Animals models for host-defense and ethics relating to use of animals in research

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Animals models for host-defense and ethics
relating to use of animals in research
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Objectives

1. Brief review of animal (inbred mice) use and
   contributions in immunology
2. Brief review of methods for measuring immune
   response in animals
3. Discuss key considerations in measuring immune
   response in animals
4. Discuss key ethical issues relating to use of
   animals in research

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Animal models of diseases

• A living, non-human animal used during the
  research and investigation of human disease.
• Allows better understanding of disease without
  the added risk of causing harm to actual human
  host
• Animal chosen will usually meet a determined
  taxonomic equivalency to humans in order to react
  to disease or treatment in a way that resembles
  human physiology

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Why animal research?

• Many similarities b/w animal and human physiology
• Immune function in mice
• Cardiovascular function in dogs
• Animals provide index for safety
• Nuremberg Code Animal studies precede human
  studies
• Helsinki Declaration medical research in humans
  must be supported by preceding animal research
• Almost all medical advances in the 19th and 20th
  centuries started with animal experimentation

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Types of animal models

• Homologous model have the same causes, symptoms
  and treatment options as would humans who have
  the same disease
• Isomorphic model share the same symptoms and
  treatments
• Predictive model animals strictly display only
  the treatment characteristics of a disease.
• commonly used when the cause of a disease is
  unknown i.e. screening

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Most commonly used animals

• Mice
• Fish
• Rats
• Rabbit
• Guinea pigs
• Dogs/pigs/chicken

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Advantages of mouse models

1. Small and cheap
2. Reagents are available
3. Inbred lines are available
4. Human disease models are easily created
5. Large, controlled crosses can be made (short
   generation time)
6. Experimental manipulations
7. Transgenics, knock-outs and knock-ins

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Inbred Mice

• Genetically identical animals produced by
  inbreeding
• Generated by sister-brother mating over
  generations
• Completely homozygous at all genetic loci
• Syngeneic to every other mouse of the same strain
  

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Advantages/Disadvantages

• Advantages
• Genetic differences are eliminated
• Permits adoptive transfer experiments
• Invaluable contribution to immunology
  transplantation
• Disadvantages
• Simplistic view of the immune system
• Relevance to human immunology?

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Inbred but different?

• Genetic contamination introduction of undefined
  genetic material
• Accidental (carefree, careless or tired animal
  handler)
• Not accidental records are lost in time or
  forgotten
• Direct mix up of distinct but genetically
  different strains e.g. same coat color
• Genetic drifts random genetic change that acts
  in concert with evolution to change species over
  time
• Constant tendency of genes to evolve even in the
  absence of selective forces
• Environmental effects
• Infections, stressors

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Transgenic Animals

• Introduction of foreign or altered gene by
• DNA microinjection
• Retrovirus-mediated gene transfer
• Embryonic stem cell-mediated gene transfer
• Methods
• Over-expression
• mis-expression
• dominant-negative
• Importance
• Agriculture (breedging, quality, disease
  resistance)
• Medicine (xenotransplantation, gene therapy,
  nutritional supplements, drugs)
• Industry

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Knockout animals

• Selective inactivation of a part of or a whole
  gene
• conventional knock-out
• knock-in/replacement
• tissue-specific knock-out
• inducible knock-out tissue-specific with
  temporal control

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Animal models of autoimmunity

• I- Experimental allergic encephalomyelitis (EAE)
• Organ specific
• Immunization with myelin basic protein (MBP) and
  adjuvant.
• Perivascular inflammation (CD4 T cells )
  phagocytes recruitment.
• Enzymes release and demyelination.
• Formation of auto Abs to MBP and proteolipid
  protein (PLP)
• Disease can be induced by adoptive transfer of
  CD4 T cells
• Human multiple sclerosis
• Symptoms shaky movements of the limbs, defects
  in speech

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Simple cutaneous leishmaniasis

• Caused by Leishmania major
• Normally self-healing
• DTH, T cell proliferation
• Low antibody responses
• Healing results in solid immunity

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Diffuse cutaneous leishmaniasis

• No DTH responses
• Antibody present
• Chronic, dissemination to many body parts
• High parasite burden
• Refractory to drug treatment

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L. major infection in mice mimics human cutaneous
disease
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CD4T helper cell cytokines regulate disease
outcome in mice
Arbitrary Units
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Mouse model of Asthma

• Mice do not spontaneously get asthma
• Models of acute and chronic allergic airway
  responses to inhaled allergens are widely used
• Type of inflammatory response is influenced by
  several factors including
• The mouse strain
• The allergen
• The sensitization challenge protocol

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Asthma in mice

• The BALB/c strain is commonly used
• Develop robust type 2 immune response associated
  with asthma
• Ovalbumin (OVA) is the most frequent allergen
• House dust mite (HDM) cockroach antigen are
  increasingly been used because OVA is not
  commonly implicated in human asthma

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Acute Asthma in mice

• Sensitization Phase
• Multiple systemic administrations of the allergen
  an adjuvant
• Aluminum hydroxide (AlOH3) ? Th2 response
• Elicitation phase
• Short-term exposure to high dose of alum

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Features of acute mouse asthma

• Similarities with human disease
• Elevated IgE levels
• Airway inflammation
• Goblet cell hyperplasia
• Epithelial hypertrophy
• AHR to specific stimuli
• In some models, early- late-phase
  bronchoconstriction in response to allergen
  challenge

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Chronic Model

• Chronic asthma models
• Attempt to model chronic AHR remodeling
• Repeated airway exposure to low allergen levels
  up to 12 wks
• Reproduces some hallmarks of human asthma
  including allergen-dependent sensitization

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Chronic asthma in mice

• Similarities with human asthma
• A Th2-dependent allergic inflammation
• Eosinophilic influx into the airway mucosa
• Airway hyper-responsiveness
• Airway remodeling
• Goblet cell hyperplasia
• Subepithelial fibrosis
• Epithelial hypertrophy

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Some Common Mouse Studies

• Adoptive transfer
• Tissue vs whole body immuno-imaging
• Transplantation
• Tumor immunology
• Cell culture systems
• Protein biochemistry
• Molecular Biology Technology

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Some in vitro studies
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Limiting Dilution Assay

• Highly sensitive technique
• Permits the measurement of frequency of
  antigen-specific lymphocyte
• Method
• Varying cell from normal or immune mice are
  plated
• Stimulate with cognate Ag and irradiated APC
  IL-2
• Measure effector cell response e.g. cytokine
  production or cytotoxicity
• Calculate frequency using Poisson distribution

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ELISA
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ELISPOT Assay

• Highly sensitive technique
• Modification of ELISA
• Permits identification of cell type secreting
  cytokine
• Determines frequency of antigen-specific cell
• Can be used for B cell (humoral) assay also
• Spots may be difficult to accurately enumerate.

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ELISA vs. ELISPOT
ELISA ELISPOT
Sensitivity
Bias due to auto-consumption of protein YES NO
Detection of parallel secretion of 2 proteins NO YES
Identification of cell type secreting cytokine NO Yes
Acquisition of numerical data Light wave extinction and std curve Computerized visual analysis
Storage time with possibility to re-analyze Hours Weeks to Months
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Intracellular detection of cytokines Flow
cytometry

• A powerful technique to detects antigen-specific
  cytokine secreting cells
• Permits identification of lymphocyte subset

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Flow cytometry contd

• Advantages
• Simultaneous detection of 2 or more cytokines in
  a single cell
• Dectection of cytokine production in a rare or
  specific cell population
• High throughput
• Easily applied in clinical studies
• Measurement of effector function
• Cytokine production
• Disadvantages
• Sophisticated equipment
• Availability and accessibility

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Flow cytometry issues

• ELISA and ELISPOT measure cytokine accumulation
  over time summation
• Flow cytometry yields results for specific time
  points
• No time point will detect all cytokine producing
  cells
• No time point may be optimal for various
  cytokines
• For multiple cytokines, kinetics recommended

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Some in vivo animal studies
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Secondary Challenge Assay

• Measures secondary memory response in immune
  host
• Method
• Animals previously exposed to pathogen (Ag) are
  challenged with the same pathogen
• Extent of pathology is compared with naïve
  controls
• DTH response
• Ab and/or cytokine response
• Survival/death

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Adoptive Transfers

• Passive
• Transfer of immune factors from immunized to a
  naïve recipient
• Immune sera
• Cytokines
• Active
• Transfer of immune cells from immunized to naïve
  recipients
• B cell
• T cells

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Adoptive Transfer Assays

• Highly sensitive technique to measure
  protective immunity
• Permits delineation of lymphocyte subset that
  mediates protection
• Method
• Cells from immune mice are adoptively
  transferred to naïve host
• Recipient is then challenged with antigen
  (pathogen)
• Effector response e.g. DTH can be measured

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Practical considerations

• A good assay to measure immune response in
  animals must be
• Highly specific
• Highly sensitive
• Very reproducible
• Utilize small sample
• Easy to perform (practical)
• Worth the effort and money (value)

• Realistic issues
• Sensitivity vs Specificity
• Herd vs individual monitoring

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Animal related factors affecting results

• Sample size
• Genetic make-up
• Inbred vs. outbred vs. mutants vs. genetically
  modified
• Physiology (Age, sex, reproductive status)
• Microbial flora
• Biological rhythms
• Presence of stress/distress
• Diseases
• Latent (subclinical or silent) infections
• Genotype-related conditions

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Determining sample size

• Step 1 Define experiment primary objective
• Step 2 Define study Design
• Step 3 Define clinically significant difference
  one wishes to detect
• Step 4. Define degree of certainty of finding
  this difference

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Important considerations

• The following affect the quality and magnitude of
  immune response
• Routes of immunization or exposure
• Antigen dose
• Low and high zone tolerance/paralysis
• Adjuvants

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Confounding Factors

• In-apparent (latent) infections
• MHV, Norovirus, Pinworms etc
• Physiological states
• Effects of hormones
• Environmental stressors
• Changes in levels of steroids and cathecolamines

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Ethical issues

• Ask the experimenters why they experiment on
  animals and the answer is 'Because animals are
  like us.
• Ask the experimenters why it is morally okay to
  experiment on animals, and the answer is
  'Because the animals are not like us.
• Animal experimentation rests on a logical
  contradiction.
• Prof. Charles R. Magel

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• Vivisection is a social evil because it advances
  human knowledge, it does so at the expense of
  human character
• George Bernard Shaw

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Critical Ethical Questions

• Should animals be used in research?
• Is there anything wrong in transferring human
  genes into other species and vice versa?
• Is it right to carry out animal research that
  involves pain, suffering and distress?
• Do we as a society want xenotransplantation as a
  medical procedure?
• Should marine mammals be kept in captivity?
• Should society permit stem cell research
  involving fusion of human-mouse embryos?

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Helsinki Declaration

• Biomedical research involving human subjects must
  conform to generally accepted scientific
  principles and should be based on adequately
  performed laboratory and animal experimentation
  and on a thorough knowledge of the scientific
  literature.
• No animal experiment shall be conducted for a
  purpose which, by expert consensus, may also be
  achieved by means other than an animal
  experiment, or by means of an experiment using
  fewer animals or entailing less distress than
  the experiment in question.

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Canadian Council on Animal Care Mandate

• The purpose of the Canadian Council on Animal
  Care is to act in the interests of the people of
  Canada to ensure through programs of education,
  assessment and persuasion that the use of
  animals, where necessary, for research, teaching
  and testing employs optimal physical and
  psychological care according to acceptable
  scientific standards, and to promote an increased
  level of knowledge, awareness and sensitivity to
  relevant ethical principles."

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The 3Rs

• Reduction to reduce the number of animals used
  to as few as possible.
• Replacement to use alternative non-animal
  methods whenever they are available.
• Refinement to refine all procedures to ensure
  that as little pain and stress as possible is
  experienced by the animals.

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To achieve 3Rs

• Researchers responsibility to prove that no
  non-animal alternatives exist and that the
  experiment has not already been conducted
• Eliminate pain and suffering caused by the
  research process
• Effect a significant annual reduction in the
  number of animals used in your research
• Avoid unnecessary repeats and control groups
• Seek, develop, validate and adopt non-animal
  techniques in your research whenever possible

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Summary

• Inbred mice highly invaluable in immunology and
  measuring immune responses
• Various methods to measure immune response in
  animals
• Key considerations in selecting a method
  specificity, sensitivity, sample type/size, cost,
  etc
• Important issues regarding use of animal for
  research
• Ethical
• Scientific etc

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Animals in Research

• Physiology/behavior rabbit, dog, monkey, ape,
  bird, rat, mouse
• Immunology mouse, rat, rabbit, monkey
• Development mouse, frog, chick, fish, sea
  urchin, fruit fly, nematode (C. elegans)
• Genetics mouse, rat, zebrafish, fruit fly,
  nematode, yeast, bacteria