BIOC 372x Study Guide Contents

Lecture L01- Cellular Immunity

Lecture L02- Inflammation and Extravasation

Lecture L03- Tolerance

Lecture L04- Autoimmunity

Lecture L05- Hypersensitivity

Lecture L06- Transplantation Immunity

Course One Study Guide: Innate Immunity and B-cell Function

Lecture L01- Introducing the Metaphors

Lecture L02- Surveying the Cells & Organs of the Immune System

Lecture L03-Innate Immunity

Lecture L04- Antigens & Antibodies

Lecture L05-Organization & Expression of Immunoglobulin Genes

Lecture L06- Development of B Cells

Course Two Study Guide: T-cell Function and Signaling

Lecture L01- Complement

Lecture L02- The Major Histocompatibility Complex

Lecture L03- Antigen Processing and Presentation

Lecture L04- T Cell Receptors

Lecture L05- T Cell Development

Lecture L06-Cytokines and Signaling

All study guides conclude with:

Glossary

Image Attribution
 Reference figure: Labeled IgG Antibody Model

This figure summarizes information on antibody construction covered in Part One. We hope this
provides you a handy reference to correlate features of antibody structure, function and synthesis
with those of T-cell Receptors

Left side labels: Antibody Structure Right side labels: Source of information coding
for structures

Internal color-coded lines indicate peptide Indicates what gene regions supplied the
backbone. information for the amino acid sequence as well as
Sequins (silver dots) indicate disulfide the locations of gene rearrangement joins, splicing
bonds. joints and leader peptide removal
 Lecture 3-1
Cellular Immunity

The future is already here. It is just not

evenly distributed. - William Gibson
 Figure 1.1, Waddington developmental landscape

Video clip 1-1

I. Orchestrating Cellular Attacks.

Hematopoietic Stem cells can develop into a myriad of cell types.

The cells responsible for orchestrating attacks on pathogen and rogue self cells at cells that
have randomly arranged the classic αβ T cell receptor, producing vast numbers of different
recognition specificities. These are cells that decided to be lymphoid in the bone marrow
relocated to the thymus, rearrange TCR genes and ultimately selected CD4 or CD8 as a co-
receptor after positive selection.

Figure 1.2, (a) TH (b) Tc Figure 1.3, Treg

A. TH - αβ T cell receptor, CD4 co-receptor, identifies antigen on MHC II.

1. TH1 - against intracellular pathogens – all-out attack

2. TH2 - against chronic threats – containment
3. TH17 - against extracellular pathogens – serious attack, but less likely to kill your
own cells than TH1
4. T follicular – T cell coordinates B cell maturation.
 B. Tc - αβ T cell receptor, CD8 co-receptor, identifies antigen on MHC I.

1. TC – requires TH signals for up-regulation, which leads to activation as CTL

2. CTL - attack and kill cells displaying specific antigens by disrupting membrane and
lysing cell.

C. Treg- αβ T cell receptor, CD4 co-receptor, identifies antigen on MHC II.

1. Prevents immune response
2. First T cell activated
3. Has CD-25 (α subunit) and thus the complete IL-2 receptor

Figure 1.4, CTL Figure 1.5, skin Figure 1.6, gut Figure 1.7, lungs

Video clip 1-2

II. Quasi Innate Cells and Border Defense (cooperate with TH17 cells)

We’re most at risk for pathogens at our borders and that’s where we concentrate our
strongest defenses. We have skin with a stratified corneum and glands that secrete
defensive compounds. Our gut has its own locales of immune differentiation, and in
addition, hydrochloric acid and digestive enzymes and mucus. Our lungs mechanically
remove dirt with cilia and secrete enzymes (lysozyme) and mucus to help remove
pathogens. We also have sets of cells that specifically work as a first alert when our borders
are penetrated.

A. γδ T cells patrol epithelia and mucosa, cooperate with Th17 cells and respond with
innate-like rapidity to threats. They attack and phagocytize pathogens. They can kill
rogue-self using same mechanisms as NK and TC cells, and they can signal.

1.  receptor genes have far fewer gene region options than do the genes for αβ
receptors, meaning they will produce limited repertoire of receptors.
2. They will thus have receptors that target a defined range of antigen. For example,
most human  T cells recognize lipid antigens characteristic of tuberculosis bacteria
and unicellular eukaryotic parasites (malaria and leishmaniasis).

3. γδ T cells- recognize lipid antigens are presented on CD1, sometimes using CD4 and
the same suite of signaling co-receptors used by αβ receptors.

4. Recognize stress signal using a NKR – a receptor similar to the one found in NK
cells.

5. Activated  T cells secrete cytokines to up-regulate responses, and, similar to TH

cells, use two different strategies:

a. IFNγ
b. IL17

6. Help with wound healing.

7. They can react with antigen directly, phagocytize and present it.

8.  T cells can down-regulate the immune response, preventing autoimmunity (Treg

function)

9. They can bind to non-classical MHC (T22), which has same over all conformation as
MHCI and CD1, but has no peptide antigen. It seems to stimulate the cells all by
itself.

10.  T cells can induce apoptosis (a Tc or NK activity)

Figure 1.8, γδ receptor and infected cell

B. NKT cells: also participate in quasi-innate recognition.

1. Similar to γδ T cells in that they are less varied and respond to primarily to lipids.

2. The T cell version is analogous to T-independent B cells:

a. Type 1 iNKT cells produce a semi-invariant  TCR. They always use the same
 option, and use one of a limited number of  options. There will still be some
variation at the regions where you put them together.

b. Type 2 NKT less specialized. Other NKT cells (type 2) recognize a wider array
of lipids and have more variation in the receptor.

c. Do not form memory cells

Figure 1.9, NKT cell interacting with infected cell

3. Like TH cells develop in the thymus, are CD4+ (or sometimes DN, but not DP or
CD8+) and bind to cells presenting lipids on CD1d. This is a typical TH function.

4. NKT cells use CD 16, the Fc receptor, a typical NK function

5. NKT cells require TLR recognition of bacterial responds to activate.

6. They attack bacterial cells with inflammatory cytokine signaling, including IL-17,
IFNγ

7. NKT cells also have NK-like stress/damage receptors.

8. They will attacks tumor with specific lipids, using FAS ligand and perforins (a typical
NK and TC function.)
Video clip 1-3

III. Out of the Bone Marrow: Innate to adaptive spectrum

While the primary agents of this form of cell-mediated (TH1) immunity are the TC and NK cells,
other cells make important contributions to the process.
A. Lymphoid – do not rearrange TCR genes

1. B cells – rearrange immunoglobulin genes, phagocytize, but do not seem to be a

primary killer of pathogens, and do not attack rogue self. The antibodies that they
produce are often important in directing attack by other cells.

2. NK (natural killer) - do not rearrange any genes. They attack and kill cells with
suspicious surface displays by disrupting their membranes and lysing the cells.

Figure 1.10, Lymphoid cell cartoons (a) B cell (b) NK cell

B. Myeloid Cells

Figure 1-11, Myeloid cell cartoons (a) Macrophage (b) Neutrophil (c) Eosinophil

1. macrophages – phagocytize, present antigen

2. neutrophils – phagocytize, do not present antigen

3. eosinophils – coordinate attack on eukaryotic parasites, do not present antigen,

weakly phagocytic
C. Summary

1. Innate to adaptive spectrum

2. Developmental Flow Chart

Video clip 1-4

IV. Attack of the TC cell

A. Activation

1. The TC cell binds a foreign antigen on MHC I (TC cells that bind self-antigen have
been killed off during negative selection.)

2. CD45R on TC and TH attaches to CD22 (sometimes called CD45L).

3. TH cell activates the cell, turning it into a CTL.

4. Cross presentation speeds this process by making it easier for the Tc cells to
encounter the foreign antigen.

5. Activation leads to
a. Cell division
b. Up-regulation of cell adhesion molecules
c. Synthesis of the complete IL-2 receptor

Figure 1.12, Lymphocyte Figure 1.13, Cytotoxic T cell

Figure 1.14, Display by Tc cells

B. CTLs on the Attack

http://www.youtube.com/watch?v=WPaRKm2-YNY

http://www.youtube.com/watch?v=FWWMOb7z5GI
 (a) Normal, uninfected cell (b) infected or malignant cell

Figure 1.15, Interaction between Tc cells and self-cells

1. CTL cell attached to target will up-regulate cell adhesion molecules, forming
conjugate

2. CTL attacks target membrane

a. cytoskeleton rearranges, directing Golgi and storage granules (perforins and

granzymes) to target cell

b. granule contents released by exocytosis (Ca2+ dependent)

c. Perforin monomers undergo conformational change and insert in the target

membrane (sequence homology with C9 of the complement system).

d. Granzymes (serine proteases) enter through pores and activate apoptosis.

Figure 1.16, Apoptosis Figure 1.17, Infected cell downregulating MHC

3. Apoptosis can also be induced by Fas ligand binding to target Fas receptor on the
target cells

a. Ipr mice are mutant for Fas (receptor) production.

 b. They laugh at Fas ligand.

c. Cells from mice that are both perforin knock-out AND Fas knock-out cannot be
killed by cytotoxic T cells.

d. Either pathway initiates apoptosis in target cells.

e. Ultimately, each sets off a pathway that activates a cascade of proteolytic

enzymes (caspases).

Table 1.1, How Mutations Affect Ability to Induce Apoptosis

4. CTL-dissociation
a. The integrin holding the cells together reverts to its less avid form after 5 to 10
minutes.

b. The CTL undocks and begins looking for another cell to hit.

c. The target cell has already received its death signal.

5. Target cell destruction

a. The target cell undergoes apoptosis.

b. The pieces are scavenged by the macrophages or neutrophils.

Video clip 1.5

V. Natural Killer Cells – yet another link between innate and immune systems
 Figure 1.18, NK cell cartoon Figure 1.19, NK cell micrograph

A. Characteristics

1. Large cells with lots of granular inclusions.

2. Lymphoid, sharing a common progenitor and surface markers with T cells, including
the use of signaling peptides similar to those in the complete CD3 complex.

3. Develop in the bone marrow and do not rearrange genes.

4. Also have TLRs and inflammatory cytokine receptors typical of those of

macrophages and neutrophils.

5. Do express the receptor of FC of IgG (CD 16) and the IL-2 receptor. This allows
them to recognize and attack antibody-coated cells during ADCC.

6. Have MHC I receptor, but it is inhibitory, and not used to recognize antigen, but
rather viral down-regulation.

7. Pre-programmed to attack cells with surface characteristics that are likely indicators
of trouble.

B. Mechanisms of Killing
http://www.youtube.com/watch?v=84MlWh1XN0Q
http://www.youtube.com/watch?v=NdxAIyn73sU
In this one, the NK cell is directed by an antibody on the surface of the cancer cell.

1. Granules containing perforins and granzymes directionally released at target cell.

2. Constitutively cytotoxic; do not require activation.

3. Do not require antigen or MHC recognition.

4. Adhesion to target triggers release.

 C. Control of Killing

Because these cells run around in the ready, there are opposing controls that act to either
unleash the NKs or rein them in.

1. Class I MHC receptor coupled to AR (inhibitory) receptor. Enough MHC and the AR
will inhibit killing no matter what the other signals.

2. However virus that down-regulate MHC I production may fool TC cells, but they will
activate NK cells.

3. Stress receptor. There are a variety of stress indications displayed by sick cells, and a
variety of receptors used by NK cells to sense them. The interaction promotes attack.

Figure 1.20, NK cell interacting with healthy and viral infected cells. In the last two infections the cells
have down regulated MHC I and in the last produced an MHC I analog

4. Cytomegalovirus not only lowers the cell’s MHC production, it attempts to fool the
NK cells by producing a fake MHC. The viral genome codes for a protein
resembling MHC I. This does not activate TC cells as it does not display foreign
antigen. However, it is recognized as MHC I by NK cells of susceptible mice. The
fake MHC binds to the same NK inhibitory receptor that real MHC binds to.

5. Some mice have evolved NK surface receptors that can distinguish fake viral MHC I
from the real thing. These mice have an NK receptor for the viral protein that
activates attack, instead of suppressing it. And these mice are quite resistant to CMV.
 Figure 1.21, Mouse NK cell with discriminating MHC I receptor interacting with viral-infected cell

Video Clip 1-6

VI. Coordinated Killing - Antibody Dependent Cell Mediated Cytotoxicity (ADCC)

A. How it Works: Targets cells displaying abnormal antigens:

1. The cells display abnormal antigen.

2. B-cells produce antibodies to this antigen
3. The antibodies attach so that their FC portion stick out from the cell.

Figure 1.22, Antibodies (a) IgG (b) IgE

Figure 1.23, NK ADCC Figure 1.24, Phagocytosis

 Table 1-2 ADCC Variations

B. Cells with Antibody (FC) Receptors

1. NK – initiated apoptosis

2. Macrophages – initiates phagocytosis

3. Neutrophils – initiates phagocytosis

4. Eosinophils – triggers attack that releases toxins and attempt to kill a much larger
target by ganging up on it.

NK cells: http://www.youtube.com/watch?v=HNP1EAYLhOs&feature=fvwrel

Leukocytes (eosinophils?) attacking worm: http://imgur.com/gallery/YQftVYv

http://directorsblog.nih.gov/2015/01/29/cool-video-cytotoxic-t-cells-on-patrol/

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4306313/
 Lecture 3-2
Inflammation

“The fact that there are unsolved problems within the

framework of an existing theory does not of itself imply that
the theory must be thrown away, or replaced by another;
unsolved problems are the essence of science, the means by
which theories are refined.” John Maddox in Nature, 1986,
vol. 320. April 17, 1986.
Video clip 3-1
I. What, Another Metaphor?!?

A. Full Red Alert

1. When the body perceives a threat, it directs defenses to meet that threat. This means
that resources used for growth, physical activity and even higher mental function may
be diverted, depending of the severity of the threat. This can save your life.

2. However, if you are on extended alert (or full lockdown) for a long period of time,
things don’t get done that need to get done. This is comparable to the dangers of
chronic inflammation.

B. Roman Insights. The military surgeons of the Roman were the best in the world of their
time. Soldiers retired with a full pension at age 40 and the majority lived to collect it,
thanks in no small part to the care they received if they were injured. The following are
specifically attributed to Celsus and Galen:

1. Hallmarks of the inflammatory response:

a. rubor (redness)
b. tumor (swelling)
c. calor (heat at tissue site)
d. dolor (pain)
e. functio laesa - loss of function

2. Cause by vascular changes

a. endothelium leaks
b. edema
c. positive feedback loop of cell signaling
d. further changes in cell adhesion molecules on both cells and endothelium.

Figure 2.1, Roman military column Figure 2.2, Bleeding

 3. Medical Background

a. Long-time recognition that fever accompanies infection. Lower fever, lower

infection?

b. Bleeding lowers fever.

c. Giving blood is associated with lower heart disease (as are statins, aspirin and fish
oils, all of which are anti-inflammatory and all of which tend to increase
bleeding).

Video clip 3-2

II. Signals – This sections reprises and extends material we introduced in Part 1 Lecture 3

A. Pattern Recognition

1. PAMPS (pattern-associated molecular proteins) - generally used to describe a variety

of molecules used to identify pathogens. The first group identifies was the toll-like
receptors, but many more types.

2. DAMPS (death or damage-associated molecular proteins) - molecules that are normal

cell components but should be inside – cytoplasmic or nuclear proteins of membrane
proteins belonging in the inner leaflet.

B. Response:

1. Activate complement, which in turn releases inflammatory signals (anaphylatoxins)

2. Attracts neutrophils, macrophages and mast cells.

C. Inflammatory Chemokines

1. Smallish (90 to 130 amino acids), with conserved cysteines

2. Receptors are seven-span membrane proteins. Cross reception common: a given

chemokine may bind more than one receptor and a specific receptor may bind more
than one chemokine

3. Binding activates G-proteins

a. large (heterotrimeric with αβγ subunits) – kicks off cytoskeletal changes –quick
change in adhesiveness

b. Switches from active to inactive as it binds GTP and then hydrolyzes it to GDP
 Figure 2.3, chemokine Figure 2.4, Seven-Span Receptor

4. G-proteins kick of several internal cascades:

a. hydrolyzes membrane phospholipids to IP3 and DAG
b. lets Ca2+ loose in the cytoplasm, polymerizing actin and promoting movement
c. kicks off Ras cascade that leads to activation of transcription factors.

Figure 2.5, GDP Figure 2.6, 7-span receptor and G protein sequence

D. The High-endothelial Venules

1. found at the end of capillaries in lymph nodes, Peyr's patches, and tonsils (not spleen)

2. The endothelial lining here is composed of cells that do not look like flattened paving
stones, but are thicker.

3. Most lymph cells that extravasate attach to these specific cells.

4. The HEVs specifically attract lymphocytes and do NOT attract neutrophils.

5. The HEV develops in response to foreign antigen exposure: you don't see this in mice
raised in a germ-free environment and you don't see this in tissues that have the
circulation to them blocked so that antigen does not enter.

6. The unique morphology is associated with the induced expression of specific

selectins, mucin-like, and Ig CAMs, and they specifically allow different classes of
lymphocytes to home to different organs and regions of the body.
Video clip 3-3
III. Cell Adhesion

A. Selectins and Mucins

1. Selectin Family, representation and ribbon diagram of lectin domain.

a. membrane glycoprotein with lectin at amino terminus that binds carbohydrates.

Compare with complement lectin pathway.

b. specific for sialic acid (mucins have a lot of these)

c. comes is L (Leukocyte), E and P (endothelium) versions

d. initiate initial sticking of leukocytes to the endothelial wall

2. Mucin-Like Family

a. protein part rich in serine and threonine (OH-containing R groups)

b. LOTS of carbohydrate linked to these OHs.

c. Carbohydrates have lots of sialic acid, interact with selectins

d. The mucin-like versions of the endothelium interact with the selectins on the
leukocytes and vice-versa.

A. B. C. D.

Figure 2.7 CAMs: A. Selectin, B. Mucin-like, C. Ig or I-CAM, D. integrin.

B. Immunoglobulins and Integrins

1. I-CAM - Immunoglobulin Super-Family

a. endothelium has several versions

b. mucosa has another, which also has a mucin-like domain

c. bind to integrin on leukocytes

d. inflammation increases their expression

2. Integrin Family – varied binding partners

a. Heterodimer ( and  chains)

b. Expressed in leukocytes

c. Different integrins bind to different immunoglobulin CAMs (cell specificity)

d. Can also bind to fibronectin

e. Cytoplasmic portion can interact with cytoskeleton and signaling proteins such as
the tyrosine kinases, Fyn and Lck.

Figure 2.8, Links to cytoskeleton Figure 2.9 Neutrophil and endothelium

Video clip 2-4

IV. Extravasation

Figure 2.10, sequence.

A. Sequence

Inner Life of the cell – Narrated: http://multimedia.mcb.harvard.edu/innerHi.swf

Note: If you would like captions, click the “cc” icon on the lower right.

1. rolling - attachment by low affinity P selectins (yellow) on the endothelium to mucin-

like molecules on the neutrophil (purple). Because the connections are loose, they
tend to break and the neutrophils kind of roll along, attaching to one endothelial cell
after the next as it is swept along by the flow of blood.

2. activation – the “stick and release” from the rolling response along the endothelium
triggers chemokine (IL-8) release by the endothelium. The neutrophils activate G-
protein cytoplasmic pathways via the 7-span receptor.

a. Video shows lipid raft. We looked at these in connection with T cell activation,
but they also form membrane compartments to organize other interactions as well.
Will return to this.

b. The mechanical pulling and tugging causes the endothelium to release a

chemokines.

3. arrest and adhesion - The G-proteins activate integrins, changing their conformation,
and increasing their affinity for Ig-related CAMs. This nails down the neutrophils.
Neutrophils cannot bind to non-inflamed endothelium.

a. The chemokine binds to a 7-span chemokine receptor.

b. This receptor activates a G protein and sets off an internal signaling cascade.
c. The integrins change conformation, enter the lipid rafts, deploy and stick Ig
tightly to the Ig CAMs of the endothelium
d. Some of the first proteins affected are those that associated with the inner side
of the plasma membrane and connect to the cytoskeleton
 4. transendothelial migration - the arrested neutrophil then finds the gap between two
adjacent endothelial cells and squeezes through it.

a. The cytoskeletal changes lead to the neutrophils changing shape and moving
by amoeboid motion.

b. The neutrophil forms a leading wedge and ootches through a gap between the
endothelial cells (recall that inflammation makes the endothelium somewhat
leaky).

Figure 2.11, HEV Figure 2.12, HEV directing lymphocyte

B. Lymphocyte Extravasation, Trafficking, and Homing

While the processes that allow the lymphocyte to leave the blood vessel (rolling,
activation, arrest, and migration) are similar, the mechanisms that control exactly where
they lymphocyte will undergo extravasation are more complex and involve specific
homing signals.

1. Naïve lymphocytes

Lymphocytes migrate in and out of secondary lymph organs where they contact
presenting sentinel dendritic cells. Recall that the chance of any one lymphocyte
recognizing any one antigen are miniscule (1 in 105), so the cells run repeated loops
through secondary organs and in and out of circulation, essentially kissing frog after
frog and looking for the rare prince.

a. T cells, for example are attracted by inflammatory signals and cruise into sites of
active infection, where they may be activated by antigen presented by innate cells
fighting the infection.

b. T cells may also be activated by dendritic cells in the lymph nodes in spleen. In
that case the dendritic cells also communicate the site of the infection by
 activating specific T-cell surface receptors for cytokines and CAMs characteristic
of particular tissues. For example retinal/retinoic acid is produced by the gut and
only gut-homing T cells have receptors for it. For skin, it’s vitamin D.

2. Effector and Memory Cells

a. Effector cells tend to head to the site of infection.

b. Memory cells tend to head to the tissues that initially had the infection.

c. Cells are directed by a combination of cytokines and cell surface receptors

(above) but can also be retrained, which is necessary if an infection spreads.

d. T cells, in particular, are programmed to home to a specific site because they

express a specific combination of cytokine and CAM receptors.

Video clip 2-5

V. Inflammatory Signaling Network

A. Inflammatory Cytokines – initiating inflammation

1. IL-1 - comprises a family of at least 11 related signaling molecules using Ig receptors.

a. The signals are smallish proteins with β-pleated sheets.

b. Synthesized as precursor in the cytosol, then clipped and secreted: leaderless
secreted proteins.
c. Most members activate TH17 cells, although an inhibitory version exists as well.

2. IL-18 – also a member of this general family, but with different controls and targets.

a. Synthesized in the cytosol, clipped and secreted.

b. Production of precursor under different controls.
c. Activate TH1 responses.
d. Promote IFN-γ production, which in turn promotes IL-18 secretion in a positive
feedback loop.
 Figure 2.13, IL-1 Figure 2.14, TLR4 plus LPS

B. PRRS- Release of IL-1 subtypes and IL-18 are in turn promoted by activation of pattern-
recognition receptors. Along with complement activation, the signaling that leads to IL-1
and IL-18 release is part of the very first responses to a new infection. Here are two
examples:

1. LPS (lipopolysaccharide) activation of TLRs – recall that these TLRs are

transmembrane proteins with a cytoplasmic domain that kicks off a complex pathway
to NFκB and AP-1 activation, which in turn up-regulate genes for inflammatory
cytokines: IL-1, IL-18, IL-6 and TNF α.

2. NOD receptors (members of a group of NLR proteins with annoying complex naming
conventions) are in the cytoplasm, recognize viral, bacterial and parasite signatures
using a leucine-rich repeat domain, as well as DAMPS and environmental irritants.
Also activate NFκB and AP-1 and thus inflammatory cytokines.

C. Activation (Again, this is a simplified example.)

1. Pattern Recognition Receptors, via NFκB and AP-1, upregulate IL-1 production in
the cytoplasm. IL-18 constitutive, but still requires this as activating signal.

2. Further stimulus (a second signal) by other danger triggers activated assembly of

inflammasome, a complex of proteins.

a. Pathogenic signatures – wide variety of proteins, nucleic acids and cell wall
components from bacteria, virus, fungi and protozoa.

b. Environmental irritants- alum (used as a vaccine adjuvant), inorganic particles

(asbestos, silica, metals), UV light.
 c. Internal signals- ATP and glucose, crystals of cholesterol and urate, β-amyloid,
hyaluronin.

Figure 2.15, NLR activation

3. NLR proteins (NLR-P3 as an example) receive signal at the leucine-rich and shift.
Since a huge variety of signals are effective here, the activation does not seem to
occur by a classic ligand-receptor interaction.

4. Conformational shift also recruits associated ASC with CARD domain

5. ASC recruits caspase-1 (yes, of apoptosis fame) orienting the hydrolytic domains
toward the center, axle region

Figure 2.16, Inflammasome assembling and activating

 6. Caspase 1 undergoes hydrolysis, activates and forms an active region at the hub.

7. Caspase-1 clips IL-1 precursor.

8. Part goes to nucleus of the cell (positive feedback loop)

9. Part is secreted from the cytoplasm

a. TLR LPS signal necessary for secretion, too.

b. Precursor enters secretory lysosome and get secreted.
c. Co-opted mechanisms used to turn over organelles and proteins and to secrete
proteolytic enzymes

10. May have originally evolved from leakage from dying cells, but is clearly a controlled
process today.

D. Inflammasome Structure in Context.

1. Inflammasome structure is a complex and interesting assembly of complex and

interesting proteins, not bound or associated with a membrane, that assembles and in
response to danger signals in the cell and can disassociate and recycle.

2. The apoptosome is a similar looking structure that also activates caspases

T

Figure 2.17, Apoptosome assembly

3. Similarities-

a. wheel-like structures, typically seven units

b. homologies in regions of the axis/assembly
c. activated by leucine rich ligand binding domains
d. have CARD (caspase recruitment) domains at axis
e. bind caspases via their corresponding CARD domains
f. Caspases active hydrolytic domains at axle region
g. Caspases then clip effector proteins
 4. Differences-

a. Basic structural proteins

b. Activation trigger
c. end result

Figure 2.18, Inflammasome and Apoptosome Compared

Table 1- Inflammasome and Apoptosome Compared

Video clip 2-6

VI. Inflammatory Mediators

A. Clotting

Central to dealing with barrier damage and raising the alarm at the first sign of a
breach.
 1. Clotting both up-regulates, and is up-regulated by, inflammation. This is why chronic
inflammation can lead to strokes and cardiac infarcts.

2. Initiated by platelets and RBCs contacting damages surfaces (collagen fibrils) and
breaking open.

Figure 2.19, RBC, platelet and leukocyte Figure 2.20, fibrinogen

3. This releases and activates Hageman factor, a large heterodimer plasma protein with
hydrolytic activity (serine protease.)

4. Activated Hageman factor (sometimes called Factor XII) initiates an expanding

hydrolytic cascade.

5. The clotting cascade ends with prothrombin activation to thrombin, which clips
fibrinogen into fibrin and peptides.

6. The fibrin forms the clot, trapping red blood cells.

7. As the wound heals, a system of fibrinolytic enzymes producing plasmin dismantles

the clot, using the enzyme plasmin.

8. The released peptides promote inflammation. Plasmin also produces inflammatory

peptides and activates complement

9. Hageman factor helps activate this process.

 Figure 2.21, clot Figure 2.22, bradykinin

B. Kinins – Signals Derived from Peptides

1. Bradykinin – 9 amino acid peptide specifically leads to pain, in addition to increased

endothelial permeability and smooth muscle contraction. This may not look like a
peptide, in part because it contains prolines at position 2, 3, and 7, which distort the
peptide backbone.

2. Cascade initiated by Hageman factor, again by proteolysis, eventually activating

kallikrein, which turns around and activates more Hageman in a positive feedback
loop.

3. Kallikrein clips kininogen to bradykinin.

4. ACE inhibitors block the breakdown of bradykinin, lowering blood pressure (but for
some reason not increasing pain.) This happens as a side effect of blocking a different
signaling pathway.

C. Lipid derivatives- these often depend of activations of specific enzymes.

1. Membrane phospholipids play a structural role but also serve as the raw material for
generating a lot of signaling compounds.

2. Phospholipids have two fatty acids linked to glycerol and the glycerol links up via a
phosphate to a polar group (ethanolamine or inositol, for example).

3. Depending on what you begin with and where you cut it, and how you modify the
results, you can get an array of different potent signaling molecules.
Figure 2.23, membrane phospholipid figure 2.24, phosphoinositol

4. Inositol-containing phospholipid

5. Clip to DAG and PIP3, used in previous pathways.

6. A different clip, can remove a free fatty acid.

7. Arachidonic acid- derived from one to the fatty acids.

Figure 2-25, Figure 2-26 Figure 2-27,

arachidonic acid thromboxane prostaglandin

8. Act on it with enzymes called cyclooxygenases and you kind of bend it into the
hairpin shape.

a. Produces thromboxane or a variety of prostaglandins

b. Inhibited by COX2 inhibitors block cyclo-oxygenases and block inflammation

and NSAIDs (aspirin, Advil) will also block prostaglandins

9. Act on it with lipoxygenase and get a variety of leukotrienes.

a. Released by mast cells in allergic response.

b. Antihistamines have been joined by anti-leukotrienes in over-the-counter (OTC)
medication.
 10. In a separate origin pathway, phospholipids can provide the raw material for Lyso-
PAF

a. Phospholipid in this case has ethanolamine polar group.

b. One fatty acid
c. Remains of the second, which used to be attached in the middle of the glycerol.
d. Now have PAF (Platelet Activating Factor)
e. Inhibitory drugs under investigation as for treating both allergies and
cardiovascular problem.

Video clip 2-7

VII. Clinical Considerations- All of this signaling and enzymatic activity provides a lot of
opportunity of diagnosis, monitoring and treating disease.

A. Systemic Acute-Phase Response – against acute infection

1. Compromised by malnutrition or starvation

2. IL-6, TNF, IFN γ

3. Act on the hypothalamus to induce fever and CRP release, which in turn causes the
pituitary to release of ACTH which causes the adrenals to produce steroids (cortisol
etc.).

4. The liver responds with the release of acute-phase proteins (a general term) - activates
complement.

5. increased leukocyte production and activation, especially neutrophils

B. Chronic Inflammatory Response – IFNγ and TNF, transcription factor NF-κB

1. persistent infection (gum disease), autoimmune response, cancer, chronic injury.

2. old age, obesity, diet high in trans-fats, triglycerides (diet high in sugar) wrong gut
flora, diabetes, sleep disorders.

3. contributes to disease processes, including cancer and cardiovascular diseases

4. Increased clotting

5. fibrosis - a type of scar tissue formed when chronic inflammation leads to excess
production of fibroblasts and collagen

6. granuloma (also called tubercle) - an attempt to wall off the problem with
macrophages and specialized TH cells.
Video clip 2-8

VIII. Clinical Therapies

A. Anti-inflammatories - Braking the System- Hey, that drug sound familiar!

1. Antibodies used to block leukocyte extravasation:

a. antibodies to integrins
b. antibodies to CAMS

2. Corticosteroids

a. Prednisone, cortisone, dihydrocortisone

b. High doses block adrenals and many immune functions

c. Side effects – mood swings, edema, glaucoma, increased susceptibility to

infections

3. NSAIDs

a. Aspirin (acetylsalicylic acid), Advil (ibuprofen) Aleve (naproxin) are OTC

b. prevent prostaglandin production from arachidonic acid. Celebrex specifically

blocks COX-2

c. NOT Tylenol/acetaminophen- acts on brain to raise pain threshold and lower

hypothalamic thermostat).

B. Cooling - Interferes with inflammatory process (recall that fever is often a helpful part of
the response to infection)

a. used to prevent excessive tissue damage following injury or stroke- exercise

raises levels of CRP and other inflammatory indicators

b. used during surgery

c. used to cut down on symptoms of autoimmune disease multiple sclerosis –

chilling via hands to allow athletic competition.

d. Harmful if you’re ill, unless your fever is dangerously high.

References:

Inner Life of the cell – Narrated: http://multimedia.mcb.harvard.edu/innerHi.swf

IL-18: http://journal.frontiersin.org/article/10.3389/fimmu.2013.00289/full

Leaderless secretory proteins: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3662868/

Structure of IL-18 binding complex

http://www.nature.com/ncomms/2014/141215/ncomms6340/full/ncomms6340.html IL-18

APAF- NLR similarities: http://www.ncbi.nlm.nih.gov/pubmed/18446235

WAY beyond the content of this course, but should you become fascinated with apoptosis
control: http://www.nature.com/nrm/journal/v9/n5/box/nrm2393_BX1.html
 Lecture 3-3
Tolerance

Tolerance is the virtue of those who don’t care

very much.
Video clip 3-1

I. Danger Assessment (A Review of Inflammatory Triggers)

A. DAMPS (death or damage-associated molecular proteins) and PAMPS (pattern-

associated molecular proteins)

1. Environmental irritants-asbestos (carcinogenic), alum (used as a vaccine adjuvant)

and UV light.

Figure 3.1, asbestos 3.2, Alum salt crystal

3.3, UV a. light b. damage

2. Signals of cell damage are things that should be inside a cell but are now outside, or
things present in a cell in small quantities, but now there’s a bunch of them:

Figure 3.3, chromatin: DNA and histone

a. nuclear proteins (transcription factors and histones) and DNA

b. cytoplasmic proteins (Heat Shock Proteins), which denature in the oxidizing

environment outside the cell
 Figure 3.4, beta amyloid precursor Figure 3.5, beta amyloid

c. β-amyloid- a breakdown product from an integral membrane protein

d. Various chemicals: ATP, high glucose, crystals of cholesterol and urate,

fragments from hyaluronin.

Figure 3.6, ATP Figure 3.7, gram negative bacterium

e. Pathogenic signatures: peptidoglycan, LPS, flagellin, strange nucleic acids, fungal

cell wall compounds.

B. Receptors – two major categories – both share leucine-rich hook

1. NOD receptors

Figure 3.8, NOD Figure 3.9, TLR Figure 3.10, TLR hook
 2. TLRs: Both these danger receptors have this hook-like structure that recognizes
danger via a somewhat mysterious mechanism. Antibodies recognize antigen in a
manner similar to enzyme-substrate binding, but here the binding of a hook to a
signal is much less specific.

Also note that the leucine-rich hook of the TLR extends outside the cell or into the
interior of the endomembrane complex. NOD receptor hooks sit in the cytoplasm.

C. Inflammation Up-regulators

Both these receptors activate inflammation, but via two very different mechanisms.

1. IL-1, the general inflammatory cytokine, activated by NOD.

Figure 3.11, IL-1 Figure 3.12, NOD activation

a. Components of the inflammasome bind a DAMP or PAMP.

b. The subunits undergo a conformational shift and assemble.
c. Assembly activates a caspase.

Figure 3.13, Inflammasome

d. The caspase clips and activates the IL-1.

e. The cell secretes the IL-1, triggering a variety of inflammatory responses.

 2. NFκB (nuclear factor kappa B)

NFκB is a transcription factor activated by TLRs (and by other mechanisms), which

then enters the nucleus and up-regulates gene that produce pro-inflammatory signals
and related proteins.

a. Has an N-terminal domain (rel) that can bind to DNA but also binds to IκB (the
inhibitor of NFκB).

b. Has a C terminal domain with ankyrin repeats that binds to spectrin, which forms
a reinforcing network just under the plasma membrane. Spectrin unit junctions
are anchored in tropomyosin, which also connects up to actin microfilaments.

c. Thus the ankyrin - spectrin node ties the inhibited factor to a region of the
cytoplasm close to the plasma membrane.

Figure 3.14, inhibited NFκB Figure 3.15, phosphorylated IκB

d. TLR activation sets off a phosphorylation path the causes IkB to phosphorylate,
pick up ubiquitin and get degraded in the proteasome.

Figure 3.16, ubiquinated IκB Figure 3.17, released NFκB

 e. This releases the NFκB, which migrates to the nucleus.

Figure 3.18, active NFκB Figure 3.19, active NFκB (ribbon mode)

Figure 3.20, comparison of danger signaling

3. Danger Signal comparisons:

Both examples are positive controls that permit or potentiate an immune response. They
have in common a receipt of a danger signal by a leucine-rich hook and differ completely
in the response mechanisms.

a. The first response assembles a structure that activates a cytoplasmic cytokine

precursor by hydrolysis, which then leaves the cell.

b. The second disassembles a structure by phosphorylating and removing an inhibitor,

allowing the migration of a transcription factor from the cytoplasm to the nucleus.
Video clip 3-2

II. Negative Selection and Central Tolerance: takes place in the primary lymphoid organs during
the production of B and T cells.

Figure 3.21, Immune Compartments

A. B cells – Negative selection

1. B cells arise in the bone marrow.

2. First the progenitor must make a productive arrangement of genes for both light and
heavy chains.

3. Then each developing B cell synthesizes the M class Ig receptor with its individual,
randomly generated recognition site at the end of the arms.

4. At this point, the receptor may recognize an antigen in the bone marrow and cross
link.

5. This causes the cell to die by apoptosis.

6. Thus B cells that can recognize self-antigen found in the bone marrow will die and no
cells recognizing these self antigens will survive and enter the circulation.

7. Notice however that the bone marrow does not express all self antigen. Some B cells
can leave the bone marrow capable of recognizing tissue-specific antigen only
produced outside the bone marrow.

8. However this initial cull deletes cells that recognize housekeeping proteins and any
specific proteins found in the marrow.
 Figure 3.22, B-cell Development Figure 3.23, thymus

B. T cells – Thymus
It turns out that the initial cull in the thymus is much more critical to preventing
autoimmune problems than is the cull in the bone marrow. And therefore the deletion
of self-reactive T cells has some extra features.

Figure 3.24, CD8+ (Tc) and CD4+ (TH) cells

1. Cells rearrange the β gene and then the α.

2. Receptors respond to a sequence of amino acids presented on an MHC molecule.

There is a vast possible number of  TCRs.

3. At this point T cells (currently at the cortex of the thymus) undergo two kinds of
selection:
a. Positive - cells that cannot recognize MHC will die.
b. Negative - cells that recognize self-antigen will die.

4. Cells then decide which MHC to respond to, and thus whether they should continue
to make CD4 or CD8.
 a. Cells that can bind MHCI will keep the CD8 and lose the CD4.

b. Cells that kind bind MHCII will keep the CD4 and lose the CD8.

5. Cells then move inward, contacting stromal cells in the medulla.

6. Stromal cells of the medulla produce a varied buffet of self-antigens, including many
proteins normally characteristic of differentiated tissues.

C. AIRE- They do this by expressing an unusual transcription factor, AIRE.

Figure 3.25, AIRE transcription factor Figure 3.26, AIRE metaphor

1. The Swiss army knife of transcription factors, having two plant Hodo Zn fingers, 4
nuclear receptor motifs, a SAND domain and N terminal similar to SP100.

2. What AIRE seems to be doing is turning on the ectopic production of certain organ-
specific antigens in presenting cells that would not ordinarily appear in the thymus.

3. This exposes developing thymocytes to many more of the body’s antigens than
developing B cells encounter in the bone marrow.

4. Mutations in the aire (autoimmune regulator) gene are inherited in humans as a

classical Mendelian autosomal recessive, the homozygous non-functioning condition
producing a type of multi-organ autoimmune disorder call APECED.

D. Fezf2: a second transcription factor promoting expression of tissue-specific antigen in the

thymic medulla.

1. Zn finger transcription factor – that is it has several domain where a Zn ion stabilizes
the association of an alpha helix with a bend in the backbone.
 Figure 3.27, single Zn “finger” Figure 3.28, transcription factor example

2. This finger attaches in the major groove to a specific base sequence.

3. Actually, in this case it attaches to a LOT of sequences - to the promoter regions of over
1000 different tissue-specific genes.

4. The mechanism of gene activation is completely distinct from that of AIRE. AIRE seems
to open up swathes of chromatin and make them accessible and Fezf2 acts like a more
typical enhancer – except for its promiscuity.

5. Between positive selection and this stringent, double-barreled negative selection, fully
98% of thymocytes die via apoptosis during development. Parenthetically this process
does NOT apply to Treg cells, which are also CD4+ T cells with αβ receptors.

6. The T cells that survive the dual selection leave the thymus via blood vessels exiting
from the medulla and enter the general circulation, where they will continue to get
monitored by peripheral tolerance.

Figure 3.29, background vocabulary

Video clip 3-3

III. Peripheral Tolerance: ongoing monitoring – apoptosis or anergy.

A. Please Don’t Hurt Me – tolerance triggers.

1. Antigen present in high levels – most likely one of your own.

2. Antigen persists– it could be you, or it could be something you have to learn to live
with.

3. Antigen not accompanied by inflammatory stimuli, as discussed in section I.

4. Antigen sequestered from immune surveillance.

a. Brain: courtesy of the blood brain barrier.

b. Testicles and uterus

c. The anterior capsule of the eye: (cornea, lens and space between)

Figure 3.30, Human Eye Figure 3.31, Golden Syrian Hamster

d. Cheek pouches: (if you’re a golden Syrian hamster.)

5. Exposure during infancy.

B. Infancy – The education of the immune system begins in the first month of life.

1. Vaginal birth

a. Exposure to microbes in birth canal promotes tolerance.

b. C- sections are associated with an 8-fold risk of developing serious allergies in

children with a genetic tendency to produce excess IgE.
 c. Lately some hospitals have placed vaginal swabs over the mouth and nose of an
infant delivered by C-section.

Figure 3.32, Newborn Figure 3.33, child nursing Figure 3.34, IgA

2. Breast feeding also helps. Newborns don’t make IgA and IgA from the mother:

a. prevents excessive bacterial growth in the gut

b. primes the immature system to accept certain antigens

c. calms the inflammatory response

d. is associated with lower risk of allergies (breast feeding for at least four months)

Recall that IgA crosses epithelia, is associated with little inflammation and is more
likely to be synthesized when Treg cells are active.

3. Infants also seem to spending the first few weeks taking some sort of self-census.

a. This isn’t necessarily limited to their own tissues, but includes a lot of antigen that
are an integral part of their environment, including gut and skin.

b. You can even preform a heart transplant with a mismatched blood type on a very
young infant.

Video clip 3-4

IV. Experiments and Mechanisms

A. Exploring Responses with Genetic Engineering C.C. Goodnow, 1991: Central Tolerance
of B Cells
 1. Hen egg-white lysozyme (HEL) is typically a strong immunogen in mice.

Figure 3.35, Raw egg showing white Figure 3.36, mating of mouse from strain # 2
(HEL on most cells) and strain #3 (makes
anti-HEL antibodies)

2. You can engineer mice with the HEL gene and they will then synthesize this protein,
expressing it in the plasma membrane of most cells. Call this strain #2.

3. You can also engineer mice with the rearranged gene that codes for an antibody that
binds to HEL, and such mice will produce B cells with anti-HEL IgG receptors and
then activate if they encounter HEL and secrete anti-HEL antibodies into the plasma.
Call this strain #3.

4. If you cross these strains, you get a mouse that’s a #2/#3 hybrid and should make
both HEL in the plasma membrane and secrete anti-HEL antibodies in the plasma,
which should attack and destroy pretty much any cell they encounter.

5. However, this doesn’t happen. The B cells with receptors that bind to the HEL either
undergo apoptosis (typically) or (more rarely) recycle through the rearrangement
process and come up with an edited L chain and a recognition arm that no longer
binds HEL.

6. Thus this F1 deletes HEL-Ig B cells in the bone marrow via central tolerance and
never makes any anti-HEL antibodies.

B. Exploring Responses with Genetic Engineering

1. Engineer a mouse to make HEL such that the protein is only secreted into the plasma
(strain #4).

2. Cross this with strain #3 and you get a mouse that’s a #4/#3 F1 hybrid and should
secrete HEL in the plasma ONLY.
 3. In this case the B cells that respond to HEL will not encounter it in the bone marrow,
and some will exit with anti-HEL Ig receptors.

4. These cells will then encounter HEL in the plasma and the receptors will cross link.

Figure 3.37, mating of mouse from strain # 4 (HEL in plasma) and strain #3 (makes
anti-HEL antibodies), showing Peripheral Tolerance.

5. However, they do not activate. These cells don’t respond, don’t divide and don’t
produce antibodies, a state called anergy.

6. Likewise, if you take a mouse making HEL and add B cells from another mouse, they
will encounter the HEL and crosslink. However, they will NOT begin to proliferate.
They become anergic when they can’t find a Th cell to get instructions from.

7. So one big reason that you don’t get activation in this situation is that absence of Th
cells to promote clonal expansion and antibody secretion.

8. We have already looked at Central Tolerance in T cells, and you would expect that
mice engineered to express HEL on all plasma membrane will delete HEL-reactive
cells in the thymus.

C. Peripheral Tolerance- T cells.

1. If you transfer a naïve TH cell capable of reacting to a HEL (that is, it has the
receptor, but has not yet activated) from one mouse into a strain #4 mouse secreting
HEL into the plasma only, it will become anergic.
 Figure 3.38, Peripheral Tolerance in T cells

2. Recall that there is one and only one kind of cell that can activate a naïve TH cell, and
that is a sentinel dendritic cell.

3. Moreover, for this cell to activate a TH cell, it must also receive a danger signal from
one of our pattern recognition receptors.

4. A dendritic cell will present antigen and bind to the TH cell receptor. However,
without a danger signal, it cannot supply the second necessary signal.

5. That signal is the binding of CD28 on the T cell to B7 on the APC.

6. This insufficient co-stimulation by B7 is a consequence of a consequence of a lack of

inflammatory responses, and probably other mechanisms as well.

7. And as a reminder CD28 – B7 stimulation can be blocked by B7 binding to CTLA 4.

The TH cells produce the CTLA 4 after they begin to develop.

(A) (B)

Figure 3.39, B7 co-stimulus Figure 3.40, dendritic cells, (A) stationary, (B) migrating
 8. Critical Importance of Dendritic Cells.

a. Only dendritic cells can activate naïve TH cells.

b. Dendritic cells will endocytose, digest and present peptides on MHC II.

c. However this presentation, but itself, will not activate TH cells (although it may
activate Treg cells.

d. Only if it also receives a danger signal will it produce enough B7 to activate a

naive TH cell.

Figure 3.41, Intro background, balancing responses

Video clip 3-5

V. Treg cells- critical to active peripheral tolerance.

A. Treg cells– Shimon Sakaguchi

Figure 3.42, Dr. Shimon Sakaguchi Figure 3.43, CD4 – TCR receptor
B. About the Cells

1. Like TH cells, they rearrange the αβ T cell receptor in the thymus and display the co-
receptor, CD4.

2. Like TH cells they respond to IL-2 by clonal expansion and activation.

3. The IL-2 cytokine is a small peptide with four α helices.

a. b.

Figure 3.44, IL-2 (a) cytokine and (b) its receptor.

4. The complete IL- 2 receptor is a Class I heterotrimer.

a. α (CD 25)
b. β (CD122)
c. γ (CD 132)

5. L-selectin (CD62L), which targets extravasation to the secondary lymphoid organs,

and binds efficiently to HEV.

6. High levels of CTLA4- do not seem to suppress them, but rather allows them to tie up
B7 that might stimulate some other T cell.

7. Unlike other CD4+T cells, they do NOT need to up-regulate the α (CD 25) subunit
upon activation.

8. This has two important consequences, producing a more deliberate response:

a. Treg cells get a head start over other T cells.

b. Treg cells will “sop up” most of the circulating IL-2 initially because their IL-2
receptors have high affinity.
 Figure 3.45, Thymus Figure 3.46, Lymph node

C. Promoting Treg development

1. There are two different sources of Treg cells:

a. “natural” ones produced in the thymus (nTreg) and released as Tregs.
b. “induced” in the secondary lymphoid organs (iTreg), by conversion from naïve TH
cells (or maybe TH17).

Figure 3.47, helix-loop-helix Figure 3.48, winged helix

2. The transcription factor FoxP3 is both necessary and sufficient to induce the
development to Treg. This is a winged helix transcription factor, a member of the
helix-loop-helix group, characterized by α helices that attach in the major groove and
extended unstructured domains reaching out from the axis. The overall structure is
reminiscent of a butterfly perched on the DNA, its feet recognizing specific base
sequences.

Figure 3.49, butterfly metaphor Figure 3.50, STAT activation

 3. FoxP3 expression is induced by STAT 5. Recall that STATS are transcription factors
that reside in the cytoplasm until they are phosphorylated, dimerize, activate and
migrate to the nucleus. Different STATs coordinate different developmental
pathways and STAT 5 is tasked with up-regulating FoxP3 and thus Treg function.

4. The STATS are activated by cytokine signals received by surface receptors. STAT5
(and Treg activation) requires TGFβ in the ABSENCE of inhibitory inflammatory
cytokine signals (IL-6 in particular, but IFNγ shuts down this pathway as well). IL-2
and retinoic acid (important in the gut) will synergize the response.

5. The differences between the development of iTreg and nTreg in some ways parallel
the differences between central and peripheral tolerance, only instead of the T cell
becoming anergic it becomes active in suppression.

b. nTreg cells are set aside in the thymus from those cells with a high affinity to
MHCII antigen (presumable self-antigen) – this these are activated by self antigen
in the primary organ (thymus).

c. iTreg cells also require antigen presentation, but in the secondary organs. If these
cells encounter antigen, TGFβ and no inflammatory cytokines, they will make the
decision not to become TH17 but rather to switch to Treg, the equivalent of
peripheral tolerance.

D. Treg signaling

1. more TGFβ
2. IL -10
3. remove IL-2
4. CTLA-4- ties up B7
5. L-selectin (CD62L) ties up extravasation attachment sites. Naïve T cells use these sites
to find secondary organs, and lose the L-selectin when they activate.

Figure 3.51, Treg cartoon Figure 3.52, γδ T cell cartoon

 E. Regulatory Gamma-delta T cells

1. Here’s a cartoon illustrating their properties (covered in the second course)

a. γδ receptor: bent, often binds lipids

b. variety of receptors and presenting peptides
c. patrols epithelia

2. Dysregulation of γδ T cells leads to autoimmunity. Typically, this results from the

pro-inflammatory poperies of γδs (shown here secreting proinflammatory cytokines)
and also their ability to down-regulate Tregs.

3. However, γδ plus retinoic acid actively promote tolerance and protective peptide
releases in the gut. Mutant γδ – mice get colitis.

4. Further studies of γδ T cells show that they are not all the same and that there is a
subpopulation with inhibitory properties. Selective depletion can serve as therapies in
disorders.

Figure 3.53, Summary of glycolysis

Video Clip 3.6

VI. Thought Questions

A. Illustration of a Basic Principle: control of glycolysis

1. Glucose provide the energy source for most ATP production.

2. The sequence of reaction in glycolysis splits glucose into pyruvate, which then enters
the mitochondria to generate most of the ATP.

3. Ironically, the first step of the process, the conversion of glucose to glucose-6-
phosphate by hexokinase, requires ATP. So the enzyme’s active site binds both
glucose and ATP.
 Figure 3.54, a. phosphofructokinase b. active c. inhibited

4. A second, allosteric, site on the enzyme also binds ATP, and when it binds ATP, the
enzymes shifts and turns off.

5. So here’s the apparent paradox you must resolve: how can an enzyme get shut off by
its own substrate and still work?

6. So stop and think a minute. Which site (active or allosteric) should have the greater
affinity, that is, have a stronger tendency to grab ATP?

a. If the allosteric site has greater affinity, it will bind to the ATP first, shut off the
enzyme and stop energy generation completely. By the time ATP levels have
dropped to the point where the allosteric site releases ATP and the enzyme turns
back on, there won’t be enough ATP left to charge up the active site.

b. However, if the allosteric site has a lower affinity, it will only bind when ATP
levels are relatively high. This means that the enzyme will keep working, feeding
carbon into the Krebs cycle, until the cell has an adequate supply of ATP. Then it
will turn off, blocking further metabolism of glucose, until the system begins to
run low on energy.

Figure 3.55 dendritic cell presenting antigen and signals to TH cell

7. Tweaking the activity of the immune system is far more complicated than this, but
let’s for the purposes of argument assume that something similar works in the
regulation of TH cell activity by B7.

a. The sentinel dendritic cells (APCs) activate the TH cell by presenting antigen on
MHC II.

b. If the APCs received a danger signal they make the second necessary second
signal, B7

c. TH cells bind B7 using CD28 (which is in the ready) in order to activate.

d. However, shortly thereafter, they produce CTLA4, which also binds B7 and shuts
down the cell, over-riding the signal produced by the CD28.

e. The strength of binding to CTLA4 is 10 times that to CD28.

f. Cells typically have 10 times the CD28 as CTLA4.

g. TH cells adjust the relative numbers in response to cytokine signals from APCs.

8. Treg cells have CTLA4, but use it to tie up B7 if there is no indication of infection.

9. Cancer cells down regulate the immune system.

a. James Allison developed a cancer therapy that interferes with this.

b. Antibodies to CTLA4 tie it up.

c. The TH cells them become more active and aggressively attack the malignant
cells.
 Figure 3.56, blocking the APC signal to the TH cell

B. Predicting Tolerance

Those wacky scientists are engineering mice again! They develop a strain (B) of mice that
has a gene for anti-HEL-antibody, and constitutively produces B cells that produce these
antibodies/Ig receptors.
In a separate experiment, they produce three different strains of mice with the gene for hen
egg white lysozyme (HEL), a protein foreign to the mouse.
 Strain 1: most of the cells secrete the protein into the interstitial fluid surrounding them.
 Strain 2: liver cells secrete the protein into the plasma, and the protein is found nowhere
else.
 Strain 3: the HEL gene is located next to the gene for δ crystalline and is thus only
deposited in the cornea.
Question 1: In the next experiment they cross strain B with each of strains 1, 2 and 3 and
then monitor function of the adaptive immune system.

Figure 3.57, Experiment summary

1. A check of the TH cells shows NO HEL-reactive cells from strains 1 and 3, but reactive TH
cells from strain 2 in the thymus, but not in the plasma. Explain.

A. All #1 reactive TH cells have been deleted by Central Tolerance, and the #2 thymic
epithelial cells express HEL and the #3 thymic epithelial cells do not.
B. All #1 reactive TH cells have been deleted by Central Tolerance, and the #2 thymic
epithelial cells do not express HEL and the #3 thymic epithelial cells do.
C. Since HEL is completely foreign, TH cells will only be exposed to HEL in the plasma.
D. Strains #1 and #3 have TH cells deleted by peripheral tolerance, however strain #2 has
encountered HEL and activated.
The correct answer is B.

Figure 3.58, Cells from Question 1

Strain B1 Strain B2 Strain B3

Deleted in thymus by
central tolerance
No reactive cells in
periphery
Reactive cell exit Deleted in thymus by
thymus central tolerance
No reactive cells in No reactive cells in
periphery: rendered periphery
anergic

Ordinarily you might expect that in hybrid B1 the Central tolerance would delete the
reactive TH cells, but not in 2B or 3B. You would then expect the 2B reactive cells to
become anergic when exposed to HEL in the plasma. No surprises.

Since the 3B cells would not be exposed to HEL in the plasma, you would expect to see
reactive cells there too. But you don’t see reactive cells anywhere! What could have
happened? Since the reactive cells were deleted by Central Tolerance, the TH cells
must have been exposed to HEL in the thymus, via AIRE induced expression.
2. The scientists then begin to look for reactive B cells, that is, those cells with an Ig receptor
for egg white lysozyme that can begin to activate and divide in its presence. In the bone
narrow, they find reactive cells from strain(s) _______. In the cornea they find reactive cells
from strain(s) ________. (Hint: it might help to draw a table.)

A. none of the mice; none of the mice

B. 3 only; 1 only
C. 2 and 3; none of the mice
D. 2 and 3; 2 only

The answer is C.

Figure 3.59, Cells in Question 2

Strain 1 will delete reactive B cells in the bone marrow by central tolerance, but strain 2
and 3 will not. Recall AIRE induction of tissue-specific proteins happens only in the
thymus. However, in normal mice NO lymphocytes enter the cornea, so you won’t
find any reactive cells of any type there at all.

Video clip 3-7

VII. Mutations

1. Mutations in Foxp3 block Treg production, resulting in immune dysregulation called

IPEX (immune-dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome
in humans and scurfy in mice). The runaway autoimmune response is fatal, attacking
multiple organs.

a. What does this suggest about the presence of self-reactive circulating B and T
lymphocytes?

b. The FoxP3 gene is part of the X chromosome, and you only need one
functioning copy of the gene for Treg production. Will you be more likely to
see IPEX in boys or girls?
 Figure 3.60, FoxP3 Figure 3.61, Fas/Fas ligand

2. Mutations in Fas or Fas ligand block apoptosis. This produces ALPS in humans and
lpr in mice, which results from excessive numbers of active lymphocytes.

Induction of apoptosis is critical in preventing an excessive or over-extended

immune response. B and T cells up-regulate their Fas and Fas ligand upon
activation so that they can be quickly apoptosed once the crisis has passed.

a. While doctors can help patient manage ALPS and lead normal lives, one extra
risk is Hodgkin’s lymphoma. Why would ALPS make a person at risk?

b. Surprisingly people with ALPS are often anemic (low red cell count), or have
too few neutrophils or platelets. Why not too many?

c. In the previous course, we looked at CTL ability to attack infected or

cancerous self cells. Mutation in FAS ligand only did not block their ability
to attack. Why not?

d. Another strange clinical finding in ALPS is the presence of double negative T

cells. What does this suggest about apoptosis during the development of T
cells?

3. Mutations in aire (autoimmune regulator) are inherited in humans as a classical

Mendelian autosomal recessive, producing a type of multi-organ autoimmune
disorder call APECED (Autoimmune Polyendocrinopathy Candidiasis-Ectodermal
Dystrophy). Also called APS (Autoimmune Polyglandular Syndrome) type I.
The functional version of the gene promotes central tolerance by up-regulating
tissue-specific proteins in the thymic medullary stromal cells and improving the
range of negative selection. The symptoms appear over a period of years with
differences in order and severity. Generally things get worse and worse with time.
 a. Would you expect to see more APECED in boys or girls?

b. Oddly the most common first symptom of the disease is candida infections in
infancy.

c. The next most common symptoms are malfunctioning adrenals and parathyroid
glands.

d. Eventually, the patient’s immune system can attack the liver, the Islets of the
pancreas, the lungs, the gut lining, red blood cell production and more. But this
varies a lot, not all patients getting all symptoms and first showing those
symptoms anywhere from infancy to adulthood.

Video clip 3-8

VIII. Implications and Analogy

A. Implications and Analogy

1. A final take-away lesson from this is that a mutation in even one element of the
system can produce autoimmunity.

a. You need both FAS and FAS ligand to interact and signal apoptosis. If you’re
missing either one, you have an autoimmune problem.

b. No Fezf2? It doesn’t matter if AIRE is present, you still have autoimmune

problems.

c. No AIRE? It doesn’t matter if the FOXP3 and FAS genes are fine, you’re sick.

d. No FoxP3? IPEX is lethal, even in humans that produce functional AIRE.

Figure 3.62, Drosophila Figure 3.63, eye-color determining genes

2. What does that indicate about the prevention of autoimmunity? The fruit fly eye color
analogy:

a. In the wild, fruit flies have dark brick red eyes.

b. Fruit flies have four pairs of chromosomes and the three largest have a number of
different genes whose product affect fruit fly color (the fourth chromosome and
the Y are both small and do not have eye-color genes)

c. There are a bunch of genes, distributed over different chromosomes, that code for
enzymes in multiple pathways contributing to pigment production. Examples:

white,
carnation,
purple,
brown,
sepia,
cardinal

d. There is no one gene that produces the red brick eye color, nor one gene that
produces tolerance. All the genes products must work together to produce the
complete phenotype.

Figure 3.64, Analogy parallels

References:

Ted Talk: Rob Wright talks about the microbiome, including its importance in tolerance.
http://www.ted.com/talks/rob_knight_how_our_microbes_make_us_who_we_are#t-
170499

Anterior Eye Capsule – Immunological Privilege:

Yasmin, Seema (2016). Ebola’s Second Coming. Scientific American: 315(1) 40-45, July.

Tolerance Experiments:
Goodnow, C. C. (1991) http://www.ncbi.nlm.nih.gov/pubmed/1944535

T Regulatory cells:

Sakaguchi, S., Wing, K., and Miyara, M. Regulatory T cells in brief history and
perspective. Eur. J. Immunol. 37:S116-123, 2007.

Sakaguchi S, Powrie F. Emerging challenges in regulatory T cell function and

biology. Science. 317:627-629, 2007.

Wing, K., and Sakaguchi, S. Regulatory T cells exert checks and balances on self-tolerance
and autoimmunity. Nat. Immunol. 11:7-13, 2010.

Sakaguchi, S., Powrie, F., Ransohoff, R. M. Re-establishing immunological self-tolerance

in autoimmune disease. Nat Med. 18:54-58, 2012.

Tolerance Overviews:

http://andersonlab.ucsf.edu/sites/andersonlab.ucsf.edu/files/publication_attach/WaterfieldN
YAS.pdf

ALPS: https://www.niaid.nih.gov/topics/ALPS/Pages/20Years.aspx

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3312674/

gamma-delta T cells: http://www.jleukbio.org/content/97/2/259.long

Fezf2 (also promote promiscuous gene expression in thymic epithelial cells):

https://webmail.rice.edu/imp/view.php?actionID=view_attach&id=2.2&muid=%7B5%7DI
NBOX41658&view_token=lbx-HRNxUGyGB0-kCxU2JQ4&uniq=1469907372675
and Takaba et al (2015) Cell 163: 975
 Lecture 3-4
Autoimmunity

Men were designed for short, nasty, brutal lives; women

are designed for long miserable ones. - Estelle Ramey,
quoted in Newsweek, January 12, 1987.
Video clip 4-1
I. Systemic

Clinicians divide autoimmune diseases into two categories – systemic and organ specific.
Like many biological categorizations, this one has fuzzy edges. We’ll look at two diseases
generally considered to be good examples of systemic diseases, that is, situations where the
attacks are wide disseminated throughout the body, and not confined to a specific organ or
tissue.

A. Systemic Lupus Erythematosus (SLE or Lupus)

1. Butterfly rash: Frequently (although not always) presents with butterfly rash on the
face. However, if someone shows up with this rash, lupus is the first thing you should
check out.

2. Antigen Spreading: People with this (typically women) produce antibodies to many
self-antigens, including histones, a variety of blood cell surface proteins, clotting
proteins, and even their own DNA and they become sensitive to a greater variety of
antigens over time.

3. Excessive antigen-antibody complexes: These can activate complement.

4. The complement activates inflammatory responses leading to widespread tissue and

blood vessel damage, commonly anemias, bleeding disorders, kidney damage and
more. Hence systemic.

Figure 4.1, Lupus “butterfly” rash Figure 4.2, rheumatoid arthritis, affected joints

B. Rheumatoid Arthritis

1. Defined by inflamed joints, hence arthritis.

2. The complexes also affect the blood, cardiovascular, and respiratory systems, hence
systemic.
 3. Rheumatoid factors, are complexes of auto-antibodies (typically IgM) to the Fc
stems of other antibodies, IgGs.

4. These clumps of IgM-IgG complexes get deposited in the joints, activating

complement, leading to inflammation and deforming the spaces.

5. The clumps may also overwhelming the ability of neutrophils and macrophages to
clear the debris, leading to a general activation of complement, inflammation and
further widespread damage.

Video clip 4-2

II. Endocrine
If antibodies or T cells are directed against an antigen specific to an organ, then this is the
only part of the body they will attack. There are a number of diseases associated with this
type of response, which can involve antibodies and/or TC cells.

A. Thyroid

Figure 4.3, Thyroid gland

1. Hashimoto's thyroiditis

a. TDTH cells attack the thyroid, bringing with them macrophages and plasma (B
lineage) cells and set up germinal follicles.

b. The whole gland gets inflamed, producing goiter.

c. The plasma cells make antibodies to a number of thyroid proteins, shutting down
its ability to take up iodine and make thyroxin.
 Figure 4.4, Healthy thyroid tissue Figure 4.5, Graves disease dysplasia

2. Grave's disease

a. Also affects the thyroid.

b. Antibodies to the TSH receptors cross-link them and permanently stimulate them.

c. This can also lead to enlargement of the thyroid.

d. This causes the thyroid to release excess thyroxin. Symptoms are similar to those
of any other hyperthyroid condition.

Figure 4.6, pancreas (islet) Figure 4.7, normal islet Figure 4.8, diseased islet

B. Insulin-Dependent Diabetes Mellitus (Type I or juvenile onset)

1. The pancreatic exocrine glands secrete digestive enzymes through a duct into the
small intestine.
 2. Scattered throughout the exocrine tissue are small clusters of cells, the Islets of
Langerhans, which secrete hormones directly into the blood.

3. Type I diabetes is a specific CTL attack against the  cells in these clusters.
a. α cells produce glucagon
b. β cells produce insulin
c. δ cells which produce somatostatin

4. Auto-antibodies contribute to the attack by initiating ADCC in macrophages and

triggering release of cytokines that attract a subset of TH1 cells, the TDTH cells.

5. The TDTH cells also produce cytokines, initiating a local inflammatory response and
calling in more macrophages and CTLs.

6. Without insulin, glucose builds up in the blood and strains the kidney’s ability to
excrete it. This results in ketoacidosis, toxicity, tissue damage and death.

7. The process often begins after some infection during the recovery period, and the
current hypothesis is that something specific to the  cells bears an unfortunate
similarity to an antigen of the pathogen.

8. However, mice have a defective gene region associated with the release of anti- cell
antigen- recognizing T cells from the thymus- a defect in negative selection.

9. Current treatment involves replacement of insulin by injection or delivery with an

insulin pump, but this is not entirely successful. Current research is aimed at
blocking the autoimmune process and allowing the Islets to regrow or to replace the 
cells by transplantation.

Video clip 4-3

III. Neuromuscular

A. Myasthenia gravis

1. Nerve cells stimulate muscles by releasing acetylcholine, which diffuses across the
neuromuscular synapse to its receptor on the surface of the muscle plasma membrane,
inducing depolarization and muscle contraction.

Figure 4.9, Myasthenia gravis

 2. In myasthenia gravis, antibodies to the acetylcholine receptor on muscle cells bind
and block them.

3. This results in muscle weakness. People with it may even need to tape up their
eyelids.

B. MS, Multiple Sclerosis

1. CTLs attack the myelin sheath of peripheral nerves.

2. This slows with nerve conduction, resulting in loss of sensation (including vision) and
paralysis.

3. The cells also enter the cerebrospinal fluid and attack the myelin in the brain and
spinal cord, which produces an array of neurological problems.

4. This is associated with genetic factors, but nothing as strong as an autosomal recessive or
dominant gene. If one identical twin has the disease there is a 2 in 3 chance the other will
have it as well.

Figure 4.10, MS attack on myelin sheath Figure 4.11, US map

5. Geographical risk factors (living north of the 37th parallel) if you live there before age
15, possibly related to vitamin D deficiencies.

6. New evidence based on experiments with mice, suggest that it is the anti-
inflammatory properties of the salates in sunscreen.
 Figure 4.12 a. homosalate b. octisalate

a. These are homosalate and octisalate, which are esters of salicylic acid.

b. Aspirin is a different ester, acetylsalicylic acid.

7. Retrovirus activation- Recently, scientists have discovered we all have the DNA from
the retrovirus HERV-W incorporated onto chromosomes 6 and 7. Its reactivation and
replication are associated with MS and schizophrenia.

8. Fundraising Comment- Most autoimmune diseases are treatable, but rarely curable.
This is also true of many genetic diseases and many cancers. Such medical
conditions are expensive, a particular burden in the US, with no universal health care.
US citizen often organize fundraisers to support research and treatment of particular
diseases.

Figure 4.13a and b, MS 150

On the one hand, I wish we didn’t need these fundraisers. But on the other, it’s very
clear that participants enjoy their rides, their fellow riders and the whole satisfaction of
making a difference.
Video clip 4-4
IV. Epithelia: The body’s defenses begin at the boundary between it and the outside world. So it
is no surprise that a number of autoimmune dysregulation disease involve the skin, mucus
membranes and gut.

Figure 4.14, skin Figure 4.15, psoriasis Figure 4.16, psoriacin

A. Psoriasis

1. Skin provides important innate immune barrier. The epidermis is composed mostly of
keratinocytes which arise from dividing layer above the dermis. They mature and
eventually die, leaving behind the layer of keratin at the outermost part of the skin.

2. 90% of the cells in the epidermis are keratinocytes, which form the framework that
holds the pigment-producing melanocytes and a collection of T cells, including
gamma delta T cells, in positions at the body’s external boundary.

3. Activated keratinocytes secrete a wide variety of proinflammatory chemokines and

other cytokines, activating T cells and macrophages.

4. Psoriasis result primarily from over-activity of T cells, antibodies not seeming to play
much of a role. Scientists have proposed defects in Treg function.

5. All the excess activity prompts the basal layer into overdrive and excess cell division.
Cells mature much faster than normal and produce a variety of inflamed, itchy
lesions, often with layers of dead skin cells on top.

6. People with an overproduction of psoriacin (antimicrobial peptide that attacks E. coli

and disrupts their membranes) are susceptible.
 7. In addition to drug therapies discussed below, treated with localized creams contain
Vitamin D analogs, retin-A and steroidal anti-inflammatories (oddly NSAIDs may
make it worse)

Figure 4.17, normal GI architecture Figure 4.18, inflamed crypts

B. Inflammatory Bowel Diseases

1. Ulcerative colitis – just the colon or large intestine and rectum.

a. T cells in epithelium: CTL directly attack tissue

b. p-ANCA (peri-anti-neutrophilic) antibodies found here target the body’s own

neutrophils – possibly cross reaction to gut bacteria, similar to one proposed
mechanism for initiation of Type I diabetes.

c. Sulfide producing gut bacteria may exacerbate the symptoms.

d. Genetic or familial predisposition.

e. Whole epithelium involved –spreads evenly from the rectum up, with damage
around the whole organ.

f. Prevents water absorption, resulting in diarrhea.

2. Crohn’s Disease – both large and small intestine.

a. Patchier

b. Deeper lesions

c. Associated with other autoimmune problems (arthritis, rashes)

d. This is the version treated with pig whip worms, but drugs promoting a Th2
response and mucus production may also help.
Video clip 4-5
V. Mechanisms – Failure of tolerance causes autoimmune diseases. Self-reactive B, TC or TH
cell evade the processes of deletion and suppression and recognize self-antigens. They then
raise an attack using antibodies and/or CTLs, abetted by TH cytokine signaling.

A. Confused Adaptive Immune Cells

1. B cells: some autoimmune responses involve ONLY antibodies.

a. TH1 cells coordinate attack.

b. ADCC prompts attack by macrophages, neutrophils and NK cells.

Figure 4.19, plasma cell cartoon Figure 4.20, TH cell cartoon

2. CTLs
a. TC cells require TH cell signals to activate to CTLs.

b. CTLs attach directly to cells, inducing apoptosis.

c. In this case, apoptosis is accompanied by inflammation.

Figure 16.21, Tc cell cartoon Figure 16.22, Treg cell cartoon

3. Treg cells – where are they when you need them?

B. Sex and Gender - Incidence rates vary among men and women.

1. For most autoimmune disorders, women are 2 to five times as likely to be affected.
Can be even higher (SLE).

2. There are some autoimmune disease that disproportionately strike men (ankylosing
spondylitis – vertebral joint fusion).

3. But why? Men are more likely to raise a TH1 response to infection, which suggests
they should get more autoimmune diseases. However, testosterone lowers the
response to infection, which suggest they would be less likely.

4. Pregnancy, which requires that the mother tolerate an immunologically foreign fetus,
results in down-regulation of certain immune responses and often an abatement of
autoimmune symptoms.

C. Infection

1. Some autoimmune responses involve attack on self-antigen that resembles epitopes

from pathogens - strep and rheumatic heart disease, Type I diabetes.

2. Some autoimmune diseases improve when the patient get antibiotics: psoriasis and
Crohn’s disease. Generally damping down inflammation may help here.

D. Genetics: MHC I and II and more

Figure 4.23, MHC II and I

1. Possession of a particular allele increases your relative risk, sometimes very

substantially.

2. Hereditary hemochromatosis results from a rare mutant autosomal recessive variant

of HLA- HFe. The gene is not a classical MHC I allele. It codes for a surface protein
on the cells of the gut, and regulates of iron uptake.

3. A variant of DQB1 (MHC II) provides better example.

 a. If you have it you are 130 times more likely to exhibit narcolepsy as a typical
member of the general public.

b. If you don’t have the variant, you can still get narcolepsy, but your risk is much
lower.

4. People with variant of HLA (B27) are 90 times more likely to get ankylosing
spondylitis. However, with or without the allele, men are more susceptible than
women.

5. An association between disease and MHC genotype doesn’t tell you much about
causation.

a. The gene might be closely linked to some other causative gene and simply
segregate with it.

b. On the other hand, a particular allele may, by dumb luck, serve as a receptor for
some disease virus.

c. Or an MHC might be very good at presenting a particular viral antigen, but alas it
happens to be nearly identical to a self-antigen, triggering adaptive immunity
against a particular tissue.

6. Just remember: possession of a particular allele does not predictably doom you to a
certain disease; environment and dumb luck also play a role.

E. Vitamin D, the sunshine vitamin

Figure 4.24, Vitamin D3

1. Cholecalciferol (D3) formed from 7-dehydrocholesterol via UV.

2. Vitamin D – decreases IL-2, IL-12 and IFNγ (proinflammatory signals) and TH17
cells.

3. Deficiencies associated with MS, Type I diabetes.

 Video clip 4-6

VI. Therapies

A. Replace what’s missing (damaged endocrine glands)

1. Thyroxine replacement for Hashimoto’s – works quite well.

Figure 4.25, thyroxine Figure 4.26, insulin pump

2. Insulin injections- not so much

3. Cholinesterase inhibitors – turn up the volume, as it were.

B. Immune Suppression by its very nature carries an assortment of dangers

1. Corticosteroids and other general suppressants will block the autoimmune response,
but leave you more vulnerable to infection generally. Prednisone – mood problems,
elevated glucose, long term neurological

Figure 4.27, prednisone Figure 1428, cyclosporine A

2. Cyclosporine A blocks T cell receptor activation and is at least somewhat specific for
only those T cells currently activated because it triggers a chain of events that blocks
IL-2 receptor binding. This strange fungal peptide, not synthesized on ribosomes and
contain a D (most are L) amino acid. Risk of adverse drug reaction include kidney
damage and more.
 3. Statins block an enzyme in the pathway that produces cholesterol. They may also
lower MHC II levels, inflammation generally and shift the response into a more TH2
attack. Lipitor, Crestor, Zocor.

4. Plasmapheresis temporarily removes antigen-antibody complexes, and clear out

inflammatory signals. This works for several weeks and then gradually your body
resupplies the antibodies and you have to do it all over again. Not as bad as dialysis,
but not a picnic, either.

C. Directed Immune Therapy

Since T cells lie at the heart of the process, coordinating the attacks of the body's tissues,
specific strategies against them can help. Many of these involve antibodies.

1. antibodies against CD4 - This attacks all TH cells.

2. CTLA4-ab ties up B7 co-stimulus. This tends to target newly activated cells.

3. Antibodies to specific T cells – much harder -have to develop for every individual.

4. Competing with auto-antigen for binding site on class II MHC with a slightly
different peptide - keeps auto-anti-Ts from getting stimulated.

D. Anti Hygiene

1. Anti-Hygiene Therapies – whipworms, farm animal dander, and even Lactobacillus

2. Promoting a TH2 response with drugs – increase mucus secretion.

3. Providing the antigen orally. This seems to induce tolerance in mice by producing
anergy in T cell clones.

Figure 4.29, Mother zebra and foal

You might be wondering, at this point, why we haven’t evolved a better sense of
discrimination between ourselves and our pathogens. At the heart of this, and any other
biological problem is the issue of optimizing the use of your resources. The adaptive
immune system currently uses a significant portion of our energy budget and improving the
system check would require even more energy. Where will that come from? Can you eat
more? Starve your brain a bit?

Both zebras and humans need an immune system able to tell self from non-self, but only
well enough, on the average, for them to live long enough to reproduce. Some 5% to 7%
of humans have some kind of auto-immune problem. Eliminating these by tinkering with
the gene pool would most likely compromise some other immune functions or the overall
resources available to the person.

References:

MS 150
http://main.nationalmssociety.org/site/TR?fr_id=27003&pg=informational&sid=13240

Crohn’s disease video.webm by Osmosis, https://en.wikipedia.org/wiki/Crohn%27s_disease

Sunscreen and MS:

http://www.pnas.org/content/early/2017/07/18/1703995114.full?sid=96ed41b4-bd7f-4b8e-
829a-ddf389158c13
 Lecture 3-5
Hypersensitivity

“Logical thought is, of course, a vital element in scientific

discovery, maybe even more so than it is in poetry, art, or
any other high exercise of intellect. But it is not the only
element.” William Broad and Nicholas Wade, Betrayers of
the Truth, page 126
Video clip 5-1

Hypersensitivity describes any inappropriate or misdirected immune response. These

reactions are normal and useful in one context, but annoying or dangerous in another.

I. Type I: Characteristics and Diseases

A. General Characteristics

1. allergic responses, including asthma and anaphylactic shock

2. After sensitization the response is immediate, occurring in seconds to minutes.

3. Immediate responses are caused by antibody interactions, in this case IgE.

4. The responding cells are mast cells, basophils and then eosinophils.

B. Barrier Flushing- The normal, useful version of this response.

Environmental toxins – wood smoke, cave environments.

Figure 5.1, parasitic worms Figure 5.2, pollution

C. Atopy - a localized version of the above response

1. Allergic rhinitis (hay fever) - an atopic (local) as opposed to systemic response

centered on the eye and nasal mucosa and upper respiratory tract. Bizarrely, no one
has figured out why some pollens are allergenic and others are not.
 Figure 5.3, pollen Figure 5.4, allergic eczema

2. Atopic dermatitis (allergic eczema) - inflammatory response in the skin, with local
pustules resulting from local edema.

3. Food allergies (e.g. peanut allergies). Recall important of breastfeeding in educating

the infant gut.

a. Gut- local (atopic) response can involve vomiting and diarrhea, rapid onset
paradoxical, because IgE should not cross the gut mucosa.

b. Circulation- If the allergen enters the circulation, you can get hives, asthma, and
worse. With some severe cases, even breathing around peanuts will do it.

4. Asthma technically atopic, of the lower respiratory tract

a. Lower tract develops edema, mucus secretion and contraction of bronchial

smooth muscles.

b. Asthma sufferers may even have increased levels of neurotransmitters that lead to
smooth muscle contraction and lowered levels of receptors for those that lead to
their relaxation.

c. Asthmatic attack can be induced by allergens, but also by exercise or cold.

Asthma has been associated with spending too much time indoors, especially if
the housing is infested with roaches and dust mites.
 Figure 5. 5, asthma Figure 5.6, lung tissue damaged by asthma

D. Systemic Anaphylaxis (anaphylactic shock) - Clinical Consequences of Excess Type I

Response

Systemic degranulation: releases vasodilators and permeability increases and smooth

muscle contraction throughout body, resulting in edema, bronchiole constriction, shock
and death.

1. Drop in blood pressure defines shock.

2. Bronchiole constriction compromises oxygenation.

3. epithelial swelling

4. GI and skin responses

5. CNS responses – loss of consciousness, confusion, anxiety

6. Opposed by epinephrine, which reduces vascular permeability, relaxes smooth

muscles, increase cardiac output, and raises cAMP levels, blocks further
degranulation.

7. The Epi-pen
 Figure 5.7, anaphylactic shock Figure 5.8, an epi-pen

Video clip 5-2

II. Type I: Mechanism of Action

A. Sensitization and Activation: Type I reactions, while immediate, still require the two or
more weeks it takes to initiate any adaptive response.

2. B cells become sensitized to a parasitic antigen or environmental irritant.

1. hapten mechanisms possibly involved

2. Plasma cells secrete IgE with a CDR that recognizes the antigen.

3. The FC (stem) ends of the IgE differ from those of the IgG, having a fourth
constant domain and a rigid bend.

4. The C terminal only binds to receptors specific to IgE.

Figure 5.9, IgE Figure 5.10, Mast Cell Figure 5.11, Basophil
 3. Capture of Fc stems by basophils and mast cells using Fc receptors. Once bound
there, the IgE may last for weeks.

a. basophils - make up less than 1% of circulating leukocytes

b. mast cells - migrate from bone marrow to tissues, particularly abundant in

connecting tissues around circulatory system and in the skin and mucus
membranes.

c. Both have Fc receptors specific to IgE.

d. Both have lots of secretory granules in the ready.

4. Light micrograph of with granule staining (basic stains attach to acidic contents), the
Fc receptors not visible.

Figure 5.12, mast cells Figure 5.13, Basophil Figure 5.14, FcεRI

5. FcεRI is an unusual receptor in that it

a. has high affinity for IgE only.
b. binds IgE Fc stems even if they are not bound to antigen.
c. will essentially tie up most of the IgE in the body.
d. Alpha subunit resembles TCR receptor peptide has 2 Ig domains, however
functions as monomer.
e. Beta subunit unique to this receptor and unusual in # membrane spans,
cytoplasmic N terminal and location of ITAM on C terminal.
f. Gamma-gamma subunits resemble TCR zeta-zeta, except they have one ITAM

6. Antigen
a. Cross-links two high affinity receptors that have already bound to IgE.
b. A tyrosine kinase on the cytoplasmic side activates ITAM.
c. Sets off several internal signals. This should sound very familiar, but note
difference between this and cross-linking B cell Ig receptors.
 Figure 5.15, antigenic activation of IgE receptors

7. Experimental manipulation. Interventions with antibodies that cross-link receptors

have the same effect.

a. Antibody to IgE antibodies

Antibody substitutes for antigen in crosslinking the IgE antibodies.

Figure 5.16, Antibody to IgE antibody Figure 5.17, Antibody to Fc receptor

b. Antibody to Fc receptors (FcεRI) crosslinks the receptors directly.

Anything that crosslinks the receptors will initiate the response.

8. internal signaling pathways

a. release of Ca2+ from the ER and through the plasma membrane

b. lead to fusion of granule (vesicle) with the plasma membrane containing

histamine and heparin. Can mimic effects with Ca2+ ionophore.

c. Massive degranulation at the plasma membrane disrupts the structure, activating

of phospholipase C to produce DAG and PIP2.

d. These signal the production of prostaglandins and leukotrienes.

 9. At the same time, activation of a kinase cascade in turn activates transcription factor
NFkB.

a. NFκB turns on genes that lead to the synthesis of pro-inflammatory genes,

including those for inflammatory cytokines.

b. TNF- may also induce shock, in a manner analogous to toxic and bacterial
shock.

c. Also up-regulates synthesis of prostaglandins and leukotrienes from arachidonic

acid.
HC N

HN CH

CH 2

H2N CH 2

histamine

Figure 5.18, histamine Figure 5.19, heparin

E. Mediators and Signals

1. Immediate release: Histamine and heparin.

a. increases smooth muscle contraction of intestine and bronchioles

b. increases permeability of venules and vasodilation

c. increases mucus secretion by goblet cells

d. negative feedback loop on further release by mast cells and basophils

Figure 5.20, prostaglandin Figure 5.21, leukotriene

2. Later: prostaglandins and leukotrienes

a. synthesized from arachidonic acid after granule release

b. broncho-constrictors
 c. does everything histamine does only more strongly and over a longer period of
time
d. Leukotrienes are a new target of antiallergenic medication.

Figure 5.22, TFNα

3. cytokines

a. TNF- - apoptosis, fever, loss of appetite. May also induce shock, in a manner
analogous to toxic and bacterial shock. Can be soluble or membrane bound.

b. IL-4 – increases IgE production

Video clip 5-3

III. Type I: Therapies and Epidemiology

A. Therapeutic Approaches

Figure 5.23, skin prick test Figure 5.24, skin patch test
1. Tests (again mostly for IgE):

a. Skin Prick
b. Skin Patch- usually for food sensitivities
c. Direct food challenge – a pill with an isolated food allergen. Can occasionally
immunize someone against a new allergen or result in a really bad reaction.
d. Blood tests assay IgE in blood.

2. Immune therapies:

a. Desensitization: Inject increasing doses of allergen into patient, sublingual, oral or

subcutaneous. If you're careful, this will cause the sufferer to make IgG4 (non-
inflammatory) against the allergen. Then, when the patient subsequently gets
exposed to the allergen, the IgG will tie up the allergen before the IgEs get to it.

Figure 5.25, immune therapy for allergy

3. Drugs that directly block the response:

Figure 5.26, therapeutic interventions

 a. Antihistamines and anti-leukotrienes work by preventing mast cell degranulation
and blocking receptors on target cells.

b. Phosphodiesterase inhibitors, which increasing the levels of cyclic AMP, directly

blocking degranulation – inhalers.

c. Epinephrine, which reduces vascular permeability, relaxes smooth muscles,

increase cardiac output, and raises cAMP levels, blocks further degranulation –
the EpiPen

4. Recently – antibiotics. Some asthmatics have low level lung infections and the
resulting inflammatory response seems to potentate an attack.

B. Hygiene Hypothesis

1. There is a negative correlation between exposure to farm animals and tendency to

develop asthma. We seem to be designed to tolerate farm antigens, especially the
endotoxins produced by bacteria characteristic of farm animals.

(A) roach (B) dust mite

Figure 5.27, source of allergenic antigens.

2. But the OPPOSITE is true of cockroach and dust mite antigens. The exposure to
these is correlated with spending a lot of time indoors.

(A) roundworm (B) roundworm egg

Figure 5.28, helminth therapy

3. Researchers in Iowa are using pig whip worms to treat individuals with Crohn’s or
inflammatory bowel disease. Worms cause mast cells to produce mucus, which
soothes the inflammation.
Video clip 5-4

IV. Type II (immediate) Antibody-Mediated Cytotoxic Hypersensitivity - misdirected ADCC

A. Overview (see also table at the end of the handout on this lecture).

2. IgM or inflammatory IgG antibodies. Subsequent exposure causes these antibodies

mark the body's own cells for cell-mediated destruction.

3. Antibodies interact with neutrophils, NK cells and macrophages, not basophils, mast
cells or eosinophils.

4. Immediate, based on pre-synthesized antibodies (prior sensitization required).

5. Th1 and not Th2 response.

6. Typically directed at red blood cells (which are quite fragile).

7. Can contribute to autoimmune disorders and transplant rejections.

B. Transfusion Reactions

Figure 5.29, red blood cell membrane with antigens

1. red blood cell antigens - The red blood cell has a wide variety of glycoproteins and
glycolipids, coded for by genes with multiple alleles: MN, Rh, Kidd, Kell, Duffy, etc.

2. ABO glycolipids are composed of a ceramide lipid with an oligosaccharide attached,

always facing to the exterior of the cell.

3. The oligosaccharide for blood type O (sometimes called H) forms the 5 sugar
foundation for all three ABO blood types.
4. If you are blood type A, you have the gene for an enzyme that adds an additional
sugar, N-acetylgalactoseamine, to the end of this.

5. If you are blood type B, you have the gene for an enzyme that adds a plain galactose
to the end of this.

Figure 17.30, ABO oligosaccharides

6. Since the glycolipids of the ABO group are also produced by intestinal flora, most
people make antibodies to either A or B unless they have A and/or B blood.

7. Usually the anti-A and anti-B antibodies are of the IgM class, probably because this
class is associated with the epithelia, including that of the GI tract.

a. type AB makes neither

b. type O makes both
c. type A makes anti-B only
d. type B makes anti-A only
e. There no such thing as anti-O because there’s nothing extra on it for an immune
cell or antibody to recognize that isn’t on either A or B.
 8. A transfusion of A blood into a O person will result in IgM binding to the cells and
complement mediated cell lysis, resulting in:

a. release of hemoglobin into the plasma

b. excretion of hemoglobin by the kidney
c. separation of the heme group and increased plasma levels of bilirubin, which is
toxic

9. Antibodies to the other surface glycoprotein antigens are usually IgG made after
exposure.

10. If a person becomes exposed on subsequent occasion to these antigens, the IgG may
activate complement, which results in:

a. partial hemolysis
b. clumping of cells
c. activation of macrophages
d. extravasation of complexes into the tissues

11. This is why you do a last-minute cross check matching blood types before you
actually dump blood or organs into people.

Video clip 5-5

V. Clinical Consequences

A. Hemolytic Disease of the Newborn (Rh Incompatibility)

1. At risk: Rh- mother with Rh+ fetus. During pregnancy the circulatory systems are
next to each other, but should not mix.

2. However, human delivery is a messy process and blood may cross over from the fetal
circulation into the maternal.

Figure 5.31, placental circulation Figure 5.32, first maternal Rh+ exposure
 3. IgM antibodies may or may not be produced, but if they are they will clear the fetal
blood from the maternal circulation.

4. You can check the levels of these antibodies by performing something called a
Coombs titer.

5. The memory cell produced by the first exposure puts subsequent pregnancies with an
Rh+ fetus at risk:

a. Memory cells when activated will produce IgG against Rh+ antigen.

b. These may cross the placenta and attack the fetal red blood cells, a condition
called erythroblastosis fetalis.

Figure 5.33, second maternal Rh+ exposure Figure 5.34, RhoGAM passive immunization

6. Passive immunization with Rh+ antibodies (RhoGAM) prevents the mother from
becoming immunized.

7. The antibodies to Rh+ antigen attach to the baby’s RBCs, kill them and get them
cleared by neutrophils before the adaptive immune system has a chance to become
sensitized.

8. If for some reason the mother has been sensitized, her blood can also be treated by
plasmapheresis.

B. Drug-Induced Hemolytic Anemia (sometime called Type V in Britain.)

1. For example, a person can develop a sensitivity to penicillin, cephalosporin, and

streptomycin.

2. Drugs act as haptens, something we looked at in Lecture 4 of the first course.

 3. So here we have a red blood cell that has picked up drugs on its surface. These can
now cross linked Ig receptors, leading to the production of antibodies that will bind to
the cell surface.

Figure 5.35, drug on surface of erythrocyte attracting antibody

4. Antibodies to the drug (or to the drug and surface protein) activate complement and
break up the cell.

Video clip 5-6

VI. Type III (immediate) - Immune Complex-Mediated Hypersensitivity

Type III Hypersensitivity is also an immediate response, based on antibody production

during prior sensitization. In this case the antibodies, instead of attaching to self-cells,
form clumps, interacting with complement proteins and overwhelming the capacity of
neutrophils to dispose of them.

A. Defined – arises from an excess of antibody-antigen complexes.

1. Produced by excess antibody-antigen complexes.

2. Immediate, again because antibodies initiate the reactions.

3. Neutrophils are the primary disposers of these complexes. If the quantity of
complexes exceeds the ability of the phagocytes to clear it, the neutrophils may break
open and die, their contents injuring the surrounding tissues.

Figure 5.36, an overwhelmed neutrophil Figure 5.37, mosquito bites

4. Clumps of antibodies activate complement, which deposits opsonins on the

complexes, producing a strong local inflammatory reaction.

5. Arthus reactions: Localized reactions occur when the complexes are deposited in the
tissues, for example mosquito bites.

6. Complexes carried through the blood may deposit in a variety of damaging places far
from the site of the initial deposition.

a. blood vessel walls, initiating clotting reactions

b. in the glomerular basement membrane initiating kidney damage
c. in the choroid plexus of the brain

Figure 5.38, sites vulnerable to Type III damage

 7. In joints- rheumatoid factors are complexes of two different types of antibodies.

Figure 5.39, rheumatoid factors

B. Localized - the Arthus Reaction

1. In nature, often caused by insect bites.

2. Localized inflammatory response associated with the production of ozone and

hydrogen peroxide, probably produced by the antibodies by an as yet mysterious
mechanism.

3. May also occur from local irritants to the lung endothelium.

a. "farmer's lung" from moldy hay

b. “pigeon fancier's disease" from protein in dried pigeon feces

C. General or Systemic – the result of large amounts of circulating antibody in the general
circulation.

1. serum sickness- a reaction to treatment by passive immunization. The patient

develops his or her own antibodies to the therapeutic antibodies, and the resulting
reaction can generate fever, kidney damage, and arthritis.

2. Autoimmune disorders - problem arise not solely from damage to specific tissues, but
also from the large number of circulating antibody-antigen complexes.

3. Drug reactions - another bad response to penicillin and sulfonamides.

4. Serious infectious disease. A person can produce such a strong antibody responses
that they overwhelm their ability to clear complexes.
 D. Therapeutic Strategies

1. avoidance

2. anti-inflammatories

3. heat – mosquito bites only

Video clip 5-7

VII. Cellular (or delayed): The fourth classical category.

A. Overview

1. Delayed-Type Hypersensitivity

2. Type IV Hypersensitivity is primarily a cell-mediated response and takes a couple of

days to develop.

3. Tasked with getting rid of compromised cells: those that are damaged malignant or
harboring intracellular pathogens.

4. Delayed means it happens after at least a day, usually 2 or 3, certainly NOT within an
hour.

5. Results from signaling by a subset TH1 cells, the TDTH cells. DTH stands for Delayed
Type Hypersensitivity, and it may be delayed, but it’s a serious response.

6. This is a Th1 response and as such, it uses IL-1, IL-12 and IFN γ signaling.

7. The TDTH cells signal to the Tc cells, activating them in to CTLs (cytotoxic T cells),
which will then attack the target cell. Macrophages that pick up a bacterial pathogen can
also gather together in a granuloma, walling off infected cells in a lump.

8. This can kill off healthy tissue, and is the principal mechanism of tissue destruction in
autoimmune disease.

9. This can lead to contact dermatitis, in addition to granuloma formation. Type IV

hypersensitive attacks on heathy self- cells and on transplanted tissue are often
accompanied by Type II attacks as well.

10. Here are cartoons representing the cells very directly involved:
Figure 5.40, TH cell Figure 5.41, Tc cell Figure 5.42, macrophage

11. And here are cartoons representing cells with indirect involvement:

Figure 5.43, sentinel dendritic cell Figure 5.44, mast cell Figure 5.45, γδ T cell

B. Process

1. Three manifestations of the response:

a. Granuloma
b. Dermatitis
c. Tissue destruction (showing myelin here) a defense that involves often
sacrificing some of the body's own cells to get rid of pathogens. This is supposed
to be used in fairly circumscribed regions of infection. If the tissue damage is
limited, this is adaptive. If the tissue damage is extensive, or if the attack
damages perfectly normal tissues, it can become pathologic and even life-
threatening.

Figure 5.46, Clinical Manifestation

2. Like all adaptive responses, it take two weeks to select and expand the T cells that
produce the response.

3. In delayed hypersensitivity, it typically takes 1 to 3 or 4 days after subsequent

exposure for a sensitized individual to exhibit a response.

Figure 5.47, poison ivy and urishiol

4. Type IV antigens are often haptens: small molecules or even metal ions. A variety of
agents can complex with skin proteins, just as we saw drugs complex with proteins on
the surface of red blood cells, and the complex initiates immune response:

a. poison oak and ivy (urishiol, pentadecacatechol)

b. other small organics (hair dyes, penicillin)
c. metals (nickel, silver)- Nickel activates TLR 4.

Figure 5.48, nickel Figure 5.49, TLR 4

 5. The sensitization process depends on phagocytosis, processing and presentation by
Langerhans (sentinel) dendritic cells (figure 5.43).

a. She’s ready to phagocytize.

b. In addition to the MHC I that all nucleated cells display, this cell presents an
antigen on MHC II, ready to activate the TH cell.

c. She also has a B7 co-stimulus in the form of a double marshmallow.

d. Her toll-like receptors extend from her hat, ready to identify the nature of the
pathogen.

e. She also has a cookie box full of cytokine signals, released in response to TLR
signals.

a. And finally her merit badge announces that she is the cell responsible for the first
up-regulation of TH cells.

6. This professional antigen presenting cell (APC) activates the TH cells, using MHC II-
antigen, B7 and IL-12, leading them to differentiate into TDTH cells.

7. The TH cells undergo clonal expansion and cell division during an initial 1 to 2 week
delay.

8. The TDTH cells secrete cytokines that activate Tc cells and macrophages, not
neutrophils.

a. IFNγ

b. TNFβ

9. The flip side of this is treatments that lower the response:

a. Anti-inflammatory steroids (oral and cream)

b. Prednisone

10. Mast cells can down-regulate this response, signaling with a local release of IL-10.
However this signal must come from the mast cells; other sources are ineffective.
Suggests an additional co-signal. FcR plays a role as well, and seems to be induced
by IgG (as opposed to IgE) antibodies.

C. Tuberculosis- when this response is the right thing to do.

1. Tuberculosis is caused by Mycobacterium tuberculosis, an intracellular bacterium.

 Figure 5.50, Tuberculosis

2. The reaction may produce large granulomas called tubercles, which gives the disease
its name.

a. produced by concentration of macrophages that form epitheloid associations

b. Macrophages may fuse to form multinucleate cells.
c. Results in palpable nodule (fibrocytes at exterior).
d. Nodule may release lytic enzymes that damage surrounding tissue.
e. The granuloma develops a necrotic center (cheesy or caseous).
f. Detection of a DTH reaction is the basis for tuberculin skin test - intradermal
injection of protein derived from the bacterial wall may cause a local swelling
(again read at 48 to 72 hours).

3. Gamma-Delta Cells are important in fighting off TB (figure 17.45)

a. Often found around the granulomas.

b. Subtypes have a specific γδ T-cell receptor gene arrangement dedicated to
producing a receptor for a tuberculosis lipid.
c. Once targeting the cells, the γδ T cell can be cytotoxic and also secrete IFNγ
d. They also secrete IL-17, part of the fundamental switch from tolerance.
e. They have a receptor for the soluble heat shock protein 60, produced from tissue
damage.
f. Targets to epithelia.

Video clip 5-8

VIII. Wrap-up and Context

A. Penicillin
 Figure 5.51, penicillin

1. Identified by Alexander Fleming, structure worked out by Dorothy Hodgkin, works

by interfering with bacterial peptido-glycan cell wall synthesis.

Figure 5.52, Alexander Fleming Figure 5.53, Dorothy Hodgkin

2. The bad news: not only did bacteria evolve resistance to the drug, many people began
to develop hypersensitive reactions.

B. Hypersensitive Reactions to Penicillin

1. Type I reaction is a classical allergy. When people experience a bad reaction after
taking penicillin, this produces a very itchy rash and may even progress to
anaphylactic shock.

2. Type II reactions may result from drug attachment to red blood cells and cause
their destruction, leading to kidney damage.

3. Type III versions have similar results, except that the anti-drug antibodies can
show up anywhere in the body, again leading to kidney damage.
 4. A type IV reaction generally produces a nasty rash, but one that takes a day or
more to manifest.

And there is no guarantee that you might not have more than one of these at the same time.

Next we have a video from Osmosis, which summarizes the Type IV response and applies it to
autoimmune destruction. This is a ve4y good review and correlation of process in this lecture
and the previous lecture.

https://en.wikipedia.org/wiki/Type_IV_hypersensitivity
References:
http://www.newscientist.com/article.ns?id=dn4852 - pig whip worms as therapeutic agents

Brandzaeg, Per (2007). Why We Develop Food Allergies. American Scientist (1) 95: 28 – 35,
January-February.

Crohn's Disease Genetics (2001) (advanced web publications) now cited as Nature 411: 599-
603 and 603-606

Fox, Douglas. (2010). The Insanity Virus. Discover Magazine, June, pages 58-64.

Grimbaldson, Michelle A., et al. (2007) Mast cell-derived interleukin 1 limits skin pathology
in contact dermatitis and chronic irradiation with ultraviolet B. Nature Immunology, advanced
online publication.

Lee, June Yong and Stephen C. Jameson (2012) Remembering to be Tolerant. Science 335:
667-668, February 10.

Stuart, Marie, et al (1978). Chronic Idiopathic Thromobocytopenia Purpura: A Familial

Immunodeficiency Syndrome? JAMA 239 (10): 939.

Von Herrath, Mathias and Leonard C. Harrison (2003). Antigen-Induced regulatory T cells in
autoimmunity. Nature Reviews Immunology 3: 233-232.

Innate regulatory cells: http://www.nature.com/nri/posters/mdscs/index.html

https://en.wikipedia.org/wiki/Food_allergy
Tick bites and immunity to meat sugars http://io9.com/5981332/the-tick-that-can-make-you-a-
vegetarian
http://bodyodd.nbcnews.com/_news/2012/11/09/15053860-rare-meat-allergy-linked-to-ticks-
found-across-us?lite
http://allergytomeat.files.wordpress.com/2013/05/tpm-and-spc-current-allergy-2013-
review.pdf
https://en.wikipedia.org/wiki/Food_allergyPollock on You Tube:
https://www.youtube.com/watch?v=3v2o9gLmU08
 Lecture 3-6
Transplant Immunity

Vision without funding is hallucination. – Edward Djerejian, 2004

Video clip 6-1

I. Assays

One of our most important tools in preventing transplant rejection is to match the organ
to the donor with respect to blood type and specific MHC I and II alleles.

A. Review of monoclonal antibodies

Tissue typing assays all depend on the technology used to make monoclonal antibodies.

1. The antibodies are made by a clonal lineage of identical plasma cells, all chucking out
the same antibody.

Figure 6.1, plasma cell Figure 6.2, hybridoma

2. The plasma cells are engineered for immortality by fusing them with myeloma cells
(a type of B cell cancer) and then selecting for fused cells HAT medium, which does
not support the growth of the myeloma cells.

a. H: hypoxanthine (purine precursor)

b. A: aminopterin (methotrexate, a folic acid analog which prevents de novo
synthesis of DNA bases)
c. T: thymine (pyrimidine precursor, also converted to cytosine)

3. This cell line can now supply an indefinite supply of copies of a defined antibody.

4. You have to go through this process every time you want a new antibody against a
different protein.

B. RIA– Employs Competitive Binding

1. Produce a supply of monoclonal antibody with a high affinity for a particular antigen,
for example TSH (thyroid stimulating hormone).

2. Get a supply of the antigen (TSH) you want to test for and radiolabel it, typically with
125
I. This has a half-life of about 60 days.
a. Bad news, you have to keep generating it from a nuclear reactor.
b. Good news: it doesn’t hang about forever once you’re done with it.
 Figure 6.3, anti-TSH antibody holding cold TSH and radioactive TSH

3. Put the antibody together with the labeled antigen. Put in enough antigen so that
essentially every antibody should attach to antigen.

4. Add the sample you wish to test. This may or may not have antigen, but if it does, the
antigen will be cold (unlabeled).

Figure 6.4, add serum with TSH Figure 6.5, cold TSH replaces radioactive

5. The unlabeled antigen will compete with the labeled (recall that the binding
resembles enzyme-substrate interactions).

6. Then you wash off the displaced radioactive TSH.

a. If there is relatively little antigen, most of the radiolabeled antigen will remain
bound.

b. It there is a lot of antigen, most of the radiolabeled antigen will get displaced.
 Figure 6.6, graph of results Figure 6.7, Rosalyn Sussman Yalow

7. Thus the more antigen in the sample, the lower the bound radioactivity remaining.

B. Technical Issues

1. Separating complexes from free antigen: Today, the labeled antibodies are bound on
walls of microtiter wells (96 wells on a plate about the size of an index card) and
screen large numbers of samples (e.g. blood supply for hepatitis B).

2. The samples are calibrated so that they are likely to have antigen in the range where
there is a linear relationship between cold antigen and displacement of hot. For TSH,
physiological ranges are usually 1 to 4 ng / ml of blood.

3. Rosalyn Sussman Yalow won a well-deserved Nobel Prize for developing this
technique.

C. ELISA Assays: Enzyme-Linked Immunosorbent Assays. This technique is every bit as

sensitive and does not require the use of radioactive reagents.

1. Basic Technique Principles

a. Again we will use monoclonal antibodies that recognize a specific antigen.

b. This antigen is often attached to the surface of a small (microtiter) well.

 Figure 6.8, antigen-recognizing monoclonal antibodies

c. However, the reporter in these reactions is the development of color, not the
amount of radioactivity.

d. Here we have a second antibody, with an enzyme attached. The enzyme will act
on a colorless substrate to produce a visible reaction.

Figure 6.9, monoclonal antibody with reporter enzyme

e. Notice we have two versions of antibodies attached to this enzyme, one specific
for the Fc stem of any antibody and one specific for a particular antigen – note
color coding.

f. The most common enzyme used in these reactions is alkaline phosphatase which
removes phosphates from a variety of compounds. Here it’s taking the phosphate
from p-nitrophenyl phosphate (pNPP), which is colorless, and generating para-
nitrophenol, a yellow compound.
 Figure 6.10, monoclonal antibody with reporter enzyme

2. Direct ELISA: assays antigen directly.

a. Make your monoclonal antibodies to the antigen you wish to monitor.
b. Link the antibody to a reporter enzyme.
c. The reporter enzyme is one that can react to a colorless substrate to produce a
colored product (alkaline phosphatase, for example).
d. Present antigen in such a way that you can separate out the complexes with
antigen from the unbound antibody-enzyme complexes that don’t have antigen.
Most techniques attach it to a well.
e. Add antibody.
f. Add substrate.
g. Intensity of the color produced is measure by sensitive spectrophotometers.

Figure 6.11, Direct ELISA Figure 6.1212, Indirect ELISA

3. Indirect ELISA: assays antibody

a. Fix antigen to the antibody (the one you want to measure) onto the sides of
microtiter well.
b. Add the antibody source, and any relevant antibody present will bind to the well.
You want to make sure there’s enough antigen for all antibody to bind.
c. Wash, to remove serum with extraneous proteins.
d. Check for bound complexes using a second antibody complexed to a reporter
enzyme. The second antibody binds to the primary antibody (the one you want to
measure) via a general recognition site that bind Fc stems.
e. Add substrate, look for color. The more antibody you’re trying to measure, the
more color. You can also screen large number using the 96 well microtiter plates.
 f. OraQuick – measures HIV-1 antibodies

i. Qualitative screening device – yes or no

ii. Fast- results in 20 minutes
iii. Can use spit as well as blood
iv. Can only be used if the person has been infected long enough to produce
antibodies.

Figure 6.13, OraQuick Figure 6.14, Sandwich ELISA

4. Sandwich ELISA: Assays antigen from a sample

a. Antibody to the antigen is immobilized in the microtiter well surface.

b. Add sample, antigen binds to well antibodies.

c. Add second antibody linked to reporter enzyme. This one binds to a different
epitope of the antigen already bound.

d. The antigen is therefore sandwiched between two antibodies, hence the name.

e. The more antigen you’re trying to measure, the more color develops.

f. Basis of pregnancy test HCG (protein antigen) in urine:

i. two places to read the test

ii. one line is control to check to see if the test if working

iii. In a second region, a line appears if the test taker is pregnant.

5. Competitive ELISA: assays antigen from a sample

a. First you incubate the sample (presumably with the antigen you wish to measure)
with a known (and presumably excess) quantity of antibody to that antigen.
 b. All the antigen should be bound to antibodies and there should be leftover
unbound antibodies.

Figure 6.15, Competitive ELISA, first step

c. Then add mixture of Ab-Ag complexes and unbound antibody to a well with
antigen immobilized on its surface.

d. The more antigen in the sample, the less free antibody is available for binding to
the antigen on the well surface.

e. Wash off sample, complexes, and unbound antibody, leaving behind antibody
bound to the fixed antigen.

Figure 6.16, Competitive ELISA, second step Figure 6.17, Competitive ELISA, third step

f. Add second enzyme-conjugated antibody to the known antibody into the well.

g. The enzyme-induced color change measures the amount of bound antibody,

which decreases with increasing antigen in the sample.

h. Therefore the more antigen in the sample, the less color develops.

6. ELISA Summary and Analysis

a. All require prior development of a line of monoclonal antibodies.

b. All add, from a sample to be tested, something that will wind up attached to a well
or substrate, so that the amount attached varies with concentration.
 c. All add, as a last step, a monoclonal antibody with a reporter enzyme attached.
This antibody will recognize whatever is stuck to the well and then cause a
reaction that develops varying amounts of color.

Video clip 6-2

II. Graft Rejection

A. Definitions

1. autograft - moving around tissue from one part of the same person to another avoids
the whole problem

a. typically skin on burn victims, but also infection and cancer- new techniques for
extending usefulness and minimizing damage

b. storing your own blood or treated bone marrow

c. moving blood vessels from your legs to replace clogged coronary arteries
(A) (B) (C)
Figure 6.18, autograft Figure 6.19, isograft

2. isograft - transfer between genetically identical individuals

a. identical twins (first kidney transplant – 1954, link in references gives details)
b. inbred mouse strains

3. allograft - between genetically different members of the same species

a. most organ transplants today

b. experimentally between mice of different inbred strains

(A) (B) (A) (B)

Figure 6.20, allografts Figure 6.21, xenografts

4. xenograft - between species.

a. first heart transplant from baboon

b. engineered valves from pigs

B. The Processes of Acceptance or Rejection of a Skin Graft

1. If it's from an autograft that takes

a. In 3-7 days the tissue vascularizes.
b. The new skin turns pink, the neutrophils move peacefully around, and all is well.

2. If it's from an allograft, rejection follows the same principle we’ve seen in
hypersensitivity. Without prior sensitization.
 a. The tissue revascularizes in 3-7 days.

b. The neutrophils and other inflammatory cells infiltrate.

c. Tissue becomes necrotic.

d. Rejection usually takes 12 to 14 days.

3. However, if the recipient is already sensitized to the graft antigens (ABO mismatch,
previous graft from that same donor), you get a second set response or hyperacute
attack:

a. The inflammatory response and necrosis begins within 3 days, and often
immediately.

b. The body has antibodies already. The rapid response begins with ADCC directed
by pre-existing antibodies.

c. Neutrophils attack.

d. Tissue is gone within a week.

C. Effector Responses: Sensitization and Attack

1. TH cells play a central role in the process. TDTH cells (subset of TH1) trigger delayed-
type hypersensitivity (type IV) upon stimulation by sentinel dendritic cells.

2. Cytokines secreted by the TH cells recruit TC cells activated by Class I MHC-antigen

complexes which become CTLs and attack the foreign cells.

3. They summon and activate macrophages which attack tissues.

4. B cells will enter the fray, secreting antibodies to the tissue surface proteins.

5. Antibodies trigger attack by neutrophils and NK cells.

6. Antibodies trigger attack by complement.

 Figure 6.22, cell-cell interactions during immune activation

D. Cell-Mediated Responses: Experiments designed to explore the roles of T cells.

1. If you don’t remove any T cells, the tissue is rejected by 15 days.

2. If you use antibodies to remove the Tc cells, rejection is also complete at about 15
days – the treatment essentially doesn’t matter.

3. If you use antibodies to remove the TH cells, rejection is complete at about 30 days-
doubles the survival time.

4. If you use antibodies to remove both the TH cells and the Tc, the graft lasts 60 days,
four times as long.

5. This suggests that if you remove Tc cells alone the TH cells can still initiate the
response, possibly partnering with other immune cells or promoting rapid expansion
of Tc populations.

6. However, if you remove the TH cells, the trigger is gone (at least initially) and must
repopulate before you can activate the process.
 7. If you remove both of them, the TH cells must regenerate and signal, and if the Tc
cells then have to regenerate, this will delay the response even further.

8. Basically this puts the TH cell – sentinel dendritic cell interaction at the base of the
reaction sequence leading to rejection.

E. The Role of the MHC in Histocompatibility

Figure 6.23, the MHC genetic locus

1. Recall that tissue compatibility is largely determined by MHC (HLA in humans, H-2
in mice) and that Class I and II MHC alleles are closely linked in a block of genes.
a. position on Chromosome 6
b. relative position of MHC I and II
c. more detailed representation showing other genes in the block (play a lesser role,
but may contribute to rejection)

2. These genes code for the proteins used to present antigen to Tc cells (MHC I) and TH
cells (MHC II), which we have shown previously in drawings and 3D models.

Figure 6.24, MHC II and I Figure 6.25, MHC II and I models

3. Each of these genes may come in many different versions, or alleles. That is, the
genes for MHC I A will all code for a similar protein structures, but there are 1000
different versions of the gene, primarily differing in the antigen binding site. Take
note that the MHC II molecule is a hetero-dimer of α and β, multiplying the
possibilities for variation in the final molecule.
 4. The blocks are typically inherited and passed on without crossing over, and the set on
each chromosome is a haplotype.

Figure 6.26, haplotype inheritance options from Mom (left) and Dad (right)

5. There is a 1 in 4 chance that someone will share a haplotype with a sibling.

6. Because of the extreme polymorphism of the genes, it’s rare to share both haplotypes
with a non-relative, but it does happen.

7. Even with what looks like a perfect match, the tissues may not be compatible because
of differences in the non-classical, or minor histocompatibility, alleles

8. On the other hand, sometimes you can tolerate a certain amount of mismatching.

a. For kidneys, MHCI A and B and MHCII DR are more important, with DR being
the most critical, C DP and DQ less so.

b. A mismatch at DR is worse than a mismatch with both A and B.

c. A mismatch with either A or B is worse than multiple mismatches at MHCI C,

MHCII DP and DQ.

d. For liver, matching seems less important.

F. Testing, testing.

1. First, even before checking MHC, you check ABO blood type. ABO antigens are
also found on most cell surfaces and a mismatch here means the recipient has
antibodies in the waiting to attack the transplant.

2. Then check the white blood cells for compatible MHC (HLA in humans). MHC
matches have become less critically important with the development of better
 immunosuppressive techniques and drugs, but surgeons still look for the best
available match.

3. Then check “immune predispositions,” which is my term for any adaptive response
that a recipient may have in the ready to recognize antigen from the transplant. Anti
A or anti-B antibodies are the most obvious threat, but not the only one. People
getting transplants have often been through a lot of previous medical procedures,
including a prior failed transplant. You have no idea what they might have gotten
sensitized to.

4. Cross-matching: Checking for antibodies- Luminex assay, which is basically a variant

of Indirect ELISA.

a. Identify antigen in the organ not found in the recipient.

b. Attach antigen on a luminex bead (as to the bottom of a well). You put each
different antigen on a bead with a different flourochrome with different
fluorescing properties

c. Add recipient serum, whose antibodies will bind to the beads.

d. Add reporter antibody with phycoerythin, which glows bright read in UV.

e. Hit with red and green laser and the different light emitting properties able you to
identify the presence and type of the antibody.

Figure 6.27, Luminex Assay

5. Mixed Lymphocyte Reaction: checking for T cells

a. Take lymphocytes from a donor a irradiate them or treat them with miromycin C
to stop them from dividing.

b. Use these cells to stimulate recipient T cells.

c. If the recipient’s T cells are stimulated they begin to divide, which you can
measure by the uptake of 3H thymidine.
 Figure 6.28, Mixed lymphocyte reaction indicating a bad match

6. Success Stories

a. first kidney transplant- from one identical twin to another

b. then-longest living kidney transplant

Figure 6.29, success stories a. first, b. 30-plus year survivor

Video clip 6-3

III. Encouraging Tolerance

A. General Immunosuppression

In order to encourage tolerance of a transplanted organ, we use many of the same

strategies and drugs we use to fight autoimmunity. However, don’t forget that a
transplant is, by definition, foreign and not self.
1. Side effects: Many of the same drugs used for autoimmune treatment can work to
prevent tissue or organ rejection.

a. elevated risk for lymphoma, liver and skin cancer

b. hypertension – direct result of calcineurin inhibitors (cyclosporine) and steroids

c. metabolic bone disease – Drugs affect calcium metabolism and interfere with
bone remodeling.

d. pre-existing infection- A patient may carry an infection into the transplant.

e. introduced infection- A problem you don’t have with autoimmunity.

2. Drugs – These are the source of most of the transplant side effects.

Figure 6.30, steroids

a. Corticosteroids: – mood problems, elevated glucose, long term neurological

damage

 Prednisone
 Dexmethazone: notice the overall structural similarities (3 six-member and
one 5-membered ring) with a fluorine substitution here.

b. Cytostatics- stop cells division. The constant production and culling of both B
and T cells makes them vulnerable to these drugs.

 Methotrexate or aminopterin (also used for psoriasis) – folate analog recall,

folic acid antagonist, blocks purine synthesis and therefore DNA synthesis.
Also used in cancer chemotherapy.
Figure 6.31, methotrexate and folic acid Figure 6.32, azathioprine

 Azathioprine or Imuran – nucleic acid analog- blocks purine production, given

right before and after transplant to suppress the immediate T-cell proliferation.

 Cytotoxic antibiotics – dactomycin (actinomycin D) – inhibits both

transcription and replication of DNA.

Figure 6.33 dactomycin Figure 6.34 cyclophosphamide

 Cyclophosphamide - disrupts DNA chain by crosslinking

Figure 6.35, IL-2 blockers

 c. IL-2 blockers - Cyclosporin (and related)

Interfere with production of IL-2 or its receptor.

Cyclosporin is a very strange fungal peptide, not synthesized on ribosomes and

contain a D (most are L) amino acid.

 cyclosporin A (CsA)
 FK506 (tacrolimus)
 Rapamycin (sirolimus)

d. Statins – block an enzyme I the pathway that produces cholesterol. Lipitor,

Crestor, Zocor.

They lower MHC II levels, shift the response into a more TH2 attack and
generally downregulate inflammation

B. T Cell Directed

1. antibodies against CD4

2. antibodies against CD40 ligand – Recall, this is a signal used by B cell and other
antigen presenter to up-regulate TH cells.

3. CTLA4-ab ties up B7 co stimulus. This targets newly activated cells.

4. Anti-IL2

5. Anti-CD3 – signaling peptide of the T cell receptor

6. Antibodies to specific T cell – much harder -have to develop for every individual.

C. Monoclonal Antibodies - often used to deplete mature T cells from a bone marrow
graft before transplant. As the new cells mature from stem cells, they do so in the
host environment and are more likely to accept it.

D. Suppressing Lymphoid Cells That Attack the Graft:

a. Total lymphoid irradiation

i. Irradiate all the lymphoid tissue (nodes, spleen, thymus) but NOT the bone
marrow.
ii. Graft.
iii. Allow the bone marrow stem cells to regenerate.
iv. This can also block B cell development via anergy, as there may not be up-
regulatory T cells.
 b. Plasmapheresis, which temporarily removes antigen-antibody complexes, and
clear out inflammatory signals. This works for several weeks and then gradually
your body resupplies the antibodies.

E. Aside on monoclonal antibody terminology.

Figure 6.36, monoclonal antibody naming conventions

1. Last syllable – mab – monoclonal antibody

2. The next to last syllable denotes the source (rat, human). For most drugs it will be zu,
which indicates a humanized antibody.

3. The next syllable up tells you the drug target – again, there’s a long list of designated
abbreviations, and tu refer to solid tumors.

4. The fourth (and first and sometimes one more) syllable in front, something arbitrary
that will supposedly to make it sound better.

F. Taking Advantage of Biology. Immune Tolerance to Allografts

1. Early exposure breeds tolerance (mice and cattle) Heart transplants in human infants.

2. Single mismatches may be tolerated if the mother has something the child does NOT,
as the child was exposed to this in utero.

3. "Immunologically Privileged" Sites: those without lymphatic vessels.

a. Corneal transplants, therefore, do not require tissue typing.
b. Suggests that you could protect Islet cells from the lymphoid tissues by
encapsulation and they would survive.

4. Differences in MHC I display level.

a. erythrocytes and sperm cells - no expression
b. liver, kidney and brain - much lower than immune cells
Video clip 6-4

IV. Examples of Transplants

A. Kidney

1. Most commonly transplanted organ, more than 30,000 in 2015. However, over
100,000 people are on waiting lists.

2. Kidneys are vulnerable. Recall how many diseases and the immune responses to the
disease result in kidney pathology. Also the kidney does not regenerate, and so
cannot recover from disease the way the liver and skin can.

3. Technically, the transplant procedure is relatively straightforward. You do not remove

the diseased kidney, but rather just hook up the new one to the ureter, and the arterial
and venous blood vessels.

4. Once a patient has rejected one kidney, he is more likely to reject subsequent
replacements. This makes careful tissue typing and cross-matching tests very
important subsequent transplants.

5. Patient must use immunosuppressant indefinitely, making them more vulnerable to

cancer, infections, and other problems.

6. “Domino Donation” - Most transplants come from cadavers or friends and relatives
giving a kidney directly to someone they know. However, sometimes no one has a
designated donor with a good match. One day someone had the bright idea of having
two donors give a kidney to the other members of a pair in situation where they each
matched the other’s recipient. Thus was born the idea for domino donation.

a. This begins with an “altruistic” or “undirected” donor, who simply decides to

make a kidney available for anyone who needs one. The family member who
wanted to donate to his relative (but couldn’t because of a bad match) then
donates a kidney to the next person. The sequence continues until you can no
longer find a good match for the next person who has a relative standing by. So
you pass the last kidney on to the next needy person on the wait list and the chain
ends.

b. So far, the longest chain of donation was 30 transplants involving 60 people.

c. Chains have gotten longer because the registries have expanded from regional to
national and because we use improved computer programs to check for good
matches that will allow continuation of al chain.
 Figure 6.37a, kidney transplant Figure 6.37b Tuomas Sandhorn

B. Corneas

1. First corneal transplant performed by Edouard Zirm in 1905

2. 40,000 per year
3. Wait time is 1-2 months
4. No tissue matching required

Figure 6.38, corneal transplant Figure 6.39, bone marrow

C. Bone Marrow

1. Requires careful HLA typing, and even then, unrelated donor tissue may have minor
differences that lead to incompatibilities.
 2. Requires immune suppression, although minor GVH can actually help kill remaining
cancer cells and recipient T cells that might produce an immune response. More
effective immune suppression has allowed doctors to experiment with some MHC
mismatches, but the whole procedure has an inherent rate of mortality of roughly
25%, so frankly, no one wants to add to the risk.

3. Marrow withdrawn from one person by multiple needle aspirations, possibly treated
in some way, and then transplanted into the patient simply by sticking it in an IV and
letting the cells find their way to the marrow site, which is currently been emptied out
by irradiation and cyclophosphamide.

D. Graft Versus Host: the transplanted bone marrow wages an immune attack on the entire
recipient.

1. Supply immuno-incompetent individual with competent lymphocytes from another

genetically different individual.

2. The Tc cells activate and begin to attack the host cells.

3. The B cells produce antibodies to the host cells surface antigen.

4. The patient experiences a cell-mediated attack from both NK and TC cells, with some
help from the macrophages, neutrophils and maybe even complement.

5. The cells under heaviest attack are GI epithelial (diarrhea), skin (which may slough
off in sheets), and spleen, (which may attack the red blood cells, resulting in
jaundice). The whole response can kill a person.

6. Spleen enlargement is generally used as a diagnostic indicator.

7. Autologous transplants may get around some of these problems.

Figure 6.40, heart Figure 6.41, heart and lungs

E. Heart : Most newsworthy and dramatic

1. Surgery is now established, and patients usually recover

2. Often not enough time and choice to do a good tissue match, so immune suppressants
are critical.

3. New hearts often get atherosclerosis, probably from inflammatory damage.

4. Hearts are often transplanted with lungs, which maintains the heart-lung circulatory
connections.

5. If you only need to replace the lungs, you can transplant a lobe from a living donor.

F. Liver: Liver transplantation comes with its own set of Good News/Bad News.

1. Liver cells have fewer MHC I molecules on their surfaces, and thus are somewhat
less likely to provoke rejection, and require less stringent tissue matching.

2. You can transfer single lobes of a liver from a living donor, and the donor will
regenerate the tissue.

3. Bad news, the surgery is very demanding, to a large degree because the circulatory
connection between the liver and the body are quite complex.

4. The operation is somewhat risky for the living donor. Serious complications are rare,
but donors have died as a result of this.

Figure 6.42, liver Figure 6.43, skin

G. Skin

1. Usually you can use autologous tissue. The means the grafts are typically successful,
although painful and taking a long time to heal.
 2. There are techniques that allow you to spread out skin from a donated region over a
larger area of wound (see figure 18.43).

3. In case of severe burns, however, allograft may be the only means of treatment. It’s
important to cover the site, even if the graft is later rejected. Dehydration and
infection are very real dangers.

4. Immunosuppression is particularly dangerous to a burn victim.

H. More experimental procedures

1. Islet cells
2. Testicles, ovaries and uterus
3. Faces
4. Hands

References:

ELISA assays: http://www.elisa-antibody.com/ELISA-Introduction/ELISA-types/direct-elisa

First Kidney transplant: http://www.npr.org/templates/story/story.php?storyId=4233669

Ross, Philip E. (2006) Putting Up with Self. Scientific American 295(6): 45-46. Denise
Faustman first transplants islets and then gets mice to regrow functioning islets.

Autologous bone marrow for people without matching donors

http://www.cancer.gov/clinicaltrials/EORTC-06861

Transplanting ABO-incompatible hearts in young infants:

http://www.ccs.ca/download/consensus_conference/consensus_conference_archives/2004_Pae
d.pdf (This links to something 101 pages long. The relevant info is on pages 16 to 18.)

Bone marrow for heart http://www.clinicaltrials.gov/ct/show/NCT00314366

Blood type switch after transplant http://www.physorg.com/news120396291.html and

http://news.yahoo.com/s/afp/20080124/ts_afp/australiahealthchildrentransplant
Glossary

· Actinomycin D- antibiotic that blocks RNA and DNA synthesis.

· Adaptive cells – those that rearrange their genes.

· ADCC- antibody dependent cell-mediated cytotoxicity. A process by which white blood

cells recognize the stems of antibodies attached to a cell and then attack it.

· Allele- a version of the gene. There are two alleles for the enzyme that produces color in
four o’clock flowers, one that codes for an enzyme used to make red pigment and a different
DNA sequence that does not produce a functional enzyme, leaving the flower white.

· Allograft – transfer of tissue or organs from a member of a species to another member of

the same species.

· ALT – associated lymphoid tissue. MALT (mucosal), GALT (gut), BALT (bronchial),
NALT (nasal).

· Aminopterin (methotrexate) – drug that blocks DNA synthesis and thus interferes with
cell division.

· Anaphylaxis – a state of shock whereby the blood pressure drops because of excess TH2
response trigger by a Type I hypersensitivity reaction.

· Anergy- a state where an adaptive immune cell is quiescent and unresponsive

· Antibody- a soluble immunoglobulin.

· Antigen- a molecule that can bind to an antibody, B cell receptor or T cell receptor.

· APC – antigen presenting cell. Cells that present antigen on MHC II to TH cells.

· Apoptosis – programmed cell death.

· Apoptosome – heptameric complex assembled to initiate apoptosis

· ATP- adenosine triphosphate, directly supplies energy to many biological reactions.

· Autograft – transfer of tissue or organs from a one part of a patient’s body to another.

· B7 – an important juxtacrine signal from APCs to TH cells.

· Basophils– myeloid lymphocytes important in initiating Th2 responses. They have

granules that pick up basic stains and IgE surface receptors, and circulate in the plasma.

· BSA – bovine serum albumin. A smallish soluble protein isolated from cow’s blood.

· Calnexin- first MHC I chaperone, displaced by beta macroglobulin.

· Calreticulin- binds to tapasin- MHC I complex

· CAM – cell adhesion molecule. Any one of a number of different molecules that help
stick cells together.

· Centromere- the region on a chromosome that attaches to the spindle during mitosis.

· CD – cluster of differentiation. Refers to the isolation of cells by flow cytometry

depending on exactly what proteins extend from a cell’s surface. This in turn influences how
the cell moves during the separation process. Recall that these are applied solely on the basis
of the order of discovery.

· CD1 (A through E) - the peptides or genes that code for them that bind lipids to present
them to T cells.

· CD3- the peptides that associate with the TCR for signaling.

· CD4- the co-receptor that helps TH cells bind to MHC II.

· CD8- the co-receptor that helps TC cells bind to MHC I

· CDR- complementarity determining region- the recognition side on the tips of the
antibody arms.

· Chagas Disease- caused by a trypanosome (unicellular eukaryote) that is spread by an

insect vector. Changes surface coats and causes down-regulated IL-2.

· Chitin – cell wall material of fungi, also an important component of insect exoskeletons.

· Cholecalciferol – vitamin D

· Chordate – member of the phylum Chordata. Includes vertebrates and invertebrates with
a dorsal nerve cord, gill slits, notochord and muscles in blocks.

· Choroid plexus- region of brain that produces cerebrospinal fluid by exchange across a
capillary to epithelia.

· Clip- portion remaining after hydrolysis of the invariant chain.

· CLP – common lymphoid progenitor. Gives rise to lymphoid cells: NK, T cells, B cells,
and more.

· Coley toxin – inflammatory material isolated from bacteria used in cancer chemotherapy
around 1900.

· Complement- a system of proteins that helps identify pathogens and debris for destruction and
phagocytosis (the landmines of the plasma).

· Crohn’s Disease- inflammatory bowel disease that can affect the entire GI tract

· CTL – Cytotoxic T cell. Activated TC cell, ready to kill rogue-self cells.

· Cyclosporin A – fungal peptide that blocks the action of IL-2.

· Cyclophosphamide – Inhibits DNA synthesis by crosslinking DNA strands.

· Cytostatic – general term for any compound that interferes with DNA replication and/or
cell division

· Dactomycin – see actinomycin D

· DAG- diacyl glycerol. Two fatty acids joined by ester condensation bonds to glycerol,
produced by clipping a phospholipid.

· DAMPS (death or damage-associated molecular proteins) - molecules that are

normal cell components but should be inside the cell, not identifiable on the exterior.

· Dexamethasone- an anti-inflammatory steroid drug.

· Down-regulate- turn off cellular processes (often a result of negative feedback).

· Delta-gamma T cell – a T cell with a quasi-innate γδ receptor. While they rearrange the
genes for these receptors while developing in the thymus, they have a wider range of function
than other T cells and usually target epithelial, in order to patrol the body’s boundaries.

· Downstream- the end of the DNA or RNA with the free 3’ carbon of the (deoxy) ribose.
Nucleic acid synthesis and translation proceeds 5’ to 3’.

· DN 1 through 4- stages of T cell development before cells express either CD4 or CD8.

· Domino donation – a system of lining up kidney transplant donors and recipients, such
that an initial unrelated donor starts gives a kidney to someone who needs it and then that
person’s donor (who doesn’t match him) gives a kidney to an unrelated person whose willing
donor doesn’t match her, but who does match a different person, and so on.

· ELISA assays- Reaction to determine the concentration of some substance that depend on
recognition by an antibody and development of a color based on subsequent reaction of an
enzyme with a substrate.

· Early genes- genes for signaling proteins and receptors activated within hours after T-cell
activation.

· Eczema – allergic skin rash cause by Type I hypersensitivity – Th2 response.

· Edema – tissue swelling due to accumulation of interstitial fluid derived from plasma.

· Epitope – the specific portion of a molecule that binds to an adaptive receptor. For
example, a viral protein is an antigen whose different epitopes bind to different antibody
idiotypes.

· ERp57- final chaperone binding to MHC I prior to antigen binding.

· Exon- the part of a gene that codes for a sequence of RNA that will wind up in a message
and get translated (expressed.) A gene or gene region may have one too many exons.
· FAS/FAS ligand- A tripartite juxtacrine signal set, used by immune cells to induce
apoptosis in damaged cells.

 Fezf2: a transcription factor promoting expression of tissue-specific antigen in the

thymic medulla

· G protein (trimeric) – an important internal signal transducer. It responds to receptor

activation by exchanging GTP for GDP and triggering a signal cascade to the cell’s interior.

· Gamma-delta T cells- those with the quasi-innate gamma-delta receptor, involved in

boundary defense.

· Gene region – a sequence of DNA coding for a specific part of the Ig or T-cell receptor.

· Glomerulus – cluster of capillaries that promotes fluid transfer from the blood to the
kidney.

· Goldilocks Principle- a general principle that in many biological system the intermediate
value of a process or level or a signal is most effective.

· Granulocytes – Cells with copious granular inclusions and that do not present antigen.
Includes neutrophils, basophils, and eosinophils (which have oddly-shaped nuclei) and mast
cells (which do not).

· Granuloma (also called tubercle) – a nodule of macrophages and specialized TH

cells surrounding a walling off macrophages infected with intracellular bacteria.

· Haplotype- a group of genes next to each other on a chromosome that tend to be inherited
as a block.

· Hapten- a molecule that could potentially bind a CDR, but by itself is not large enough to
kick off an immune response.

· HLA- human lymphocyte antigen, the MHC types of humans.

· Helix-loop-helix – a general category of transcription factors that function as dimers. The

monomers have two runs of alpha helix, separated by a loop of random coil of varying
lengths.

· HLA-DM – helps load MHC II by displacing clip.

· HSC- hematopoietic stem cell. Can divide and regenerate of develop into any type of
blood cell. Found in bone marrow.

· Humoral response – Immune defense found in the plasma, the word humor derived from
the ancient Greek medical theory of body fluids; it really just refers to antibodies. Stupid
term. If people stop using it, maybe it will go away.

· Hybridoma- a cell or cell line derived for the fusion of a blood cell cancer (myeloma) and
a normal, antibody-producing plasma (B lineage) cell.
· Hygiene hypothesis – suggests that growing up in a very clean environment potentiates
allergic reactions

· Idiotype – a category of antibodies that all have the same recognition region

· IL (-1, -2, -4, -5, etc.) – interleukins- a general name for a heterogeneous collection of
paracrine signaling molecules. IL-1 is the principle initiator of the overall immune response.
IL-2 initiates the adaptive immune response.

· Immediate genes- T cell activation gene for transcription factors, activated in minutes.

· Inflammasome – heptameric protein complex assembled to activate inflammatory

cytokines via hydrolysis.

· inflammation- a state of immune up-regulation characterized by changes in blood vessels

adhesion and permeability, increased temperature, phagocyte activation and often pain.

· iNKT cells – quasi –innate T cells that bind to lipids.

· Integrin- proteins that spans the plasma membrane, connecting to the cytoskeleton at the
interior and capable of binding a variety of extracellular proteins at the exterior.

· Innate cells – those that do not rearrange genes.

· Introns- that DNA sequences of the gene that code for RNA sequences that get clipped out
during processing.

· Invariant chain- MHC II chaperone.

· IP2 or IP3- inositol with two or three phosphates. Produced by clipping inositol from a
phospholipid and adding one or two phosphates to it (It has one to begin with).

· Isograft – transfer of tissue or organs from one individual to different individual of the
genotype.

· Isotype- a category of antibodies of the same class.

· Janus kinase- two-faced receptor. The signaling domains phosphorylate each other during
signaling.

· Kinase- and enzyme that adds phosphate to proteins or other molecules.

· Late genes- gene for CAMS up-regulated by T cell stimulus. These take days to activate.

· Leprosy- a relatively non-contagious bacterial disease. An ineffective immune response

results in the disfiguring loss of extremities.

· Lipid raft- a patch of plasma membrane with a distinct lipid composition and a more rigid,
thicker structure. Helps to separate different functional membrane protein from each other.

· LMP 2 and 7- proteins that determine the peptide length of peptide hydrolyzed in the
proteasome.
· Luminex assay- test for likelihood of organ rejection by measuring the presence of
antibodies to the transplant present in the recipient prior to transplant.

· Lymphoid cells – white blood cell types (innate and adaptive) found in the lymph and
(and blood and immune organs as well).

· MAC- membrane attack complex- terminal complement pathway produces this, which
punches holes in plasma membranes.

· MASP- mannose associated protein. Associated with lectin pathway of complement.

· Mast cells – myeloid lymphocytes important in initiating Th2 responses. They have
granules contain histamine and heparin and signal mucus release and tearing, usually found
localized in boundary tissues and around the circulatory system.

· MBL- mannose binding lectin. Associated with lectin pathway of complement.

· Methotrexate (aminopterin) – drug that blocks DNA synthesis and thus interferes with cell
division.

· MHC- Major Histocompatibility Complex. Includes the genes and the proteins they code
for. These include the proteins (groups I and II) that hold small peptides so that T cells can
recognize them. They also include a variety of other proteins, including enzymes important in
immune recognition and promotion. The human versions are named HLA molecules for
human lymphocyte antigen.

· Mixed lymphocyte reaction- test for likelihood of organ rejection by measuring the
sensitivity of T cells present in the recipient that react to the cells from the donor.

· Monoclonal- refers to a cell line of (theoretically) identical cells derived from the division
of a single cell.

· Mucin- CAM with proteins attached to extensive amounts of branched carbohydrate

chains, ending in sialic acid.

· Myasthenia gravis- autoimmune disease characterized by production of antibodies to

muscle acetylcholine receptors, resulting in muscle weakness.

· Myelin – cells associated with the axons of nerve cells, insulating them and improving
their rate of impulse conduction.

· Myeloid cells – innate white blood cells rarely found in the lymph.

· Necrosis- cell death from disease or injury.

· NFAT- inflammatory transcription factor activated by phosphatases in turn activated by

MAP kinases.

· NFκB- inflammatory transcription factor activated by phosphatases in turn activated by

MAP kinases.
· NK cell- Natural Killer cell. Kills rogue-self cells, recognizing them by innate
mechanisms. Does not require TH activation.

· N-nucleotide addition- During gene rearrangement, when enzymes add nucleotides at

random in the palindromic regions of the joint.

· NOD – nucleotide oligomer detectors. Soluble pattern recognition receptors found in the
cytoplasm of cells. Despite the name, they often recognize cell wall materials.

· NSAID- non-steroidal anti-inflammatory.

· Nucleic acid – RNA or DNA.

· PAMPS (pattern-associated molecular proteins) - generally used to describe a

variety of molecules characteristic of proteins- recognized by PRRs such as TLRs.

· Penicillin – a small organic molecule that functions as an antibiotic by interfering with

bacterial cell wall production.

· Peptidoglycan – mesh-like macromolecules that compose the basic structure of the

bacterial cell wall.

· Perforins- monomer secreted by NK and Tc cells that assemble into pores that construct
holes in the plasma membranes of self-cells under immune attack. Resemble the pores
produced by complement C9.

· Phagocytosis – when a cell engulfs large particles.

· Pinocytosis - when a cell gathers fluid in a vesicle and engulfs the vesicle.

· Plasma cell- activated antigen-secreting B cell.

· Plasmapheresis- technique for removing proteins, particularly antibodies from whole

blood

· P-nucleotide addition- During gene rearrangement, when enzymes fill in the missing
nucleotides at the joint by copying the palindromic nucleotides on the other strand.

· Prednisone- an anti-inflammatory steroid drug.

· Proteasome- organelle responsible for hydrolyzing and recycling cytosolic proteins.

· Psoriasis- skin lesion caused by excessive cell division in epidermis. Characterized by

inflammation.

· Psoriacin – innate defense compound secreted by the skin to protect from E. coli.

· Receptor-mediated endocytosis – when a cell binds material at its surface using a proteins
receptor and then internalized the complex into a vesicle that enters the cell.

· RAG enzymes- those responsible for gene rearrangement (related to gene used during
meiosis for gene recombination).
· Rheumatoid arthritis – autoimmune disease characterized by the production of antibodies
to antibodies, resulting in widespread tissue damaged and deformed joints.

· RIA (radioimmune assay) - assay based on displacement of a radioactive molecule from

an antibody CDR by the cold version of this molecule found in a patient’s blood or urine.

· RSS – recombination signal sequence. The sequence of 28 or 40 nucleotides that the

upstream or downstream end of a gene region providing the signals for gene rearrangement.

· Selectin- class of CAMS that have a lectin domain that bind to mucins carbohydrates.

· Simple sugar – single sugar unit, includes glucose, mannose and galactose. May be
modified into sugar units as sialic acid (NANA) or N-acetyl glucosamine.

· STAT- transcription factor activated by phosphorylation and subsequent dimerization

(Signal Transduction Activator of Transcription). Typically activated by Janus kinases.

· Statins- drugs designed to lower blood cholesterol. The also lower inflammation.

· Systemic Lupus Erythematosus (SLE or lupus) – autoimmune disease characterized by

production of antibodies to a wide variety of self antigens.

· TAP 1 and 2- transport peptides into the ER using ATP.

· Tapasin- bring TAP to MHC I.

· TC cell- cells that recognize rogue self-cells by antigen they present on MHC I. They
develop into CTLs after instructions from TH cells.

· TCR – T-cell receptor. Found extending from the surface of both TC and TH cells.
Recognizes antigen, coded for by rearranged genes.

· TH cell- thyroid helper cell. Coordinates immune responses. TH 1 cells promote a serious
response; TH 2 promotes a containment response, TH17 promotes inflammation and boundary
defense, and Treg tolerance. There are additional types as well.

· TLR – Toll-like receptors. Pattern recognition receptors that recognize molecules

characteristic of pathogens. Found embedded in plasma membrane and endomembranes of
many white blood cells.

· Tolerance – active, specific suppression of immune responses against harmless antigens,

as opposed to immune deficiency, which is a general non-strategic immune impairment.

· Transcription factor – a protein that either up- or down- regulates the copying of RNA
(transcription) from DNA. They often have domains that attach to specific sequences of
DNA nucleotides. Some attach to other proteins that attach to the DNA. Or both.

· Trastuzumab (Herceptin) – a monoclonal antibody used to treat breast cancer by blocking

the cells’ growth factor receptors.
· Ubiquitin – protein attached to other proteins to target them for degradation in the
proteasome.

· Ulcerative colitis - inflammatory bowel disease affecting only the colon

· Up-regulate- turn on pathways (sometimes related to positive feedback).

· Upstream- the end of the DNA or RNA with the free 5’ carbon of the (deoxy) ribose.
Nucleic acid synthesis and translation proceeds 5’ to 3’.

· Winged helix transcription factor- DNA-binding protein that recognizes nucleotide

sequences with alpha helices and extends random coil loops from the DNA axis. Also called
“forked – head” because of the phenotypic effects of the mutant form on fruit flies.

· Xenograft – transfer of tissue or organs from a member of a species to another member of

a different species.

· Zymosan – cell wall material of fungi

 Figure Attributions
Owl Title figures: Alma Novotny
Page 3 overleaf, Alma Novotny © 2015
Figure 1-1 developmental landscape C. Waddington
Figure 1-2 a and b Yan Jun (Daisy) Chung
Figure 1-3 Yan Jun (Daisy) Chung
Figure 1-4 Yan Jun (Daisy) Chung, modified by Alma NovotnyFigure 2 - 9 Daisy Chung © 2013
Figure 1-5 skin Shutterstock
Figure 1-6 lung Bill Blaus wiki
Figure 1-7 gut lining Anna Troshkina
Figure 1-8 composite, immunology artists
Figure 1-9 composite, immunology artists
Figure 1-10 Yan Jun (Daisy) Chung
Figure 1-11 Yan Jun (Daisy) Chung
Figure 1-12 WikiCommons
Figure 1-13 Daisy Chung © 2013
Figure 1-14 Anna Troshkina © 2014
Figure 1-15 Daisy Chung © 2013
Figure 1-16 Anna Troshkina © 2014
Figure 1-17 Daisy Chung © 2013
Figure 1-18 Daisy Chung © 2013
Figure 1-19 Jordan Orange
Figure 1-20 Daisy Chung © 2013
Figure 1- 21 Daisy Chung © 2013
Figure 1- 22 Daisy Chung © 2013
Figure 1- 23 Satchmo2000 WikiCommons https://
commons.wikimedia.org/wiki/File:ADCC.svg
Figure 1-24 NIH WikiCommons https://commons.
wikimedia.org/wiki/File:MRSA,_
Ingestion_by_Neutrophil.jpg
Lecture 3-2, background figures Anti-inflammatory medications
Tashatuvango Shutterstock292005323
Figure 2 - 1 Soota Shutterstock# 50611630
Figure 2 - 2 Burns Archive WikiCommons
https://en.wikipedia.org/wiki/Bloodletting#/media/
File:BloodlettingPhoto.jpg
Figure 2 - 3 Ramin Herati WikiCommons
http://en.wikipedia.org/wiki/Chemokine#mediaviewer/File:
IL8_Solution_Structure.rsh.png
Figure 2 - 4 Bensaccount WikiCommons
http://en.wikipedia.org/wiki/G_protein-coupled_receptor#
mediaviewer/File:7TM4_(GPCR).png
Figure 2 - 5 Neurotiker WikiCommons https://en.wikipedia.
org/wiki/G_protein#mediaviewer/File:
Guanosindiphosphat_protoniert.svg
Figure 2 – 6 Sven Jähnichen
http://en.wikipedia.org/wiki/File:GPCR-Zyklus.png
Figure 2-7. A, B, C and D Anna Troshkina © 2014
Figure 2 - 8 Anna Troshkina © 2014
Figure 2 - 10 Daisy Chung © 2013
Figure 2-11 Anna Troshkina
Figure 2-12 AnnaTroskina
Figure 2 - 13 boghog2 WikiCommons
http://en.wikipedia.org/wiki/Interleukin_1#mediaviewer/ File:2ILA.png
Figure 2 - 14 Daisy Chung © 2013
Figure 2- 15 Edward Novotny © 2015
Figure 2 - 16 Edward Novotny © 2015
Figure 2 – 17 Edward Novotny © 2015
Figure 2 - 18 Edward Novotny © 2015
Figure 2 - 19 WikiCommons https://en.wikipedia.org/wiki/
Platelet#/media/File:Red_White_Blood_cells.jpg
Figure 2 - 20 molekuul.be Shutterstock 120616438
Figure 2 - 21 Somersalt1824 Shutterstock 156803315
Figure 2 - 22 Yikrazuul WikiCommons https://en.wikipedia.org/wiki/
Bradykinin#/media/File:Bradykinin_structure.svg
Figure 2 - 23 Phospholipid_TvanBrussel.jpg WikiCommons
Figure 2 - 24 WikiCommons
Figure 2 - 25 Edward the Confessor WikiCommons
https://en.wikipedia.org/wiki/Arachidonic_acid#/media/File:Arachidonic
acid.svg
Figure 2 - 26 Foobar WikiCommons https://commons.wikimedia.org/
wiki/File: Thromboxane_A2.png
Figure 2 - 26 Bimatoprost.svg WikiCommons
Figure 2 - 27 WikiCommons
Lecture 3-3 Background figures:
Apoptosis Shutterstock #119629432
Trypanosome, www.royaltystockophoto.com Shutterstock
# 245322619
Bone marrow harvest, Li Wa, Shutterstock #5649271
Apoptosis Shutterstock #119629432

Figure 3-1 Pumbaa80
Figure 3-2 en:User:AudioPervert (Wiki Commons)
Figure 3-3a UV – NASA stereo project
Figure 3-3b UV damage Mouagip
Figure 3-4 pixabay – science pics2016
Figure 3-5JonSDSUGrad
Figure 3-6 jeff brender (biophysik)
Figure 3-7 Alma Novotny
Figure 3-8 Shutterstock. ID
#106535432 Credit: Alila Medical Media
Figure 3-9 MLGProGamer123
Figure 3-10 Alma Novotny from Yan Jun (Daisy)
Chung
Figure 3-11 The cat~commonswiki
Figure 3-12 Boghog2 (talk)
Figure 3-13 Edward Novotny 2016
Figure 3-14 Edward Novotny 2016
Figure 3-15 Edward Novotny 2016
Figure 3-16 Edward Novotny 2016
Figure 3-17 Edward Novotny 2016
Figure 3-18 Edward Novotny 2016
Figure 3-19 Edward Novotny
Figure 3-20 BogHog
Figure 3-21 upload.wikimedia.org/
wikipedia/commons/1/1b/1SVC.png
Figure 3-22 Alma Novotny 2016
Figure 3-23 Yan Jun (Daisy) Chung
Figure 3-24 designua, shutterstock 186874943
Figure 3-25 MadanaM WikiCommons http:
//en.wikipedia.org/wiki/File:63-T-CellReceptor-
MHC.tif
Figure 3-26 Gonn WikiCommons
http://en.wikipedia.org/wiki/
File:PBB_Protein_AIRE_image.jpg
Figure 3-27 Volodymyr Krasyuk
Shutterstock 118547506
Figure 3-28 Thomas
Splettstoesser (www.scistyle.com
Figure 3-29 Thomas Splettstoesser WikiCommons
Figure 3-30 Alma Novotny
Figure 3-31 pixabay public domai
Figure 3-32 raining128
Figure 3-33 Tom Adriaenssen –
http://www.flickr.com/photos/inferis/110652572/
Figure 3-34 Stanisław Wyspiański – http:
//cyfrowe.mnw.art.pl/dmuseion/docmetadata?id=3983
Figure 3-35 Yan Jun (Daisy) Chung
Figure 3-36 pixabay public domain
Figure 3-37 Edward Novotny 2016
Figure 3-38 Edward Novotny 2016
Figure 3-39 Edward Novotny 2016
Figure 3-40 Anna Troshkina
Figure 3-41 (A) Allison Skinkle 2016
Figure 3-41 (B) NIH, WikiCommons
Figure 3-42 Alma Novotny 2016
Figure 3-43 Shimon Sakaguchi 2016
Figure 3-43 Anna Troshkina 2014
Figure 3-43 (A) Ramin Herati
Figure 3-44 (B) Anna Troshkina 2014
Figure 3-45 designua, shutterstock186324788
Figure 3-46 Alila Medical Media
shutterstock_100625044
Figure 3-47 A2-33
Figure 3-48 Jawahar Swaminathan and MSD staff
Figure 3-49 Lucia Lee
Figure 3-50 Anna Troshkina
Figure 3-51 Yan Jun (Daisy) Chung
Figure 3-52 Yan Jun (Daisy) Chung
Figure 3-53 Alma Novotny
Figure 3-54 a. Richard Wheeler (Wiki Commons)
b and c Alma Novotny
Figure 3-55 Anna Troshkina
Figure 3-56 Anna Troshkina
Figure 3-57 Alma Novotny
Figure 3-58 Alma Novotny
Figure 3-59 Alma Novotny
Figure 3-60 Jawahar Swaminathan and MSD staff
Figure 3-61 Anna Troshkina 2014
Figure 3-62 Tomatito, Shutterstock 134872160
Figure 3-63 Alma Novotny
Figure 3-64 Alma Novotny
Figure 4-1 Doktorinternet
Figure 4-2 James Heilman, MD
Figure 4-3 joshya, shutterstock
Figure 4-5 KGH. Wiki Commons
Figure 4-6 Bruce Blausen
Figure 4-7 bruker.com
Figure 4-9 ellepigraphia, Shutterstock 253141705
Figure 4-10 designua, pixaby 239380201
Figure 4-11 Alma Novotny
Figure 4-12a homosalate – Edgar 181
Figure 4-13b octisalate - Jü, wiki commons
Figure 4-13 Demitri Dale
Figure 4-14 Shutterstock. ID
#79599691 Credit: Alex Luengo
Figure 4-15 Hans Braxmeier, Pixabay
Figure 4-16 Emw, CC BY-SA 3.0
Figure 4-17 BallenaBlanca
Figure 4-18 KGH, CC BY-SA 3.0
Figure 4-19 Yan Jun (Daisy) Chung
Figure 4-20 Yan Jun (Daisy) Chung
Figure 4-21 Yan Jun (Daisy) Chung
Figure 4-22 Yan Jun (Daisy) Chung
Figure 4-23 Alma Novotny
Figure 4-24 Calvero
Figure 4-25 wiki common public domain
Figure 4-26 David-i98
Figure 4-27 Bryan Derksin, Wiki commons
Figure 4-28 Hans Braxmeier, Pixabay
Figure 5-1 hookworm, CDCs
Figure 5-2 Pixabay public domain
Figure 5-3 Juan Gaertner
Figure 5-4 public domain
Figure 5-5 NIH, Wiki Commons
Figure 5-6 Yale Rosen, Wiki Commons
Figure 5-7 Mikael Häggström, Wiki Commons
Figure 5-8 CC BY-SA 3.0, Wiki Commons
Figure 5-9 Yan Jun (Daisy) Chung
Figure 5-10 Yan Jun (Daisy) Chung
Figure 5-11 Yan Jun (Daisy) Chung
Figure 5-12 Kauczuk, Wiki Commons
Figure 5-13 User CS99 at German Wikipedia
Figure 5-14 Alma Novotny
Figure 5-15 Edward Novotny
Figure 5-16 Edward Novotny
Figure 5-17 Edward Novotny
Figure 5-18 Alma Novotny
Figure 5-19 wiki public domain
Figure 5-20 Bimatoprost.svg WikiCommons
Figure 5-21 Calvero Wiki
commons
Figure 5-22 Ramin Herati
Figure 5-23 Wolfgan Ihloft, Wiki Commons
Figure 5-24 Jan Polak wiki Commons
Figure 5-25 SabanSari
Figure 5-26 public domain
Figure 5-27 (A) fotoarwin, pixabay
 Figure 5-27 (B) Sebastian Kaulitz, Shutterstock
43984414
Figure 5-28 Wiki, public domain
Figure 5-29 Ernst Hempelmann Wiki, public
domain
Figure 5-30 Alma Novotny
Figure 5-31 OpenStax College with Lucia Lee
Figure 5-32 OpenStax College with Lucia Lee
Figure 5-33 OpenStax College with Lucia Lee
Figure 5-34 OpenStax College with Lucia Lee
Figure 5-35 Shutterstock 152927564
Figure 5-36 Alma Novotny from Yan Jun (Daisy)
Chung and Lucia Lee
Figure 5-37 EmBaSy, Shutterstock 327441755
Figure 5-38 (A) Anatomist90, Wiki Commons
Figure 5-38 (B) from Henry Vandyke Carter,
Wiki Commons
Figure 5-39 Edward Novotny from Yan Jun
(Daisy) Chung
Figure 5-40 Yan Jun (Daisy) Chung
Figure 5-41 Yan Jun (Daisy) Chung
Figure 5-42 Yan Jun (Daisy) Chung
Figure 5-43 Yan Jun (Daisy) Chung
Figure 5-44 Yan Jun (Daisy) Chung
Figure 5-45 Yan Jun (Daisy) Chung
Figure 5-46 Mutleysmith Wiki Commons
Figure 5-47 Alborz Fallah, Wiki Commons
Figure5-48 Marvin 101, Wiki Commons
Figure 5-49 Yan Jun (Daisy) Chung
Figure 5-50 (A) Dr. Ray Butler; Janice Carr, Wiki
Commons
Figure 5-50 (B) Mutleysmith, Wiki Commons
Figure 5-50 (C) Greg Knobloch, Wiki Commons
Figure5-51 Yikrazuul, Wiki Commons
Figure 5-52 Calibuon at English Wikibooks
Figure 5-53 Nobelprize.org
 Background 18- intro Ziko van Dijk Figure 6-34 NEUROtiker, Wiki Common
Figure 6-1 Yan Jun (Daisy) Chung Figure 6-35 (C) Fvasconcellos, Wiki Commons
Figure 6-2 Alma Novotny from Yan Jun (Daisy) Figure 6-36 Alma Novotny
Chung Figure 6-37a Bruce Blaus
Figure 6-37b – Carnegie Mellon University
Figure 6-3 Alma Novotny from Lucia Lee Figure 6-38 ARZTSAMUI, Shutterstock
Figure 6-4 Alma Novotny from Lucia Lee 255479821
Figure 6-5 Alma Novotny from Lucia Lee Figure 6-39 Li Wa, Shutterstock 99952475
Figure 6-6 Alma Novotny Figure 6-40 Africa Studio, Shutterstock
355383209
Figure 6-7 Public Domain
Figure 6-41National Heart Lung and Blood
Figure 6-8 Alma Novotny from Lucia Lee Institute (NIH), Public Domain
Figure 6-9 Alma Novotny from Lucia Lee Figure 6-42 Suseno, Wiki Commons
Figure 6-10 Alma Novotny Figure 6-43 MedlinePlus, Wiki Commons
Figure 6-11 Edward Novotny from Lucia Lee
Figure 6-12 Edward Novotny from Lucia Lee
Figure 6-13 OraSure Technologies
Figure 6-14 Edward Novotny from Lucia Lee
Figure 6-15 Edward Novotny from Lucia Lee
Figure 6-16 Edward Novotny from Lucia Lee
Figure 6-17 Edward Novotny from Lucia Lee
Figure 6-18 (A) MedlinePlus
 Figure 6-18 (B) LiWa, Shutterstock 99952475

Figure 6-18 (C) Blausen Medical

Communications, Inc
Figure 6-19 Pixabay public domain
Figure 6-20 (A) Pixabay public domain
Figure 6-20 (B) , kontur-vid Shutterstock
#57341071
Figure 6-21(A) Pixabay
Figure 6-21 (B) HIA Wiki Commons
Figure 6-22 Alma Novotny from Yan Jun
(Daisy) Chung and Anna Troskina
Figure 6-23 Alma Novotny
Figure 6-24 Alma Novotny
Figure 6-25 Alma Novotny
Figure 6-26 Alma Novotny
Figure 6-27 Alma Novotny
Figure 6-28 Edward Novotny
Figure 6-29 (A) Francis A. Countway Library of
Medicine, Wiki Commons
Figure 6-29 (B) Alma Novotny
Figure 6-30 Edgar 181, Wiki Commons
Figure 6-31 Yikrazuul, Wiki Commons
Figure 6-32 Ayacop – PubChem, Wiki Commons
Figure 6-33 Edgar 181, Wiki Commons