Part V: Generation of bioluminescent Pseudomonads
2. Mouse models of CF
Although CF is considered to be a rare disease a large number of patients rely on science to provide a cure or at least alleviation of their condition. As for all diseases new drugs or treatment options cannot immediately be tested in humans for obvious reasons. As animal testing is not regarded favorable by the general population, and should indeed also be questioned by the researcher, alternatives or improvements of
Conclusions and outlook
C-3
existing models should be performed whenever possible. This concept is commonly known as the 3R’s – Reduce, Refine, Replace [Russell and Burch 1959]. In the context of CF various approaches to improve existing models have been undertaken.
Replacement has been attempted by using cell culture models and refinement by investigating a wide variety of species (mice, ferrets, pigs) for their ability to serve as a meaningful model. A complete elimination of animal models is unrealistic as they still are too important for the study of disease mechanisms and the testing of new treatment options. This thesis therefore focused on the refinement of existing models, by utilizing a very sensitive method which is also non-invasive, thus in turn reducing the number of animals that need to be tested. Two mouse models of CF, which are available at the central animal laboratory of Hannover Medical School, were assessed for their potential to serve as animal models of CF. The results from chapter III were quite encouraging, showing small initial differences in the breathing pattern of CF and wild type mice of either genotype. Faster, shallower breathing was observed apparently to overcome pulmonary limitations. In addition lower weight gain was observed in one mouse model – a characteristic feature of CF.
However, the findings of Teichgräber and coworkers [Teichgräber et al 2008]
reporting a higher susceptibility to infection in Cftr-deficient mice could not be confirmed. Reasons for this originate most likely from the mode of infection. They utilized an intranasal infection, which leads to a distribution of the inoculate preferentially in the large conducting airways, while only a small amount penetrates into the deeper airways. We utilized an intratracheal inoculation, therefore bypassing the large conducting airways and distributing more bacteria in the lower airways. In this setting no age-dependent difference between Cftr-deficient and wild type mice could be observed
Further reasons might be attributed to the leaky expression of CFTR in the lungs of both models. New mouse models of CF would therefore be desirable for the study of CF related pulmonary issues. As the method is now established further mouse models of CF could be assessed for their usability, since lung function measurements using non-invasive head-out spirometry are very rare and not commonly performed for all models.
Conclusions and outlook
C-4 3. Bioluminescent Pseudomonads
With the emergence of systems able to measure luminescence in whole animals (mice and rats) without the need to sacrifice them, a new methodology became available to study the course of infections utilizing bioluminescent bacteria. Not only could they be used for the study of CF-related pulmonary issues but also in other contexts where P. aeruginosa is involved, e.g. burn wounds. Consequently, a representative panel of bioluminescent strains with various origins/properties would be useful. Bioluminescent Pseudomonads were created by a rather simple and easy procedure using the vector system from Herbert P. Schweizer. As long as the target site is not degenerated virtually all Pseudomonas strains could be made bioluminescent, including strains not utilized in this study, like environmental strains (Pseudomonas putida) that degrade pollutants, or important plant pathogens (Pseudomonas syringae).
Luminescence was about a factor 4 lower than for the firefly system, albeit with certain advantages in terms of kinetics and cost. Therefore, the bioluminescent strains hold great promise for the study of infections. However, a more sensitive analysis system than the IVIS® 200 would be needed, as the sensitivity limit in mice is very quickly reached. Furthermore measurements take quite long, resulting in high background and lower sensitivity. Sufficient luminescence can currently only be achieved through the application of high doses leading to unrealistic models.
Clearance by the immune system proved to be very efficient and resulted in a marked reduction of luminescence below the sensitivity limit of the apparatus within 24 hours post inoculation. Investigating a more chronic infection model, like the agar bead model reviewed by van Heeckeren [van Heeckeren and Schluchter 2002] could be worthwhile. Topical infections with P. aeruginosa, however can be easily investigated, e.g. in the study of burn wound models. Other in vitro applications of the strains are possible as well.
In summary, both newly established methods aimed at the non-invasive, non-lethal investigation of bacterial pulmonary infections hold great promise for future experiments.
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Picture sources Taken from:
1. http://upload.wikimedia.org/wikipedia/commons/e/ef/Autorecessive_en_01.png 2. http://www.cfgenetherapy.org.uk/CFTR/cftr1.htm
3. http://www.electromedsys.com/index.html 4. http://www.hugo-sachs.de/frame1.htm
5. Taken from the IVIS® 200 Series Product brochure available at http://www.caliperls.com/products/ivis-imaging-system-200-series.htm
Abbreviations
A-1 Abbreviations
aa Amino acid
ABC ATP-binding cassette
AHL N-acyl homoserine lactone
AM Alveolar macrophage
ASL Airway surface liquid
Asm Acid sphingomyelinase
attB Gene locus, host attachment site for phage
bp Base pairs
CCD Charge-coupled device
CF Cystic fibrosis
CFF Cystic fibrosis foundation
CFTR Cystic fibrosis transmembrane conductance
regulator
CFU Colony forming units
ddH2O Double destilled water
DMSO Dimethyl sulfoxide
e.g. exempli gratia (for example)
EF50 Midtidal expiratory flow at 50% expiration
EF50 Midtidal expiratory flow at 50% expiration