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Establishing a gnotobiotic facility

Timothy Hand PhD


R.K. Mellon Institute for Pediatric Research
Childrens Hospital of Pittsburgh
University of Pittsburgh

Gastrointestinal Immune Homeostasis


mucus
epithelium
SIgA
Commensal/foo
d antigens

IgA+

Th17

CD103+ DC
Treg

commensalspecific Tregs

MLN
Treg

Lamina
Propria

CD103+ DC

mucosal firewall

commensalspecific plasma
B cell

Physical Barrier
- Epithelia/Tight Junctions
- Mucus Layer
- Anti-Microbial Peptides
Active Barrier
- Tolerogenic DCs
- Tregs
- IgA+ B cells

peripheral
lymphoid
organs

MacPherson, Powrie, Rudensky, Littman, Elson, Fagarasan, Hsieh, Belkaid et al.

Dysregulated immune responses against the microbiota contribute


to multiple chronic inflammatory diseases
Gut:
Environmental Enteropathy
Crohns Disease
Ulcerative Colitis
Colon cancer
Necrotizing Enterocolitis
B

B
B

Teff

N
Teff

Teff
Teff

Stomach
Ulcers and stomach cancer
Lung
Cystic Fibrosis
Skin:
Psoriasis
Atopic dermatitis

Infection at mucosal sites blurs the distinction between pathogen and


commensal
Health

Inflammation
1

Molloy et al. (2013) Cell Host Microbe

TNF

Dendritic
cell

IFN

Inflammatory
Monocyte

Neutrophil
T cell
Lamina propria

TNF

Adipose tissue
Effector
T cell

2
Fonseca, Hand et al.
(2015) Cell
3

mesLN

Hand et al. (2012) Science

Objectives: Establishing a gnotobiotic


community
Education and training
Training for facility
Education of the community (both users and
administration)

Steps to building a new gnotobiotic center


Design of the facility
Types of isolators

Building a gnotobiotics infrastructure


Sequencing (16S and metagenomics)
Bioinformatics
Microbiology

Education: Administration
Need to get buy-in from the Senior Administration
- Will provide you with start-up for facility as well as subsidy to
keep facility running

Need to make strong case for the benefits of the facility


(Cost controlled)
Need to be clear on the necessity for point-of-contact
facility versus collaboration
Fit gnotobiotics into framework of microbiome and
metagenomics effort

Education: Community
What are gnotobiotic animals?
How are they made? (This one is critical)
What are the advantages and limitations of these
animals?
How do we physically carry out the experiments and
how does that affect experiment design? (need to
explain the isolators etc.)
This is also an ad hoc recruitment effort because we
need people to use the facility

Education: Microbiome Symposium

Ran full day symposia this past Spring


Double effect of education and discovering peoples needs in the facility
Have BootCamp on the microbiome planned for next month
Result: Pitt has unique interest in pulmonary microbiome and relationship
of microbiome to cancer/therapies. Also significant interest in looking at
shifts in the microbiota (mutations, transcription) as response to chronic
disease
Needs: long-term housing, ability to handle BSL2+ pathogens

Training for facility


Staff
Staff >>>>>>>> Facilities
Training and hiring is incredibly important
1. Hire details-oriented personnel with
experience working in gnotobiotic setting
2. Send carefully selected staff to train in
another facility for a few weeks (We hope
to send out staff to the NIH)
3. Adherence to SOP

Designing a gnotobiotic facility (big thanks to Dr.


Alexander Chervonsky and Univ. of Chicago)
Components of facility:
Redundancy

protects against whole


facility contamination
and strain loss

Autoclaves outside room


Decreases noise
contamination and
allows for easy
expansion
HVAC/forced air
Positive to the
hallway
Compressed air for
fogging the isolators
Security

Gnotobiotic Isolators

CDC vinyl isolator


Advantages
Can breed (will use the larger isolators)
Can house mice for long periods of time (6
mos+ @ NIAID/NIH facility)
SOP is well developed
Disadvantages
Footprint limits size of experiments/facility
Turnover time of isolators
Breaking down/inflating isolators is labour
intensive
Large experiments must be done out of
isolation

Tecniplast Caging system


Advantages
Efficiency (34 experiments at once in small
footprint)
Short turnover time
Disadvantages
Max time without contamination is about 46 weeks
Will still need vinyl isolators for breeding
and re-derivation
Changing cages is extremely labourious

Sourcing the mice


Taconic
Reliable source for C57BL/6, but costly
(>$400/mouse with shipping)
Genetically-modified strains
Either have to be re-derived in house or sourced
from pre-existing facility (UNC)
Whether we can do re-derivations immediately
will depend upon the experience of the staff and
the needs of the community

Building a gnotobiotic infrastructure


Anaerobic chamber

Sequencing

MiSeq x 2

NextSeq x 2

Building a gnotobiotic infrastructure


Bioinformatics expertise:
Kyle Bibby
Vaughn Cooper
Kelvin Li
Barbara Meth
Mike Morowitz
Alison Morris

Welfare of gnotobiotic animals


Gnotobiotic animals have unique health requirements
We will focus on hiring/training people who are
concientious, detail-oriented and experienced
We will implement the same SOP that has decades of
proven success at UAB, U of Chicago and the NIH

Education of researchers on what is a feasible and


controllable experiment

Acknowledgements
Pitt
Mark Shlomchik
Mark Gladwin
Terry Dermody
Art Levine
Alison Morris
Joe Newsome
Jay Kolls
NIH
Yasmine Belkaid

Funding:

UAB
Trent Schoeb
Chicago
Alexander Chervonsky

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