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

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

2. Objectives • Review of animal (inbred mice) use and contributions in immunology • Methods for measuring immune response in animals • Key considerations in measuring immune response in animals • Ethical issues relating to use of animals in research

3. Animal models of diseases: what and why • 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

4. 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 must 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

5. Types of animal models • Homologous model: has same causes, symptoms and treatment options as would humans who have the same disease • Isomorphic model: shares the same symptoms and treatments (but not same cause) • 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

6. Most commonly used animals • Mice • Fish • Rats • Rabbit • Guinea pigs • Dogs/pigs/chicken • Monkeys/non-human primates

7. 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

8. Mice: most commonly used animal

9. Advantages of mouse models • Small and cheap • Reagents are available • Inbred lines are available • Human disease models are easily created • Large, controlled crosses can be made (short generation time) • Experimental manipulations • Transgenics, knock-outs and knock-ins

10. 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

11. 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 physiology/immunology?

12. Inbred but different? • Genetic contamination: introduction of undefined genetic material • Accidental (carefree, careless or tired animal handlers) • 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 changes that act 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

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

14. Knockout/Knock-in 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

15. Mouse model 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… 15

16. Mouse model of cutaneous leishmaniasis

17. Mouse model of cutaneous leishmaniasis Simple cutaneous leishmaniasis • Caused by Leishmania major • Normally self-healing • DTH, T cell proliferation • Low antibody responses • Healing results in solid immunity

18. Diffuse cutaneous leishmaniasis • No DTH responses • Antibody present • Chronic, dissemination to many body parts • High parasite burden • Refractory to drug treatment

19. 5 BALB/c 4 Parasite load (log) 3 Lesion size (mm) 2 C3H, B6 1 B/c B6 2 4 2 4 6 8 10 12 Time (wks) L. major infection in mice mimics human cutaneous disease

20. 5 CD4+Th2 BALB/c 4 Lesion Size (mm) 3 2 CD4+ Th1 1 B6 Weeks CD4+T helper cell cytokines regulate disease outcome in mice Arbitrary Units

21. Mouse model of airway inflammation • Mice do not spontaneously develop asthma or asthma-like symptoms • 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 • Environment • Microbiota

22. Mouse model of airway inflammation • The BALB/c strain is commonly used • Develop robust type 2 (Th2) 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

23. Acute airway inflammation • Sensitization Phase: • Multiple systemic administrations of the allergen (OVA) + an adjuvant (Alum) • Aluminum hydroxide (AlOH3)  Th2 response • Elicitation phase: • Short-term exposure to high dose of allergen (intranasal/aerosol)

24. Features of acute mouse airway inflammation • 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

25. Chronic airway inflammation • 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

26. Similarities of chronic mouse airway inflammation 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

27. Some Common Mouse Studies • Adoptive transfer • Tissue vs whole body immuno-imaging • Transplantation • Tumor immunology • Cell culture systems • Protein biochemistry • Molecular Biology Technology

28. Some in vitro studies

29. 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

30. ELISA

31. 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.

33. ELISA vs. ELISPOT

34. Intracellular detection of cytokines: Flow cytometry • A powerful technique to detects antigen-specific cytokine secreting cells • Permits identification of lymphocyte subset

35. Flow cytometry contd: • Advantages • Simultaneous detection of 2 or more cytokines in a single cell • Detection 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

36. 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

37. Some in vivo animal assays

38. 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

39. 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

40. 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

41. 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

42. Determining sample size • Step 1: Define experiment’s primary objective/goal • Step 2: Define study design and margin of error • Step 3: Define clinically significant difference you wish to detect, i.e. confidence level • Step 4. Define degree of certainty of finding this difference, i.e. degree of variability

43. 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 • etc

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

45. 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 animals are not like us.’ • “Animal experimentation rests on a logical contradiction.” —Prof. Charles R. Magel

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

48. 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?

49. 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 lessdistressthan the experiment in question.