी 25 फ्लास्क में माइकोबैक्टीरियम पैराट्यूबरकुलोसिस के -10 स्ट्रेन को उगाने से शुरू करें। दृश्य निरीक्षण के माध्यम से नियमित रूप से कॉलोनी के विकास का आकलन करें। एक सप्ताह के लिए हिलने वाले इनक्यूबेटर में 500 मिलीलीटर एर्लेनमेयर फ्लास्क में 200 मिलीलीटर संवर्धन संस्कृति विकसित करें।बैक्टीरियल सस्पेंशन को 70 डिग्री सेल्सियस पर पांच से 10 मिनट के लिए रखें ताकि उन्हें निष्क्रिय किया जा सके और उन्हें 10 मिनट के लिए 3000 जी पर सेंट्रीफ्यूज किया जा सके। 20 मिलीलीटर पीबीएस में छर्रों को दो बार धोएं और निलंबन को सेंट्रीफ्यूज करें। गोली को एक बाँझ कंटेनर में स्थानांतरित करें और इसे तरल नाइट्रोजन के साथ फ्रीज करें।जमे हुए गोली को तुरंत एक लियोफिलाइज़र कक्ष में स्थानांतरित करें। लियोफिलाइजेशन के बाद इसे बायो क्लीनिंग बेंच में स्थानांतरित करें। स्टेनलेस स्टील स्पैटुला का उपयोग करके कंटेनर की आंतरिक दीवारों का पालन करने वाले सूखे एमएपी गांठों को हटा दें और उन्हें एक महीन पाउडर में बदल दें।पल्वराइज्ड कोशिकाओं का वजन करें और मैन्युअल रूप से उन्हें एक सड़न रोकनेवाला 10 मिलीलीटर शीशी में रखें।

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 ...