alternative models for studying aspergilli dr peter warn school of translational medicine university...

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

http://www.aspergillus.org.uk/

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 areasUnderstanding Innate and adaptive immunityPathogenesisVirulenceDrug discovery

Desirable attributes of animal Desirable attributes of animal models 1models 1Mirror 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 Desirable attributes of animal models 2models 2Amenable to studies including

* Evaluation of therapeutics

* Evaluation of host response

* Evaluation of pathogen virulence

factors

* Assessment of in vivo gene

expression


Weaknesses of Animal ModelsWeaknesses 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


Potential sites of infection in mammals

Intravenous/disseminated

Intranasal/sinus

Air Pocket Subcutaneous chamber

Oral

GI tract

Peritoneal?

Vaginal

Claw/nail

Inhaled or tracheal

Skin and hair

Eyes

BladderFootpad

Heart valve

http://www.first-school.ws/t/cpmouse.html

Modulators of fungal infection – host factors• Age of animal – in general younger animals more susceptible

• Genetic background inbred v outbred – only mice

• Immune statusImmunocompetent:

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 HusbandryClean 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 modelsReview 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

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% S

urv

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Systemic infections



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Systemic infections



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2.2 x 10(6)/mL

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8.7 x 10(6)/ml

2.5 x 10(7)/mL

6.6 x 10(7)/mL

Systemic infections

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


ReviewReview of the available modelsof the available modelsMice 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

Mouse

Rat G Pig Rabbit

Cost +++ ++ +

Mouse

Rat G Pig Rabbit

Cost +++ ++ + Housing +++ ++ ++

Mouse

Rat G Pig Rabbit

Cost +++ ++ +

Housing +++ ++ ++

Availability in bulk +++ +++ + +

Mouse

Rat G Pig Rabbit

Cost +++ ++ +

Housing +++ ++ ++


Ease of handling +++ ++ ++ +

Mouse

Rat G Pig Rabbit

Cost +++ ++ +

Housing +++ ++ ++



Daily blood samples - ++ - +++

Mouse

Rat G Pig Rabbit

Cost +++ ++ +

Housing +++ ++ ++




Human lung structure

- + ++ ++

Mouse

Rat G Pig Rabbit

Cost +++ ++ + Housing +++ ++ ++ Availability in bulk +++ +++ + +



Human lung structure

- + ++ ++

Transgenics/KO strain

+++ - -

Models of localized infectionsModels of localized infections

a) Invasive Pulmonary aspergillosisMost 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


a) Invasive Pulmonary aspergillosisMost 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

Piggott and Emmons Adapted Inhalation chamber

Hinners Inhalation Chamber

SIDRANSKY and FRIEDMAN chamber

SIDRANSKYand FRIEDMAN. 1959 Am.J.Pathol. 35:169-183.

Models of localized infectionsModels of localized infectionsa) Invasive Pulmonary aspergillosis

There have been several attempts to standardize delivery of spores but none have been widely accepted

• 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 Manchesterhttp://www.sacmm.org/iaam.html

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

Suitable for:

40 mice

12 rats

8 guinea pigs

Difficult to clean after and between runs

We use vaporized formaldehyde OR VHP

Multiple strains = chambers needed


a) Pulmonary – Neutropenic Mice/Guinea pigs

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Cyclophosphamide + Cortisone

Cyclophosphamide + Cortisone

Infect

Time Course of Immunosuppression for acrylic chamber

Cyclophosphamide + Cortisone if required

Animals are severely immunocompromised

Antibiotic prophylaxis is essential – in water if possible

Severe weight loss is common

Immature animals do not tolerate immunosuppression

http://www.sacmm.org/pdf/Murine%20Inhalational%20Pulmonary%20Aspergillosis.pdf

http://www.sacmm.org/pdf/Murine%20Inhalational%20Pulmonary%20Aspergillosis.pdf

Key features of the neutropenic Key features of the neutropenic mouse/guinea pig modelmouse/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 Models of localized infectionsinfections

a) Pulmonary - Mice

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101

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103

104

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1 2 3Experiment

100

Reproducibility of infection excellent both between experiments and inter-lab


a) Pulmonary – Neutropenic Mice

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UNINFECTED

INFECTED

Murine Inhalational Model - Outcomes

Note- This model does not lead to 100% mortality

Note- There is occasionally loss of controls (steroids)


a) Pulmonary – Neutropenic Rats

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Cyclophosphamide +Long acting steroid

Cyclophosphamide

Infect by aerosol

Time Course of IPA Models

Treatment

Tissue burden mice euthanized

90-100% Untreated mice die

Tissue burden rats euthanized

100% of untreated rats die

Prednisolone in a depo formulation is used IM

Daily tail vein bleeds are possible (~1ml)



Rats need a long acclimatization period

Key features of the neutropenic rat Key features of the neutropenic rat ModelModel

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 pre-infection

Post Infection Burden: 3 x 104 cfu/g lung 48 hours post infection

Survival: Untreated Animal succumb 4-6 days post infection (100% mortality)


a) Pulmonary – Neutropenic Rat

Rats immunosupressed with 75mg/kg cyclophosphamide and 10mg/kg depo-medrone

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Days post Infection

% S

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Infected

Uninfected

CFU of infected untreated rats showed little difference in the burden over time Serial lung histopathology shows progressive Afu hyphae invading lung parenchyma

Aspergillus Burden Changes During Infection

Log CFU/gm Lung Over Time

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Characteristic disease progression in ratsCharacteristic disease progression in rats

Experimental endpoints: Major causes of death

Weight loss >25%

Laboured breathing

Bloody nasal discharge

Unable to reach food and water


a) Pulmonary – Non-neutropenic Rats

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WBC

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200mg/kg

Cortisone acetate

Infection by aerosol

200mg/kg

Cortisone acetate

Antibacterial prophylaxis

The non-neutropenic model is similar in rats and mice

The dose of cortisone is limited by toxicity



Uninfected animals have large numbers of white cells in lungs at the end of the study

*Danger of Pneumocystis in rats*

Lung Burden Progression (NON-Neutropenic)

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4h 24h 48h 72h 96h

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/gm

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Average Log CFU/gm Lung(NON)

Disease Progression – Non-neutropenic ratsDisease Progression – Non-neutropenic rats

The lung pathology following infection in non-neutropenic hosts is dominated by white cell recruitment resulting in loss of lung function

Neutropenic vs. Non-neutropenicCharacteristic 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


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

Osmotic membrane

Chambers (1cm x 0.3cm) inserted subcutaneously

Aspergillus is separated from cellular responses and unable to invade beyond the chamber.

Sampling possible though silicon membrane

Complex ‘biofilms’ develop in chamber.

Suitable for antifungal efficacy/ development of resistance/ host adaption studies

Silicon rubber membrane

1cm

Animal need ~1 week recovery post surgery

Antibiotic prophylaxis post-op

Chambers can remain in situ for up to 6 weeks

Volume recovered during sampling is small

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

alternative models for studying aspergilli dr peter warn school of translational medicine university...

Documents

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models waste disposal

weaknesses of animal

alternative models

models of aspergillosis

infection site time

clinical research models