HC: Introduction to animal free innovations
Clinicians still require new drugs and medical devices:
- Many clinical problems -> currently no optimal solution
- Many potential drugs don’t reach phase 1 testing
- Only 1 in 10 drugs entering phase 1 is finally approved
- Need to improve preclinical testing
- Challenges scientists to develop alternative methods
Drugs development pipelines:
Discovery (animals) -> lead optimization (cell based assays) -> preclinical trials (animals)
Phase 1 (small amount of people, safety) -> phase II (more humans, efficacy) -> phase III (more
humans) -> submission -> patients market
Its not about ethics but for scientific accuracy.
Instead of all the animals used at the discovery stage we need single organ models. For the
preclinical phase we need multi organ human models. For patient market we need a combination of
both.
Aim: Pre-phase 1 invitro -> need physiologically relevant human models
- Determine whether the substance is safe for human exposure (predict an adverse event)
- Identify mode of action of new drugs
- Identify the target group of patients (patient variation, personalized medicine)
o E.g. each clinical trial focuses on a specific disease and patient inclusion criteria.
Aim: Post market approval in vitro -> need physiologically relevant human models
- Determine whether the substance which has already been proven to be safe and effective for
treating one disease can also be used for other diseases
o E.g. is a drug for treating hypertrophic scar also suitable for treating keloid fibrosis
- Identify the target group of patients (patient variation, personalized medicine)
Transition to animal free methods for testing consumer products and
drugs:
Red line -> animal models
Blue line -> in vitro models
Over time the number of animal models is decreasing, and the animal
free methods are increasing. Depending on what organ you are looking at, there are differences in
how far they are in transition (brain is not far)
,The scientific challenges requires collaboration between:
•Cell biologist
•Immunologist
•Technical engineer
•Computational modeling/ big data (correlating in vitro – animal– human data)
•Clinician
•Industry
•Patient organizations
•Working together to solve a problem
SkinLab uses organotypic skin/mucosa models for:
•Barrier function
•Chronic toxicity
•Systemic toxicity (acute)
•Immunotoxicity
•Allergy vs tolerance
•Fibrosis
•Melanoma
•Safety and efficacy of dental medical devices
- Ideal test requirements are ->
o Long term, stable culture: >28 days
o Tissue complexity: organotypic, micro environment
o Bio-distribution; multi-organon chip; vasculature
HC INTRODUCTION TO THE INNATE AND ADAPTIVE IMMNUNE SYSTEM
1st line of defense: body barriers
- Physical barriers:
o Skin, hairs and nails
- Chemical barriers:
o Low pH, mucus, enzymes
- Microbiological barriers:
o Normal flora of the skin and the intestine
2nd line of defense: innate immune system
- Phagocytosis
- Complement
- Interferon
- Inflammation
- Fever
3rd line of defense: adaptive immune system
- Lymphocytes
- Antibodies
Innate Vs adaptive immune system:
, Extracellular innate response: Complement
activation
complement are plasma proteins made by
the liver and are present in the blood, lymph
and extracellular fluids. Complement coats
surface of bacteria and extracellular virus
particles for phagocytosis. Many
complement components are proteolytic
enzymes or proteases that circulate in
inactive form (zymogens). Infection ->
complement activation. Enzymes involved in
the activation of complement are serine
proteases including chymotrypsin and
trypsin. Complement component 3 (C3) is the most important. Upon infection C3 is cleaved into a
small C3a and large C3b fragment -> C3B binds to the pathogen -> phagocytosis. C3a acts as
chemoattractant to recruit effector cells including phagocytes from blood to site of infection. 3
pathways of complement activation all leading to c3 activation, deposition of c3b on pathogen
surface and recruitment of similar effector mechanisms:
1) Alternative pathway -> works at start of infection
2) Lectin pathway -> induced by infection but requires time
3) Classical pathway -> part of both innate and adaptive and requires binding of either antibody
or innate immune system protein -> c reactive protein to pathogen surface
1) First step is binding of amino or hydroxygroup to C3 -> iC3 (C3(H2O)) -> environment near
surface of bacteria increases rate at which C3 is hydrolyzed to iC3 -> binds factor B -> making
factor B susceptible to be cleaved by protease factor D -> small Ba is released and big Bb
binds to iC3 -> iC3bBb = protease -> cleaves C3 into C3a and C3b.
2) PAMP are recognized by lectins -> C4/2 are made -> used in the conversion of C3 and C5
3) Antigen: antibody immune complexes are the initiation -> amplification of C4/2 -> used in
conversion of C3/5
C3 and C5 are side products that are highly inflammatory
Function of complement:
1. Formation of the Membrane Attack Complex -> C6, C5B, C7, C8, C9 ->
causing bacterial lysis.
2. Initiation of the inflammatory response -> especially C3a and C5a
3. Opsonization and phagocytosis
Cells of the innate immune system:
Neutrophils, basophils, eosinophils, NK cells, DC, macrophages and mast cells.
Neutrophils are the most abundant in blood of all other leukocytes.
Granulocytes:
- Neutrophils -> phagocyte and kill microorganisms -> these cells die in the process.
- Eosinophils -> kill antibody-coated parasites through release of granule contents
- Basophils -> control immune response to parasites
, Killing of bacteria
by neutrophils:
Neutrophils deliver multiple anti-microbial molecules:
Azurophilic granules, specific and tertiary granules, nets that trap the bacteria and neutrophil
elastase
Neutrophil life cycle:
1. Phagocytosis, killing and degradation of microorganisms in phagolysosomes
2. Exocytosis of granule proteins
3. Proinflammatory response
4. Apoptosis and phagocytosis by macrophages
NK cells:
Kill cells infected with certain viruses. NK cells have both inhibitory and activating receptors. The NK
cell has either lectin like receptors or immunoglobulin-like receptors. NK cells recognize transformed
virus or tumor cells by recognizing MIC & ULBP (human) (RAE-1 in mouse) on these cells cytokine
production and lysis.
‘’Missing self’’ hypothesis:
NK cells detect MHC I normal expression on normal cells -> inhibitory receptor of NK cell binds to it ->
no lysis. When cells lack MHC I -> “missing self” hypothesis -> lysis.
Dendritic cells and macrophages:
Are the bridge between innate and adaptive.
Macrophages phagocytose and kill microorganisms,
activate T cells and initiate immune response. DC activate
T cells and initiate adaptive immune response. Both are
APC and sense the environment for pathogens.
Macrophages are highly plastic cells -> cause tissue
regeneration, pro-inflammatory response and anti-
inflammatory response. Macrophages and DC have TLR
(PAMP or pathogen) and endocytic PRR (pathogen).
DC are the only cells that can activate naïve T cells and
have MHC and CD80 to activate T cells. Immature DC
don’t provide signal to prime T cell response. They are matured by PAMPS, DAMPS and cytokines.
Mature DC provide signal 1 and signal 2 to prime T-cell response.
Clinicians still require new drugs and medical devices:
- Many clinical problems -> currently no optimal solution
- Many potential drugs don’t reach phase 1 testing
- Only 1 in 10 drugs entering phase 1 is finally approved
- Need to improve preclinical testing
- Challenges scientists to develop alternative methods
Drugs development pipelines:
Discovery (animals) -> lead optimization (cell based assays) -> preclinical trials (animals)
Phase 1 (small amount of people, safety) -> phase II (more humans, efficacy) -> phase III (more
humans) -> submission -> patients market
Its not about ethics but for scientific accuracy.
Instead of all the animals used at the discovery stage we need single organ models. For the
preclinical phase we need multi organ human models. For patient market we need a combination of
both.
Aim: Pre-phase 1 invitro -> need physiologically relevant human models
- Determine whether the substance is safe for human exposure (predict an adverse event)
- Identify mode of action of new drugs
- Identify the target group of patients (patient variation, personalized medicine)
o E.g. each clinical trial focuses on a specific disease and patient inclusion criteria.
Aim: Post market approval in vitro -> need physiologically relevant human models
- Determine whether the substance which has already been proven to be safe and effective for
treating one disease can also be used for other diseases
o E.g. is a drug for treating hypertrophic scar also suitable for treating keloid fibrosis
- Identify the target group of patients (patient variation, personalized medicine)
Transition to animal free methods for testing consumer products and
drugs:
Red line -> animal models
Blue line -> in vitro models
Over time the number of animal models is decreasing, and the animal
free methods are increasing. Depending on what organ you are looking at, there are differences in
how far they are in transition (brain is not far)
,The scientific challenges requires collaboration between:
•Cell biologist
•Immunologist
•Technical engineer
•Computational modeling/ big data (correlating in vitro – animal– human data)
•Clinician
•Industry
•Patient organizations
•Working together to solve a problem
SkinLab uses organotypic skin/mucosa models for:
•Barrier function
•Chronic toxicity
•Systemic toxicity (acute)
•Immunotoxicity
•Allergy vs tolerance
•Fibrosis
•Melanoma
•Safety and efficacy of dental medical devices
- Ideal test requirements are ->
o Long term, stable culture: >28 days
o Tissue complexity: organotypic, micro environment
o Bio-distribution; multi-organon chip; vasculature
HC INTRODUCTION TO THE INNATE AND ADAPTIVE IMMNUNE SYSTEM
1st line of defense: body barriers
- Physical barriers:
o Skin, hairs and nails
- Chemical barriers:
o Low pH, mucus, enzymes
- Microbiological barriers:
o Normal flora of the skin and the intestine
2nd line of defense: innate immune system
- Phagocytosis
- Complement
- Interferon
- Inflammation
- Fever
3rd line of defense: adaptive immune system
- Lymphocytes
- Antibodies
Innate Vs adaptive immune system:
, Extracellular innate response: Complement
activation
complement are plasma proteins made by
the liver and are present in the blood, lymph
and extracellular fluids. Complement coats
surface of bacteria and extracellular virus
particles for phagocytosis. Many
complement components are proteolytic
enzymes or proteases that circulate in
inactive form (zymogens). Infection ->
complement activation. Enzymes involved in
the activation of complement are serine
proteases including chymotrypsin and
trypsin. Complement component 3 (C3) is the most important. Upon infection C3 is cleaved into a
small C3a and large C3b fragment -> C3B binds to the pathogen -> phagocytosis. C3a acts as
chemoattractant to recruit effector cells including phagocytes from blood to site of infection. 3
pathways of complement activation all leading to c3 activation, deposition of c3b on pathogen
surface and recruitment of similar effector mechanisms:
1) Alternative pathway -> works at start of infection
2) Lectin pathway -> induced by infection but requires time
3) Classical pathway -> part of both innate and adaptive and requires binding of either antibody
or innate immune system protein -> c reactive protein to pathogen surface
1) First step is binding of amino or hydroxygroup to C3 -> iC3 (C3(H2O)) -> environment near
surface of bacteria increases rate at which C3 is hydrolyzed to iC3 -> binds factor B -> making
factor B susceptible to be cleaved by protease factor D -> small Ba is released and big Bb
binds to iC3 -> iC3bBb = protease -> cleaves C3 into C3a and C3b.
2) PAMP are recognized by lectins -> C4/2 are made -> used in the conversion of C3 and C5
3) Antigen: antibody immune complexes are the initiation -> amplification of C4/2 -> used in
conversion of C3/5
C3 and C5 are side products that are highly inflammatory
Function of complement:
1. Formation of the Membrane Attack Complex -> C6, C5B, C7, C8, C9 ->
causing bacterial lysis.
2. Initiation of the inflammatory response -> especially C3a and C5a
3. Opsonization and phagocytosis
Cells of the innate immune system:
Neutrophils, basophils, eosinophils, NK cells, DC, macrophages and mast cells.
Neutrophils are the most abundant in blood of all other leukocytes.
Granulocytes:
- Neutrophils -> phagocyte and kill microorganisms -> these cells die in the process.
- Eosinophils -> kill antibody-coated parasites through release of granule contents
- Basophils -> control immune response to parasites
, Killing of bacteria
by neutrophils:
Neutrophils deliver multiple anti-microbial molecules:
Azurophilic granules, specific and tertiary granules, nets that trap the bacteria and neutrophil
elastase
Neutrophil life cycle:
1. Phagocytosis, killing and degradation of microorganisms in phagolysosomes
2. Exocytosis of granule proteins
3. Proinflammatory response
4. Apoptosis and phagocytosis by macrophages
NK cells:
Kill cells infected with certain viruses. NK cells have both inhibitory and activating receptors. The NK
cell has either lectin like receptors or immunoglobulin-like receptors. NK cells recognize transformed
virus or tumor cells by recognizing MIC & ULBP (human) (RAE-1 in mouse) on these cells cytokine
production and lysis.
‘’Missing self’’ hypothesis:
NK cells detect MHC I normal expression on normal cells -> inhibitory receptor of NK cell binds to it ->
no lysis. When cells lack MHC I -> “missing self” hypothesis -> lysis.
Dendritic cells and macrophages:
Are the bridge between innate and adaptive.
Macrophages phagocytose and kill microorganisms,
activate T cells and initiate immune response. DC activate
T cells and initiate adaptive immune response. Both are
APC and sense the environment for pathogens.
Macrophages are highly plastic cells -> cause tissue
regeneration, pro-inflammatory response and anti-
inflammatory response. Macrophages and DC have TLR
(PAMP or pathogen) and endocytic PRR (pathogen).
DC are the only cells that can activate naïve T cells and
have MHC and CD80 to activate T cells. Immature DC
don’t provide signal to prime T cell response. They are matured by PAMPS, DAMPS and cytokines.
Mature DC provide signal 1 and signal 2 to prime T-cell response.
