Alternative models for studying Aspergilli Dr Peter Warn School of Translational Medicine University of Manchester [email protected] First The Good News • This should be the only slide.
Download ReportTranscript Alternative models for studying Aspergilli Dr Peter Warn School of Translational Medicine University of Manchester [email protected] First The Good News • This should be the only slide.
Alternative models for studying Aspergilli Dr Peter Warn School of Translational Medicine University of Manchester [email protected] First The Good News • This should be the only slide you need to take notes from. • This presentation will be available at http://www.aspergillus.org.uk/ • Any SOPs referred to will be available through the same link • Additional SOPs will be available through the IAAM website http://www.sacmm.org/iaam.html Why do we need models of aspergillosis? To provide a bridge between in vitro studies and clinical research – Models have been the bedrock of research under pinning many research areas Understanding Innate and adaptive immunity Pathogenesis Virulence Drug discovery Desirable attributes of animal models 1 Mirror diseases seen in humans as closely as possible Predictive of clinical outcomes Models are standardized Reproducible Easy to set-up and require little specialist equipment Reasonable cost Chamilos et al. Lancet Infect Dis 2007; 7: 42 -55. Clemons & Stevens. Med Mycol 2005; 43: S101-10. Desirable attributes of animal models 2 Amenable to studies including * Evaluation of therapeutics * Evaluation of host response * Evaluation of pathogen virulence factors * Assessment of in vivo gene expression Chamilos et al. Lancet Infect Dis 2007; 7: 42 -55. Clemons & Stevens. Med Mycol 2005; 43: S101-10. Weaknesses of Animal Models Will never fully replicate human disease No single model answers all questions May not mimic all structural features e.g. the structure of mouse lung Additional effort with drug studies to ‘humanize’ PK and metabolic effects Animal models can be acute and expensive Chamilos et al. Lancet Infect Dis 2007; 7: 42 -55. Clemons & Stevens. Med Mycol 2005; 43: S101-10. Potential sites of infection in mammals Air Pocket Subcutaneous chamber Eyes Intranasal/sinus Skin and hair Heart valve Inhaled or tracheal Peritoneal? Oral GI tract Vaginal Claw/nail Bladder Footpad Intravenous/ disseminated Modulators of fungal infection – host factors • Age of animal – in general younger animals more susceptible • Genetic background inbred v outbred – only mice • Immune status Immunocompetent: Immunocompromised: neutropenic vs. non-neutropenic •Tissue damage • Sex - Hormone status • Site of infection - route of infection/ method of infection • Pre exposure to whole fungi- hyphae or spores – immune status • Sensitization with fungal allergens Modulators of fungal infection – fungal factors • Inoculum level • Stage of growth • Lag / log /stationary • Infection form •Spore v hyphae • Intrinsic virulence factors of the fungus • Virulence factors suitable for infection site • Time between infection and treatment Housing and Husbandry Clean dedicated animal housing Day/Night light cycles Controlled temperature/humidity Room sterilization possible between models Waste disposal Housing and Husbandry Immunosuppression Normally required to establish an infection at the site of interest Make a model more ‘reproducible’ More closely replicate human disease Cytotoxic drugs (render animals neutropenic) Steroids (inhibit functions of immune cells) Hormones (change conditions at site of infection) Irradiation (render animals neutropenic) “Knock-out” / transgenic strains (potential to effect immune function/receptors/ cytokine response etc) Model types Lethal v non-lethal models Lethal models: Animals challenged with increasing inocula till death occurs. Outcome is time of death Death can be due to multiple causes Non-lethal models: Animals infected with lower doses to develop a persistent high level infection with very mild symptoms Samples can be collected at defined time-points allowing multiple surrogate markers of disease Infected at a site which avoids systemic dissemination. Review of the available models Disseminated infection Intravenous: The “unnatural” model Easy model for lethal infection in mice and other species Targets kidneys and spleen, much less the lungs – some strains invade brain – 2o effects can occur Easy model for antifungal therapy Can be easily modified to examine pathogen specific virulence factors Bypasses many stages in the infection process Systemic infections •Lethal v non-lethal models Survival after infection with Aspergillus fumigatus AF91 infection 100 % Survival 80 60 1.0x10(6)/mL 40 20 0 0 1 2 3 4 5 6 Days post infection 7 8 9 10 Systemic infections •Lethal v non-lethal models Survival after infection with Aspergillus fumigatus AF91 infection 100 % Survival 80 60 1.0x10(6)/mL 2.2 x 10(6)/mL 40 20 0 0 1 2 3 4 5 6 Days post infection 7 8 9 10 Systemic infections •Lethal v non-lethal models Survival after infection with Aspergillus fumigatus AF91 infection 100 % Survival 80 1.0x10(6)/mL 2.2 x 10(6)/mL 3.4 x 10(6)/mL 8.7 x 10(6)/ml 2.5 x 10(7)/mL 6.6 x 10(7)/mL 60 40 20 0 0 2 4 6 Days post infection 8 10 Systemic infections •Lethal v non-lethal models Endpoints Death Surrogate marker of imminent death (hypothermia/ torticollis/renal failure) Euthanize animals at specific time-points Organ culture (quantitative) over a predefined time range Measurement of fungal products e.g. Chitin, Galactomannan Measurement of fungal burden by qPCR (either DNA or RNA)/ assessment of fungal gene expression Review of the available models Mice versus other rodents Advantages: Can study disease in mice with specific host immune defects…potentially identifying the most critical Can study disease in large numbers of fairly uniform inbred animals … increasing reproducibility of results Less space for housing Cost Ease of handling Disadvantages: Serial sampling not usually possible Lung remodelling/airway narrowing differs from larger animals Drugs are cleared from mice far more rapidly than in humans Course of disease generally very acute, leading to death or recovery Cost Mouse Rat G Pig Rabbit +++ ++ + Mouse Rat G Pig Rabbit Cost +++ ++ + Housing +++ ++ ++ Mouse Rat G Pig Rabbit Cost +++ ++ + Housing +++ ++ ++ Availability in bulk +++ +++ + + Mouse Rat G Pig Rabbit Cost +++ ++ + Housing +++ ++ ++ Availability in bulk +++ +++ + + Ease of handling +++ ++ ++ + Mouse Rat G Pig Rabbit Cost +++ ++ + Housing +++ ++ ++ Availability in bulk +++ +++ + + Ease of handling +++ ++ ++ + - ++ - +++ Daily blood samples Mouse Rat G Pig Rabbit Cost +++ ++ + Housing +++ ++ ++ Availability in bulk +++ +++ + + Ease of handling +++ ++ ++ + Daily blood samples - ++ - +++ Human lung structure - + ++ ++ Mouse Rat G Pig Rabbit Cost +++ ++ + Housing +++ ++ ++ Availability in bulk +++ +++ + + Ease of handling +++ ++ ++ + Daily blood samples - ++ - +++ Human lung structure - + ++ ++ Transgenics/KO strain +++ - - Models of localized infections a) Invasive Pulmonary aspergillosis Most models of IPA use infection by direct intranasal/intratracheal inoculation • Mice are anaesthetized and conidia suspension inhaled • Rats, Guinea pigs & Rabbits infected via tracheostomy/ intubation Advantages Relatively cost effective Little specialist equipment required Possible to infect large numbers from a single organism stock Possible to test multiple strains in a single model Models of localized infections a) Invasive Pulmonary aspergillosis Most models of IPA use infection by direct intranasal/intratracheal inoculation • Mice are anaesthetized and conidia suspension inhaled • Rats, Guinea pigs & Rabbits infected via tracheostomy/ intubation Drawbacks Enormous mouse-mouse variation - direct methods better Inter-laboratory studies difficult Distribution may not be equal between lobes Inoculum delivered in liquid – assumption that all of the inoculum delivered to lungs Some animals develop bacterial pneumonia Animals develop disease in trachea or sinuses Therapeutic studies difficult Models of localized infections a) Invasive Pulmonary aspergillosis There have been several attempts to standardize delivery of spores but none have been widely accepted SIDRANSKY and FRIEDMAN chamber Piggott and Emmons Adapted Inhalation chamber SIDRANSKYand FRIEDMAN. 1959 Am.J.Pathol. 35:169-183. Hinners Inhalation Chamber • Development and standardization of aerosol challenge model of invasive pulmonary aspergillosis • Mouse, rat, guinea pig • Provide samples and resources to other investigators • Supported by NIH / NIAID • UTHSCSA / Harbor-UCLA / University of Manchester http://www.sacmm.org/iaam.html IPA Inoculation Chambers Acrylic chamber • Conidia delivered via small particle nebulizer • Consistent inoculum level • 1 hour exposure Madison chamber • Sealed chamber • Simultaneous exposure of large number of different species • Adjust inocula sizes and exposure period IPA Inoculation Chambers – Mice, rats and guinea pigs IPA Inoculation Chambers – Mice, rats and guinea pigs Difficult to clean after and between runs We use vaporized formaldehyde OR VHP Suitable for: 40 mice 12 rats 8 guinea pigs Multiple strains = chambers needed Models of localized infections a) Pulmonary – Neutropenic Mice/Guinea pigs Cyclophosphamide + Cortisone Cyclophosphamide + Cortisone Cyclophosphamide + Cortisone if required 4000 WBC / mm3 Animals are severely immunocompromised 3500 3000 Antibiotic prophylaxis is essential – in water if possible Infect 2500 Severe weight loss is common 2000 Immature animals do not tolerate immunosuppression 1500 1000 500 0 -2 -1 0 1 2 3 4 5 6 7 8 Days Time Course of Immunosuppression for acrylic chamber http://www.sacmm.org/pdf/Murine%20Inhalational%20Pulmonary%20Aspergillosis.pdf Key features of the neutropenic mouse/guinea pig model Animals: CD1 mice (>22g) BalbC mice>18g/ Hartley guinea pigs>450g Housing: HEPA filtered cages with sterile food and water Antibacterial Prophylaxis: Several antibiotics are suitable. Best if given in water Infection: Exposure to fungal spores as an aerosol (1-2 x 109 spores) Immunosuppression: Cyclophosphamide 250mg/kg, i.p., 2 days pre- and 200mg/kg 3 days post-infection plus cortisone acetate* 250mg/kg, s.c., 2 days pre- and post-infection Post Infection Burden: 1-2 x 104 cfu/g lung 48 hours post infection Survival: Untreated Animal succumb 4-6 days post infection 60-80% (mice) 100% (guinea pigs) mortality) *Cortisone acetate is given as a suspension. Has batch variability. Remains as solid beneath skin throughout model Models of localized infections a) Pulmonary - Mice 10000 104 Cfu per mouse 1000 103 100 102 10 101 1001 11 22 33 Experiment Reproducibility of infection excellent both between experiments and inter-lab Models of localized infections a) Pulmonary – Neutropenic Mice 100% 90% Note- There is occasionally loss of controls (steroids) 80% 70% 60% 50% 40% UNINFECTED 30% INFECTED Note- This model does not lead to 100% mortality 20% 10% 0% 0 2 4 6 8 10 12 14 Murine Inhalational Model - Outcomes Models of localized infections a) Pulmonary – Neutropenic Rats Time Course of IPA Models Cyclophosphamide Cyclophosphamide + Long acting steroid 4000 Prednisolone in a depo formulation is used IM 3500 WBC / mm3 Infectbleeds are possible (~1ml) Daily tail vein 3000 by aerosol 2500Antibiotic prophylaxis is essential – in water if possible Tissue burden Tissue burden 2000Severe weight loss is commonrats euthanized mice euthanized 1500Rats need a long acclimatization period 1000 500 0 -2 -1 0 1 2 3 Treatment Days 4 5 6 7 8 100% of untreated 90-100% Untreated rats die mice die Key features of the neutropenic rat Model Animals: Sprague Dawley rats, Male 225-250g Housing: HEPA filtered cages with sterile food and water Antibacterial Prophylaxis: Baytril (enrofloxacin), 4 days pre-infection to prevent secondary bacterial pneumonia & urinary tract infection. Infection: Exposure to fungal spores as an aerosol (1 x 109 spores) Immunosuppression: Cyclophosphamide 75mg/kg, i.p., 2 days pre- and post-infection plus Depo-medrone (prednisolone) 15mg/kg, i.m., 2 days preinfection Post Infection Burden: 3 x 104 cfu/g lung 48 hours post infection Survival: Untreated Animal succumb 4-6 days post infection (100% mortality) Models of localized infections a) Pulmonary – Neutropenic Rat Rats immunosupressed with 75mg/kg cyclophosphamide and 10mg/kg depomedrone 100 % Survival 80 60 Infected Uninfected 40 20 0 0 1 2 3 4 5 Days post Infection 6 7 8 Aspergillus Burden Changes During Infection Log CFU/gm Lung Over Time Log CFU/gm Lung 12 10 8 6 Log CFU/gm Lung 4 2 0 Day0 Day1 Day2 Day3 Day 5 CFU of infected untreated rats showed little difference in the burden over time Serial lung histopathology shows progressive Afu hyphae invading lung parenchyma Characteristic disease progression in rats Experimental endpoints: Major causes of death Weight loss >25% Laboured breathing Bloody nasal discharge Unable to reach food and water Models of localized infections a) Pulmonary – Non-neutropenic Rats 200mg/kg 200mg/kg Cortisone acetate Cortisone acetate 4000 Infection by aerosol 3500 The non-neutropenic model is similar in rats and mice WBC/mm3 3000 2500The dose of cortisone is limited by toxicity 2000Antibiotic prophylaxis is essential – in water if possible 1500Severe weight loss is common 1000 Uninfected animals have large numbers of white cells in 500 lungs at the end of the study 0 *Danger of -1Pneumocystis in rats* -4 -2 0 1 2 3 4 Days Antibacterial prophylaxis 5 6 7 8 Disease Progression – Non-neutropenic rats Log CFU/gm Lung Lung Burden Progression (NON-Neutropenic) 8 7 6 5 4 3 2 1 0 Average Log CFU/gm Lung (NON) 4h 24h 48h 72h 96h Hours Post-Infection The lung pathology following infection in non-neutropenic hosts is dominated by white cell recruitment resulting in loss of lung function Neutropenic vs. Non-neutropenic Characteristic Glucocorticosteroids Neutropenia Cellular trafficking BAL Rapid and extensive increase in PMN No PMN influx Cytokines BAL TNF-α and IL-10 low to undetected TNF-α and IL-10 high Histological features Diffuse and extensive consolidation and inflammation Limited consolidation, necrosis with hyphae Fungal elements Small numbers of conidia and poorly germinated hyphae Extensive angioinvasive hyphae Amphotericin efficacy No survival improvement Survival improvement with high dose AmB/AmBisome Dominant mechanisms Adverse host inflammation Unimpeded fungal growth / invasion Berenguer et al. Am J Resp Crit Care Med 1995; 152: 1079. Balloy et al. Infect Immun 2005; 73: 494. Wiederhold N TIMM 2009 Models of localized infections a) Pulmonary – Chronic infection mice C57BL/6 mice infected intratracheally with 1 x 105 spores of A. fumigatus embedded in agarose. Disease is restricted to the lungs with no tissue invasion. Infections possible for >20 days Don Sheppard IAAM Workshop 2008 Chronic Aspergillus Models - Tissue Chambers Chambers (1cm x 0.3cm) inserted subcutaneously Silicon rubber membrane 1cm Osmotic membrane Animal need ~1 recoveryfrom post cellular surgery responses and Aspergillus is week separated unable to invade beyond the chamber. Antibiotic prophylaxis post-op Chambers can remain in situ forsilicon up to 6membrane weeks Sampling possible though Volume recovered during sampling is small Complex ‘biofilms’ develop in chamber. Suitable for antifungal efficacy/ development of resistance/ host adaption studies No time to discuss other models • Rabbits – great for drug and imaging studies • Transgenic/knockout mouse models – fantastic for understanding disease mechanisms • Non-mammalian hosts • Sinus models • Allergy/Asthma Acknowledgements • • • • • • • • Andrew Sharp Raghdaa Shrief Jayesh Majithiya Joanne Slater David Denning University of Manchester IAAM Contract team Fungal Research Trust