Human TB history
- killed ~1billion people in the last two centuries
- BCG vaccine developed in 1921
- reoccurrence of TB mainly because of HIV/AIDS and resistances
- ~¹/₃ of world population is infected with Mtb
Pathology of TB
- Mtb in macrophage
- formation of granuloma -> latency
- old granuloma can become calcified (eradication) or necrotic
- necrotic granuloma can burst open -> caseous granuloma -> spread of Mtb
Mtb
- obligate human pathogen
- acid fast bacteria
- intra- & extracellular
- granuloma induction by Mtb in Mincle-/- mice
What is TDM (Trehalose-6,6-dimycolate)?
- TDM is most abundant lipid in cell wall & cord factor -> only virulent Mtb
- TDM is important for granuloma induction (Mincle is needed as factor for inflammation)
- Mincle recognises TDM
- no granuloma induced by TDM in Mincle-/- mice
Name the three animal models of TB and challenges
- mouse
- non.human primate
- zebrafish
challenge: many different stages -> no model mimics all stages
Name (dis-)advantages and routes of infection of the mouse model
advantages:
- many genetically altered strains
- easy to manipulate
- easy to handle
- drug & vaccine research
disadvantages:
- no latent infection
- death due to progressive primary TB
- no real granulomas
routes of infection:
- intraperitoneal
- intratracheal
- intravenous (high inoculation, systemic)
- aerosolization (delivery into lungs, more physiologically appropriate)
What murine parameters are analysed?
- bacterial replication in different organs
- lung pathology
- immunophenotyping using flow cytometry
- gene/protein expression
What role plays TNF in TB protection (mouse model)?
- key factor
- increases TB progression risk when receiving anti-TNF therapy (bedside observation)
- TNF is involved in maintenance and integrity of granulomas
- TNF can be soluble (sTNF) and membrane bound (memTNF)
- TNF and TNFRp55 important for early iNOS production
- granuloma necrosis only in TNF-/- or TNFRp55-/- mice
Where does a TNF antagonist play a role?
- treatment of inflammatory/autoimmune diseases (Crohn, arthritis,…)
- TNF-ABs with different variable regions
- chimeric AB -> murine vRegion and human Fc part
- humanized mAB -> human constant and variable region
- 2 extracellular domains of human TNFR2 fused to Fc part
- TNF-targeted treatment worsens TB in mice
Name (dis-)advantages of the non-human primate model
advantages:
- most useful model for latent TB
- wide range of pathology
- low dose infection 50/50 active/latent cases -> reactivation, more chronic course in active TB
disadvantages:
- costly
- lack of host genetic variants
- few tools to study immune response
Which role does TNF play in the non-human primate TB model?
- early infection -> TNF-neutralisation results in excessive, disseminated disease
- latent TB -> TNF-neutralisation causes reactivation
- similar granuloma in anti-TNF treated and non treated monkeys
- TNF-neutralisation results in dysregulation of critical cytokines
-> TNF might play a role in cellular integrity and localisation within the granuloma
Name (dis-)advantages of the zebrafish TB model
advantages:
- natural pathogen (m. marinum)
- sequenced genome
- short generation time
- easy generation of mutations (random & targeted)
- dissection of innate (embryo model) and adaptive (adult model) immunity
disadvantages:
- M. marinum not part of Mtb complex
- M. marinum doesn‘t induce pulmonary TB in humans
How to infect zebrafish embryos?
- injection in hindbrain ventricle -> recruitment of macrophages
- injection in caudal vein -> blood stream infection
- systemic infection
- multiple infection outcomes- bacteria per macrophage
- granuloma
- drug efficacy
What is the morpholino technique and how does it work?
main antisense knockdown technology in zebrafish
- splice blocking: binding to splice donor/acceptor site prevents splicosome assembly -> decay of immature mRNA
- translation blocking: binding to AUG or 5‘ UTR hinders scanning of 40S ribosome -> blocks translational initiation and elongation
What role does TNF play in the zebrafish TB model?
TNF maintains granuloma integrity indirect by restricting mycobacteria’s growth within macrophages and prevents their necrosis
What’s the role of TNF in TB?
TNF seems to be important for granuloma maintenance rather than formation (seen in every animal model and at bedside)
