CHAPTER 16

HYPERSENSITIVE REACTIONS
CLINICAL FOCUS QUESTION: Discuss why IL-4 and FcεRIβ are excellent candidate
genes involved in the genetic susceptibility to asthma.
A. IL-4 and FcεRIβ are considered excellent candidate genes in the genetic
susceptibility to asthma because they play important roles in the immune response and
allergic reactions. IL-4 is a cytokine that is involved in the production of IgE antibodies,
which are central to the development of allergic reactions. FcεRIβ is a subunit of the
high-affinity IgE receptor, which is expressed on mast cells and basophils and is
responsible for the release of inflammatory mediators in response to IgE binding.
Variations in these genes can affect the production and function of IgE antibodies,
leading to an increased risk of developing asthma.

1. Indicate whether each of the following statements is true or false. If you think a
statement is false, explain why.

a. Mice infected with Nippostrongylus brasiliensis exhibit decreased production of

IgE.
b. IL-4 decreases IgE production by B cells.
c. The initial step in the process of mast-cell degranulation is crosslinking of Fc
receptors.
d. Antihistamines are effective for the treatment of type III hypersensitivity.
e. Most pollen allergens contain a single allergenic component.
f. Babies can acquire IgE-mediated allergies by passive transfer of maternal
antibody.
g. Transfusion reactions are a manifestation of type II hypersensitivity.

A.

a. False. Mice infected with Nippostrongylus brasiliensis typically exhibit

increased production of IgE, not decreased production. IgE is associated with
immune responses against parasitic infections.
 b. False. IL-4 actually stimulates IgE production by B cells. It is a key cytokine
involved in promoting class switching to IgE in B cells.
c. True. The initial step in mast-cell degranulation is the crosslinking of Fc
receptors on the mast cell surface by the binding of allergen-specific IgE
antibodies.
d. False. Antihistamines are more commonly used to treat type I hypersensitivity
reactions, where histamine release is a major factor. Type III hypersensitivity
involves immune complex formation and is not typically treated with
antihistamines.
e. False. Most pollen allergens are complex and contain multiple allergenic
components. These components can vary in their allergenic potential, leading to
different reactions in individuals.
f. True. Babies can acquire IgE-mediated allergies by passive transfer of
maternal IgE antibodies through the placenta or breast milk. This can predispose
them to allergic reactions until their own immune systems mature.
g. False. Transfusion reactions are usually associated with type II
hypersensitivity, where antibodies target antigens on the surface of transfused
cells, leading to cell destruction.

2. In an immunology laboratory exercise, you are studying the response of mice injected
intradermally with complete antibodies to the IgE Fc receptor (FcεR1) or with Fab
fragments of such antibodies.

a. Predict the response expected with each type of antibody.

b. Would the responses observed depend on whether the mice were allergic?
Explain.

A.

a. PREDICTED RESPONSES:

Complete Antibodies to FcεR1: Injecting mice with complete antibodies against the
IgE Fc receptor (FcεR1) could lead to crosslinking of the receptors on mast cells and
basophils, triggering degranulation and release of histamines, cytokines, and other
mediators. This response is similar to an allergic reaction and could cause symptoms
like itching, redness, and inflammation.

Fab Fragments of Antibodies to FcεR1: Injecting mice with Fab fragments of

antibodies against FcεR1 might not lead to extensive crosslinking of receptors since
Fab fragments lack the Fc region responsible for binding to other antibodies. This may
result in a milder or even no allergic-like response.

b. The responses observed could be influenced by whether the mice are allergic. In
allergic individuals, exposure to an allergen can lead to the sensitization of their immune
system, including increased production of IgE antibodies specific to that allergen. These
IgE antibodies bind to FcεR1 receptors on mast cells and basophils. Upon subsequent
exposure to the allergen, the allergen binds to the IgE antibodies, causing crosslinking
of FcεR1 receptors and triggering degranulation.

If the mice are allergic to the antigen recognized by the FcεR1 antibodies, they would
likely exhibit a stronger and more immediate response upon injection of either complete
antibodies or Fab fragments, since they already have a pool of IgE antibodies specific to
the allergen. This would lead to degranulation and allergic-like symptoms. In non-
allergic mice, the response might be weaker or absent, particularly with Fab fragments,
as there may be fewer or no specific IgE antibodies to trigger the reaction.

In summary, the responses observed would likely be more pronounced and allergy-like
in mice that are allergic to the antigen recognized by the antibodies being injected.

3. Serum sickness can result when an individual is given a large dose of antiserum such
as a mouse antitoxin to snake venom. How could you take advantage of recent
technological advances to produce an antitoxin that would not produce serum sickness
in patients who receive it?
A. Recent technological advances can be utilized to produce an antitoxin that does not
cause serum sickness in patients who receive it by using genetic engineering
techniques. One approach is to humanize the antibodies by replacing the mouse
framework regions with human framework sequences. This creates a mouse/human
chimeric monoclonal antibody that is less likely to be recognized as foreign by the
human immune system. These antibodies can bind to free IgE and down-regulate IgE
production, reducing the sensitivity of basophils and preventing serum sickness in
patients.

4. What immunologic mechanisms most likely account for a person’s developing each of
the following reactions after an insect bite?

a. Within 1–2 min after being bitten, swelling and redness appear at the site and
then disappear by 1 h.
b. 6–8 h later, swelling and redness again appear and persist for 24 h.

c. 72 h later, the tissue becomes inflamed, and tissue necrosis follows.

A.
a. Immediate Hypersensitivity (Type I): The rapid onset of swelling and redness within
1-2 minutes after the insect bite, followed by its disappearance within an hour, suggests
an immediate hypersensitivity reaction. In this case, the insect bite triggers the release
of pre-formed mediators like histamine from mast cells and basophils, leading to the
initial localized swelling and redness. The response subsides quickly due to the short-
lived nature of these mediators.

b. Delayed Hypersensitivity (Type IV): The appearance of swelling and redness 6-8
hours later, which persists for 24 hours, points towards a delayed hypersensitivity
reaction. This type of reaction involves the activation of T cells, specifically CD4+ T
helper cells and CD8+ cytotoxic T cells. The delayed response can be attributed to the
time it takes for T cells to migrate to the site of the bite and for immune mechanisms to
be activated, leading to inflammation and the observed symptoms.

c. Type III Hypersensitivity and Inflammation: The development of tissue

inflammation and subsequent tissue necrosis 72 hours (3 days) later suggests a type III
hypersensitivity reaction. This involves the formation of immune complexes between
antigens from the insect bite and antibodies, followed by deposition of these complexes
in tissues. This deposition triggers an inflammatory response involving neutrophils and
complement activation, leading to tissue damage and necrosis.
It's important to note that multiple immune mechanisms can interact in response to an
insect bite, and the described reactions might involve a combination of different
mechanisms.

5. Indicate which type(s) of hypersensitive reaction (I–IV) apply to the following

characteristics. Each characteristic can apply to one, or more than one, type.

a. Is an important defense against intracellular pathogens.

b. Can be induced by penicillin.
c. Involves histamine as an important mediator.
d. Can be induced by poison oak in sensitive individuals.
e. Can lead to symptoms of asthma. f. Occurs as result of mismatched blood
transfusion.
g. Systemic form of reaction is treated with epinephrine.
h. Can be induced by pollens and certain foods in sensitive individuals.
i. May involve cell destruction by antibody-dependent cellmediated cytotoxicity.
j. One form of clinical manifestation is prevented by Rhogam.
k. Localized form characterized by wheal and flare reaction.

A.

a. Is an important defense against intracellular pathogens. - Type IV

b. Can be induced by penicillin. - Type I, Type IV
c. Involves histamine as an important mediator. - Type I, Type III
d. Can be induced by poison oak in sensitive individuals. - Type IV
e. Can lead to symptoms of asthma. - Type I
f. Occurs as a result of mismatched blood transfusion. - Type II
g. Systemic form of reaction is treated with epinephrine. - Type I
h. Can be induced by pollens and certain foods in sensitive individuals. - Type I
i. May involve cell destruction by antibody-dependent cell-mediated cytotoxicity. - Type
II
j. One form of clinical manifestation is prevented by Rhogam. - Type II
k. Localized form characterized by wheal and flare reaction. - Type I
6. In the table below, indicate whether each immunologic event listed does (+) or does
not (–) occur in each type of hypersensitive response

Hypersensitivity
Immunologic event
Type I Type II Type III Type IV
IgE-mediated degranulation of mast cells + - - -
Lysis of antibody-coated blood cells by
- + + -
complement
Tissue destruction in response to poison oak - - - +
C3a- and C5a-mediated mast-cell degranulation + - - -
Chemotaxis of neutrophils + - + -
Chemotaxis of eosinophils + - - -
Activation of macrophages by IFN- γ - - - +
Deposition of antigenantibody complexes on
- - + -
basement membranes of capillaries
Sudden death due to vascular collapse (shock)
+ - - -
shortly after injection or ingestion of antigen
 CHAPTER 20
AUTOIMMUNITY
CLINICAL FOCUS QUESTION: What are some of the possible reasons why females
are more susceptible to autoimmune diseases than males?
A. There are several possible reasons why females are more susceptible to
autoimmune diseases than males. One reason is that there are significant gender
differences in immune responses. Females tend to mount more vigorous immune
responses, producing higher levels of antibodies and CD4+ T cells. In mice, female
mice are more likely to develop pro-inflammatory TH1 responses, which can be
beneficial in certain infections but can also be deleterious in some cases. Another factor
is the role of sex hormones. Estrogen and testosterone can alter the outcome of
infections and play a role in regulating immune responses. Pregnancy also affects the
immune system, with women mounting more TH2-like responses during pregnancy.
Additionally, the presence of fetal cells in the maternal circulation and the exchange of
cells during pregnancy may contribute to the development of autoimmune disease.
However, the exact reasons for the increased susceptibility of females to autoimmune
diseases are not fully understood.

1. For each of the following autoimmune diseases (a–m), select the most appropriate
characteristic (1–13) listed below.

Diseases

a. Experimental autoimmune encephalitis (EAE)

b. Goodpasture’s syndrome
c. Graves’ disease
d. Systemic lupus erythematosus (SLE)
e. Insulin-dependent diabetes mellitus (IDDM)
f. Rheumatoid arthritis
g. Hashimoto’s thyroiditis
h. Experimental autoimmune myasthenia gravis (EAMG)
 i. Myasthenia gravis
j. Pernicious anemia
k. Multiple sclerosis
l. Autoimmune hemolytic anemia

Characteristics

(1) Auto-antibodies to intrinsic factor block vitamin B12 absorption

(2) Auto-antibodies to acetylcholine receptor
(3) TH1-cell reaction to thyroid antigens
(4) Auto-antibodies to RBC antigens
(5) T-cell response to myelin
(6) Induced by injection of myelin basic protein plus complete Freund’s adjuvant
(7) Auto-antibody to IgG
(8) Auto-antibodies to basement membrane
(9) Auto-antibodies to DNA and DNA-associated protein
(10) Auto-antibodies to receptor for thyroid-stimulating hormone
(11) Induced by injection of acetylcholine receptors
(12) TH1-cell response to pancreatic beta cells

A.

a. Experimental autoimmune encephalitis (EAE) - (6) Induced by injection of

myelin basic protein plus complete Freund’s adjuvant
b. Goodpasture’s syndrome - (8) Auto-antibodies to basement membrane
c. Graves’ disease - (10) Auto-antibodies to receptor for thyroid-stimulating
hormone
d. Systemic lupus erythematosus (SLE) - (9) Auto-antibodies to DNA and DNA-
associated protein
e. Insulin-dependent diabetes mellitus (IDDM) - (12) TH1-cell response to
pancreatic beta cells
f. Rheumatoid arthritis - (7) Auto-antibody to IgG
g. Hashimoto’s thyroiditis - (3) TH1-cell reaction to thyroid antigens
h. Experimental autoimmune myasthenia gravis (EAMG) - (11) Induced by
 injection of acetylcholine receptors
i. Myasthenia gravis - (2) Auto-antibodies to acetylcholine receptor
j. Pernicious anemia - (1) Auto-antibodies to intrinsic factor block vitamin B12
absorption
k. Multiple sclerosis - (5) T-cell response to myelin
l. Autoimmune hemolytic anemia - (4) Auto-antibodies to RBC antigens

2. Experimental autoimmune encephalitis (EAE) has proved to be a useful animal model

of autoimmune disorders.

a. Describe how this animal model is made.

b. What is unusual about the animals that recover from EAE?
c. How has this animal model indicated a role for T cells in the development of
autoimmunity?

A.

a. Creation of Experimental Autoimmune Encephalitis (EAE) Model:

EAE is typically induced in animals, such as mice, by injecting them with myelin
antigens, often myelin basic protein (MBP) or proteolipid protein (PLP), combined with
an adjuvant, like complete Freund's adjuvant (CFA). This mixture stimulates an immune
response, particularly involving T cells, to target and attack myelin, the protective
covering of nerve fibers in the central nervous system (CNS). This immune response
leads to inflammation, demyelination, and neurological symptoms similar to those seen
in human autoimmune diseases like multiple sclerosis.

b. Unusual Recovery in EAE Animals:

In some cases, animals that experience the initial symptoms of EAE, which mimic
neurological deficits, can exhibit a phenomenon known as "spontaneous recovery." This
recovery involves a reduction in symptoms and even a return to normal neurological
function despite the autoimmune attack that occurred. This recovery is unusual and not
completely understood but may involve regulatory T cells and other immunomodulatory
mechanisms.
c. Role of T Cells in Autoimmunity:

The EAE model has provided strong evidence for the involvement of T cells in the
development of autoimmunity. Researchers have observed that transfer of autoreactive
T cells from EAE-induced animals to naïve animals can induce the disease in the
recipient animals, even without the initial injection of myelin antigens. This indicates that
T cells specific to myelin antigens can initiate the autoimmune response. Additionally,
manipulating T cell populations in EAE animals has demonstrated that both CD4+ T
helper cells and CD8+ cytotoxic T cells play critical roles in driving the autoimmune
response, inflammation, and tissue damage observed in EAE and, by extension, in
human autoimmune disorders like multiple sclerosis.

In summary, the EAE model has significantly contributed to our understanding of

autoimmune diseases, highlighting the involvement of T cells in driving autoimmunity
and providing insights into potential therapeutic interventions.

3. Molecular mimicry is one mechanism proposed to account for the development of

autoimmunity. How has induction of EAE with myelin basic protein contributed to the
understanding of molecular mimicry in autoimmune disease?
A. The induction of experimental autoimmune encephalitis (EAE) with myelin basic
protein (MBP) has contributed to the understanding of molecular mimicry in autoimmune
disease by providing evidence that infection with certain viruses expressing epitopes
that mimic self-components, such as MBP, can induce autoimmunity to those
components. Studies have shown that immunization with a peptide from the polymerase
enzyme of the hepatitis B virus, which exhibits homology with the encephalitogenic MBP
peptide, can induce the formation of antibodies and T cells that cross-react with MBP.
This suggests that molecular mimicry between viral and self-antigens can lead to the
development of autoimmunity. EAE models have provided valuable insights into the
mechanisms of molecular mimicry and its role in autoimmune diseases.

4. Describe at least three different mechanisms by which a localized viral infection might
contribute to the development of an organ-specific autoimmune disease.
A.
1. Molecular mimicry: The viral infection can trigger an immune response that
produces antibodies or T cells that recognize both viral antigens and self-antigens in the
affected organ. This cross-reactivity can lead to an autoimmune response against the
organ.

2. Bystander activation: The immune response against the viral infection can cause
inflammation and tissue damage in the affected organ. This tissue damage can release
self-antigens, which can then activate autoreactive immune cells and initiate an
autoimmune response.

3. Epitope spreading: The initial immune response against the viral infection can lead
to the presentation of new self-antigens to the immune system. This can result in the
activation of autoreactive immune cells specific to these self-antigens, leading to an
autoimmune response against the affected organ.

5. Transgenic mice expressing the IFN- transgene linked to the insulin promoter
developed diabetes.

a. Why was the insulin promoter used?

b. What is the evidence that the diabetes in these mice is due to autoimmune
damage?
c. What is unusual about MHC expression in this system?
d. How might this system mimic events that might be caused by a localized viral
infection in the pancreas?

A.

a. Insulin Promoter Usage: The insulin promoter was used to drive the expression of
the IFN- (interferon-gamma) transgene specifically in pancreatic beta cells. The insulin
promoter is active in beta cells and directs gene expression primarily in these cells,
making it a suitable tool to investigate the effects of the IFN- transgene on pancreatic
function.
b. Evidence of Autoimmune Damage: The development of diabetes in transgenic
mice expressing the IFN- transgene linked to the insulin promoter suggests that
autoimmune damage is involved. Autoimmune diabetes, also known as type 1 diabetes,
is characterized by the destruction of insulin-producing beta cells by the immune
system. The presence of IFN- likely stimulates immune responses that target and attack
the beta cells. This is further supported by the fact that the diabetes occurs in mice with
the transgene expression directed specifically to the pancreas.

c. Unusual MHC Expression: In this system, MHC (major histocompatibility complex)

expression on beta cells may be upregulated due to the IFN- transgene. MHC
molecules are involved in presenting antigens to immune cells. Increased MHC
expression on beta cells could lead to their recognition by immune cells, triggering an
autoimmune response.

d. Mimicking Effects of Localized Viral Infection: In a localized viral infection in the

pancreas, viral particles and infected cells release signals, such as interferons, which
can stimulate immune responses. The IFN- transgene mimics this viral infection
scenario by causing the continuous production of interferon-gamma in the pancreas.
This sustained immune activation could lead to immune responses targeting pancreatic
beta cells, similar to what might occur during an autoimmune response following viral
infection.

Overall, this transgenic mouse model provides insights into the mechanisms underlying
autoimmune diabetes and how immune responses triggered by factors like interferon-
gamma can lead to the destruction of insulin-producing beta cells.

6. Monoclonal antibodies have been administered for therapy in various autoimmune

animal models. Which monoclonal antibodies have been used and what is the rationale
for these approaches?

A.

1. Monoclonal antibodies specific for the Vβ 8.2 T-cell receptor: These antibodies have
been used in mice to prevent the induction of experimental autoimmune
encephalomyelitis (EAE) by myelin basic protein (MBP) in adjuvant. They have also
been shown to reverse the symptoms of autoimmunity in mice with induced EAE.

2. Monoclonal antibodies to class II MHC molecules: In some autoimmune diseases,

there is evidence for increased or inappropriate MHC expression. Monoclonal
antibodies against specific class II MHC molecules have been used to block the
development of autoimmunity. For example, injecting mice with monoclonal antibodies
to class II MHC molecules before injecting MBP blocked the development of EAE.

The rationale for using these approaches is based on the understanding that
autoimmune diseases involve abnormal immune responses. By targeting specific
receptors or molecules involved in the autoimmune response, monoclonal antibodies
can potentially modulate or suppress the immune response, leading to a reduction in
autoimmune symptoms. These approaches offer the possibility of more targeted and
specific therapies for autoimmune diseases.

7. Indicate whether each of the following statements is true or false. If you think a
statement is false, explain why.

a. TH1 cells have been associated with development of autoimmunity.

b. Immunization of mice with IL-12 prevents induction of EAE by injection of
myelin basic protein plus adjuvant.
c. The presence of the HLA B27 allele is diagnostic for ankylosing spondylitis, an
autoimmune disease affecting the vertebrae.
d. Individuals with pernicious anemia produce antibodies to intrinsic factor.
e. A defect in the gene encoding Fas can reduce programmed cell death by
apoptosis.

A.

a. True. TH1 cells are associated with the development of autoimmunity. TH1
cells are involved in cell-mediated immune responses and can promote
inflammation and tissue damage. In some autoimmune diseases, such as
multiple sclerosis and type 1 diabetes, TH1 cells are implicated in driving the
immune response against self-antigens.
b. False. Immunization of mice with IL-12 would likely enhance the induction of
EAE (experimental autoimmune encephalitis) rather than prevent it. IL-12
promotes the differentiation of TH1 cells, which are involved in the immune
response against myelin antigens and can exacerbate autoimmune reactions in
EAE.
c. False. The presence of the HLA B27 allele is associated with an increased risk
of developing ankylosing spondylitis, but it is not diagnostic on its own. Many
individuals with the HLA B27 allele do not develop ankylosing spondylitis, and the
disease can occur in individuals without the allele.
d. True. Individuals with pernicious anemia have an autoimmune response
against intrinsic factor, a protein necessary for vitamin B12 absorption. This leads
to vitamin B12 deficiency and anemia.
e. True. A defect in the gene encoding Fas (CD95) can reduce programmed cell
death by apoptosis. Fas is a cell surface receptor involved in triggering apoptosis
(programmed cell death). Mutations or defects in the Fas gene can lead to
impaired apoptosis, which can contribute to autoimmune diseases and
lymphoproliferative disorders.
 CHAPTER 21
TRANSPLANTATION IMMUNOLOGY
CLINICAL FOCUS QUESTION: What features would you include in an ideal animal
donor for xenotransplantation? How would you test your model prior to doing clinical
trials in humans?
A. An ideal animal donor for xenotransplantation would have the following features:

1. Feasibility in a nonhuman primate model: The animal should be able to serve as a

model for testing the transplantation procedure before it is used in humans.

2. Proven benefit to the patient: The animal organs should provide a significant
benefit to the patients in need of transplants, such as improving their quality of life or
increasing their chances of survival.

3. Lack of infectious-disease risk: The animal should not carry any infectious
diseases that could be transmitted to the human recipient. This is important to ensure
the safety of the transplant procedure.

4. Rapid breeding and large litters: The animal should have a high reproductive rate
to ensure a steady supply of organs for transplantation.

5. Ability to be housed in pathogen-free environments: The animal should be able

to be raised in controlled environments that are free from pathogens to minimize the risk
of disease transmission.

6. Anatomic and physiologic similarity with humans: The animal should have
similarities in anatomy and physiology to humans to increase the chances of successful
transplantation and organ function in the recipient.

Pigs are often considered as potential animal donors for xenotransplantation because
they meet many of these criteria. They breed rapidly, have large litters, can be housed
in controlled environments, and share some anatomical and physiological similarities
with humans. However, there are still barriers to overcome before xenotransplantation
can be widely used in clinical practice.
1. Indicate whether each of the following statements is true or false. If you think a
statement is false, explain why.

a. Acute rejection is mediated by preexisting host antibodies specific for antigens

on the grafted tissue.
b. Second-set rejection is a manifestation of immunologic memory.
c. Passenger leukocytes are host dendritic cells that migrate into grafted tissue
and act as antigen-presenting cells.
d. All allografts between individuals with identical HLA haplotypes will be
accepted.
e. Cytokines produced by host TH cells activated in response to alloantigens play
a major role in graft rejection.

A.

a. False. Acute rejection is not mediated by preexisting host antibodies. It is primarily a

cellular immune response involving T cells recognizing foreign antigens on the grafted
tissue.

b. True. Second-set rejection is a manifestation of immunologic memory. It occurs when

a previously sensitized immune system mounts a faster and more aggressive rejection
response upon encountering the same antigens again.

c. True. Passenger leukocytes are host dendritic cells that migrate into the grafted
tissue. They can act as antigen-presenting cells, helping to initiate and amplify the
immune response against the graft.

d. False. While individuals with identical HLA haplotypes are more likely to have better
compatibility for organ transplantation, there are still other factors involved in graft
acceptance, such as minor histocompatibility antigens and non-HLA factors. Even with
matching HLA haplotypes, the immune system can recognize and respond to other
differences.
e. True. Cytokines produced by host TH (T helper) cells activated in response to
alloantigens play a major role in graft rejection. These cytokines can recruit and activate
various immune cells, leading to inflammation and graft destruction.

2. You are a pediatrician treating a child who needs a kidney transplant. The child does
not have an identical twin, but both parents and several siblings are willing to donate a
kidney if the MHC match with the patient is good.

a. What is the best possible MHC match that could be achieved in this situation?
b. In which relative(s) might you find it? Why?
c. What test(s) would you perform in order to find the bestmatched kidney?

A.

a. The best possible MHC match in this situation would be a haploidentical match. This
means that one of the child's parents shares one haplotype (half of the MHC genes)
with the child. While a full MHC match would be ideal (as in the case of an identical
twin), a haploidentical match provides the closest degree of genetic similarity after an
identical twin.

b. You might find a haploidentical MHC match in one of the child's parents. Since each
child inherits one MHC haplotype from each parent, there's a 50% chance that one of
the child's parents will share one haplotype with the child.

c. To find the best-matched kidney, several tests would be performed:

 HLA Typing: This involves testing the MHC genes of the potential donors and
the child to determine if there's a haploidentical match. HLA typing involves
assessing the compatibility of specific HLA alleles between the donor and
recipient.

 Crossmatching: A crossmatch test is done to check for compatibility between

the recipient's antibodies and the potential donor's cells. This test helps to identify
any pre-existing antibodies that might cause rejection of the graft.
  Tissue Compatibility Tests: Other tests to assess tissue compatibility, such as
blood type compatibility and testing for minor histocompatibility antigens, might
also be performed.

Ultimately, the goal is to find a kidney donor who has the best possible MHC match with
the child to minimize the risk of rejection and improve the success of the kidney
transplant.

3. Indicate in the Response column in the table on page 500 whether a skin graft from
each donor to each recipient listed would result in a rejection (R) or an acceptance (A)
response. If you believe a rejection reaction would occur, then indicate in the right-hand
column whether it would be a first-set rejection (FSR), occurring in 12–14 days, or a
second-set rejection (SSR), occurring in 5–6 days. All the mouse strains listed in the
table have different H-2 haplotypes

TYPE OF
DONOR RECIPIENT RESPONSE
REJECTION
BALB/c C3H R FSR
BALB/c Rat R FSR
BALB/c Nude mouse A N/A
C3H, had previous BALB/c
BALB/c R SSR
graft
C3H, had previous C57BL/6
BALB/c R FSR
graft
BALB/c BALB/c A N/A
BALB/c (BALB/c C3H)F1 A N/A
BALB/c (C3H C57BL/6)F1 R FSR
(BALB/c C3H)F1 BALB/c A N/A
BALB/c, had previous F1
(BALB/c C3H)F1 R SSR
graft
4. Graft-versus-host disease (GVHD) frequently develops after certain types of
transplantations.

a. Briefly outline the mechanisms involved in GVHD.

b. Under what conditions is GVHD likely to occur?
c. Some researchers have found that GVHD can be diminished by prior
treatment of the graft with monoclonal antibody plus complement or with
monoclonal antibody conjugated with toxins. List at least two cell-surface
antigens to which monoclonal antibodies could be prepared and used for this
purpose, and give the rationale for your choices.

A.

a. Mechanisms of Graft-Versus-Host Disease (GVHD): GVHD occurs when

immune cells from the transplanted graft (graft T cells) recognize and attack the
tissues of the host recipient. This typically occurs in situations where the donor's
immune cells, particularly T cells, recognize the host's tissues as foreign due to
differences in their human leukocyte antigens (HLA). The activated donor T cells
attack the host's skin, gastrointestinal tract, and other tissues, leading to
inflammation, tissue damage, and clinical symptoms.
b. Conditions Favoring GVHD Occurrence: GVHD is more likely to occur under
the following conditions:

 When the donor and recipient are mismatched in their HLA antigens, leading to
recognition of host tissues as foreign by donor T cells.

 When the transplant includes mature, functional T cells from the graft that can
initiate immune responses against host tissues.

 When the recipient's immune system is compromised, either due to

immunosuppressive treatment or a weakened immune system.

c. Diminishing GVHD with Monoclonal Antibodies: Researchers have explored

strategies to diminish GVHD using monoclonal antibodies (mAbs) that target specific
cell-surface antigens. Two potential targets for mAbs are:
 1. CD3: CD3 is a molecule found on T cells and is involved in T cell
activation. Targeting CD3 with mAbs can deplete T cells from the graft,
reducing the potential for graft T cells to attack the host.

2. CD52: CD52 is expressed on T cells and other immune cells. Targeting

CD52 with mAbs, which can be conjugated with toxins, leads to the
depletion of these immune cells, reducing the immune response that
causes GVHD.

The rationale for choosing these antigens is that both CD3 and CD52 are expressed on
T cells, the primary mediators of GVHD. By targeting these antigens, researchers aim to
selectively eliminate or suppress the donor T cells responsible for attacking the host
tissues, thereby diminishing the severity of GVHD.

5. A child who requires a kidney transplant has been offered a kidney from both parents
and from five siblings.

a. Cells from the potential donors are screened with monoclonal antibodies to the
HLA-A, -B, and -C antigens in a microcytotoxicity assay. In addition, ABO blood-
group typing is performed. Based on the results in the table on page 500, a
kidney graft from which donor(s) is most likely to survive?

ABO type HLA-A type HLA-B type HLA-C type

Recipient
O A1/A2 BB/B12 Cw3

Mother A A1/A2 BB/B12 Cw1/Cw3

Father O A2 B12/B15 Cw3

Potential Sibling A O A1/A2 BB/B15 Cw3

Donors Sibling B O A2 B12 Cw1/Cw3

Sibling C O A1/A2 BB/B12 Cw3
Sibling D A A1/A2 BB/B12 Cw3
Sibling E O A1/A2 BB/B15 Cw3
 b. Now a one-way MLR is performed using various combinations of mitomycin-
treated lymphocytes. The results, expressed as counts per minute of [3
H]thymidine incorporated, are shown in the table on page 500; the stimulation
index (ratio of the experimental value to the control in which identical leukocytes
are mixed) is listed below in parentheses. Based on these data, a graft from
which donor(s) is most likely to be accepted?

Mytomycin C-treated stimulator cells

Respondent Cells
Patient Sibling A Sibling B Sibling C Sibling D Sibling E

Patient 1,672 1,800 13,479 5,210 13,927 13,808

(1.0) (1.1) (8.1) (3.1) (8.3) (8.3)

Sibling A 1,495 933 11,606 8,443 11,708 13,430

(1.6) (1.0) (12.4) (9.1) (12.6) (14.4)

Sibling B 25,418 26,209 2,570 13,170 19,722 4,510

(9.9) (10.2) (1.0) (5.1) (7.7) (1.8)

Sibling C 10,722 10,714 13,032 1,731 1,740 14,365

(6.2) (5.9) (7.5) (1.0) (1.0) (8.3)

Sibling D 15,988 13,492 18,519 3,300 3,151 18,334

(5.1) (4.2) (5.9) (1.1) (1.0) (5.9)

Sibling E 5,777 8,053 2,024 6,895 10,720 888

(6.5) (9.1) (2.3) (7.8) (12.1) (1.0)

A.

a. We need to match the HLA antigens as closely as possible between the donor and
the recipient. Among the potential donors, sibling C has the same ABO type, HLA-A,
HLA-B, and HLA-C antigens as the recipient (O, A1/A2, BB/B12, and Cw3).
Based on both ABO blood group compatibility and HLA antigen compatibility, it appears
that sibling C is the most suitable donor. Their HLA antigens closely match those of the
recipient, increasing the chances of graft survival.

b. To determine which donor's graft is most likely to be accepted based on the results of
the one-way mixed lymphocyte reaction (MLR) data, we need to identify the
combination of donor and recipient that exhibits the lowest stimulation index. A lower
stimulation index suggests that the recipient's immune cells are less reactive to the
donor's treated stimulator cells, indicating a better match and a lower likelihood of graft
rejection.

Here are the stimulation indices for each combination of recipient and mitomycin-treated
stimulator cells:

 Patient vs. Sibling A: 1.6

 Patient vs. Sibling B: 9.9

 Patient vs. Sibling C: 6.2

 Patient vs. Sibling D: 5.1

 Patient vs. Sibling E: 6.5

Based on the data, the graft from Sibling A is most likely to be accepted by the patient.
The stimulation index of 1.6 between the patient and Sibling A indicates a lower level of
immune response, suggesting a better HLA match and a decreased risk of graft
rejection.

6. What is the biologic basis for attempting to use soluble CTL4A or anti-CD40L to block
allograft rejection? Why might this be better than treating a graft recipient with CsA or
FK506?

A. Using soluble CTLA-4 or anti-CD40L to block allograft rejection may be considered

better than treating a graft recipient with CsA or FK506 because it specifically targets
the co-stimulatory pathways involved in graft rejection. CTLA-4 and CD40L are
molecules involved in T-cell activation and immune response. By blocking the
interaction between these molecules and their ligands, the immune response against
the graft can be suppressed without inhibiting the immune response to other antigens.
This targeted approach may result in better control of graft rejection while minimizing
the side effects associated with broad immunosuppressive drugs like CsA and FK506.
 CHAPTER 23
EXPERIMENTAL SYSTEMS
CLINICAL FOCUS QUESTION: How has microarray technology changed disease
diagnosis and how is it likely to influence treatment of diseases in the future?
A. Microarray technology has revolutionized disease diagnosis by allowing researchers
to analyze the expression levels of thousands of genes simultaneously. This has led to
the identification of specific gene expression patterns that are associated with different
diseases, including cancer and autoimmune disorders. Microarray analysis has also
been used to classify tumors into subgroups, which can help in designing more effective
treatments.

In the future, microarray technology is likely to have a significant impact on disease

treatment. By identifying specific gene expression patterns associated with different
diseases, microarrays can help in developing targeted therapies that are tailored to
individual patients. This personalized approach to treatment is expected to improve the
effectiveness of therapies and ultimately enhance survival rates.

However, it is important to note that microarray technology is still evolving, and there are
challenges to overcome, such as standardization of protocols and data analysis.
Additionally, the cost and complexity of microarray analysis may limit its widespread use
in clinical settings. Nonetheless, as the technology continues to advance, it holds great
promise for improving disease diagnosis and treatment.

1. Explain why the following statements are false.

a. The amino-acid sequence of a protein can be determined from the nucleotide

sequence of a genomic clone encoding the protein.
b. Transgenic mice can be prepared by microinjection of DNA into a somatic-cell
nucleus.
c. Primary lymphoid cultures can be propagated indefinitely and are useful in
studies of specific subpopulations of lymphocytes.
A.

a. The amino-acid sequence of a protein cannot be determined directly from the

nucleotide sequence of a genomic clone encoding the protein. This is because
the genomic sequence includes both coding and non-coding regions, such as
introns, which are not translated into amino acids. The process of transcription
and translation is required to convert the genomic sequence into the amino-acid
sequence of a protein.
b. Transgenic mice cannot be prepared by microinjection of DNA into a somatic-
cell nucleus. Transgenic mice are typically generated by injecting DNA into a
fertilized mouse egg, which then develops into a transgenic mouse.
Microinjection of DNA into a somatic-cell nucleus is a technique used for cloning
animals, not for generating transgenic animals.
c. Primary lymphoid cultures cannot be propagated indefinitely. Primary lymphoid
cells have a limited lifespan in culture and eventually stop dividing. They are
useful for studying specific subpopulations of lymphocytes, but they cannot be
propagated indefinitely like immortal cell lines.

2. Fill in the blanks in the following statements with the most appropriate terms:

a. In inbred mouse strains, all or nearly all genetic loci are homozygous; such
strains are said to be homozygous.
b. SCID mice have a genetic defect that prevents development of functional B
and T cells.
c. B-cell hybridomas are formed by fusion B-cell of with myeloma cells. They
are capable of indefinite growth and are used to produce monoclonal
antibodies.
d. A normal lymphoid cell that undergoes can give rise to a cell line, which has an
indefinite life span.

3. The gene diagrammed below contains one leader (L), three exons (E), and three
introns (I). Illustrate the primary transcript, mRNA, and the protein product that could be
generated from such a gene.
A. Here is the gene diagrammed below:
DNA: 5' L E1 I1 E2 I2 E3 I3 3'

The primary transcript is the RNA molecule that is produced directly from the gene. It is
a copy of the entire gene, including the leader, exons, and introns.

Primary Transcript: 5' L E1 I1 E2 I2 E3 I3 3'

During mRNA processing, the introns are removed and the exons are spliced together.
This produces the mature mRNA, which is the RNA molecule that is used to make a
protein.

Mature mRNA: 5' L E1 E2 E3 3'

The protein product that is generated from this gene is a polypeptide chain that is made
up of the amino acids encoded by the exons. The order of the amino acids in the
polypeptide chain is determined by the order of the exons in the mRNA.

In this example, the protein product would be a polypeptide chain with three amino
acids, A, B, and C. The order of the amino acids would be determined by the order of
the exons E1, E2, and E3 in the mRNA.

Here is a diagram of the primary transcript, mRNA, and protein product:

DNA: 5' L E1 I1 E2 I2 E3 I3 3'

Primary Transcript: 5' L E1 I1 E2 I2 E3 I3 3'

Mature mRNA: 5' L E1 E2 E3 3'

Protein Product: A B C

4. The term transfection refers to which of the following?

a. Synthesis of mRNA from a DNA template

b. Synthesis of protein based on an mRNA sequence
c. Introduction of foreign DNA into a cell
d. The process by which a normal cell becomes malignant
e. Transfer of a signal from outside a cell to inside a cell

A. (c) Introduction of foreign DNA into a cell

5. Which of the following are required to carry out the PCR?

 a. Short oligonucleotide primers
b. Thermostable DNA polymerase
c. Antibodies directed against the encoded protein
d. A method for heating and cooling the reaction mixture periodically
e. All of the above

A. (e) All of the above

6. Why is it necessary to include a selectable marker gene in transfection experiments?

A. The purpose of including a selectable marker gene in transfection experiments is to
identify and select the cells that have successfully taken up the transfected DNA. The
selectable marker gene confers resistance to a specific drug or antibiotic, allowing only
the transfected cells to survive in the presence of that drug or antibiotic. This allows
researchers to easily identify and select the cells that have incorporated the desired
DNA and study their characteristics or properties.

7. What would be the result if a transgene were injected into one cell of a four-cell
mouse zygote rather than into a fertilized mouse egg before it divides?
A. If a transgene were injected into one cell of a four-cell mouse zygote instead of a
fertilized mouse egg before it divides, the transgene would only be present in a subset
of the cells in the resulting embryo. This means that only a portion of the cells in the
mouse would carry the transgene, and it would not be present in all tissues and organs.
This could result in mosaic expression of the transgene, with some cells expressing it
and others not.

8. A circular plasmid was cleaved with EcoRI, producing a 5.4- kb band on a gel. A 5.4-
kb band was also observed when the plasmid was cleaved with HindIII. Cleaving the
plasmid with both enzymes simultaneously resulted in a single band 2.7 kb in size.
Draw a diagram of this plasmid showing the relative location of its restriction sites.
Explain your reasoning.
A. Imagine the circular plasmid as a circle, and let's represent the EcoRI and HindIII
restriction sites on this plasmid.
 1. EcoRI Site: This site is cleaved by EcoRI, producing two fragments. When the
plasmid is cleaved with EcoRI, it produces a 5.4-kb band. This indicates that the
EcoRI site is located at the center of the plasmid's DNA strand.

2. HindIII Site: This site is cleaved by HindIII, producing two fragments. When the
plasmid is cleaved with HindIII, it produces a 5.4-kb band. Similar to the EcoRI
site, this suggests that the HindIII site is also located at the center of the
plasmid's DNA strand.

3. EcoRI and HindIII Sites Simultaneously: When the plasmid is cleaved with both
EcoRI and HindIII simultaneously, it produces a single band of 2.7 kb. This
suggests that the EcoRI and HindIII sites are located close to each other, likely
on opposite sides of the plasmid's DNA strand.

Here's a rough representation of the plasmid:

In this representation, the "HindIII" and "EcoRI" labels indicate the positions of the
respective restriction sites. The 2.7-kb region between the restriction sites represents
the DNA fragment that is cleaved when both enzymes are used together.

The reasoning behind this is that the plasmid's cleavage patterns with EcoRI and HindIII
indicate that these sites are symmetrically placed on the plasmid's circular DNA strand.
When cleaved simultaneously, the 2.7-kb fragment forms because the enzymes cut the
DNA at these adjacent sites.

9. DNA footprinting is a suitable technique for identifying which of the following?

a. Particular mRNAs in a mixture

b. Particular tRNAs in a mixture
 c. Introns within a gene
d. Protein-binding sites within DNA
e. Specific DNA sites at which restriction endonucleases cleave the nucleotide
chain

A. (d) Protein-binding sites within DNA

10. Explain briefly how you might go about cloning a gene for interleukin 2 (IL-2).
Assume that you have available a monoclonal antibody specific for IL-2.
A. To clone the gene for interleukin 2 (IL-2) using a monoclonal antibody specific for IL-
2, you would follow these general steps:

a. Extract RNA from IL-2-producing cells.

b. Create cDNA from RNA using reverse transcription.
c. Amplify IL-2 cDNA via PCR.
d. Insert cDNA into a cloning vector.
e. Transform bacteria with vector, select with antibiotics.
f. Purify plasmids from bacteria.
g. Sequence to confirm accuracy.
h. Optionally, insert cDNA into an expression vector for protein production.

Throughout, the IL-2-specific antibody can help validate IL-2 presence and specificity at
various stages.

11. You have a sample of a mouse DNA-binding protein and of the mRNA that encodes
it. Assuming you have a mouse genomic library available, briefly describe how you
could select a clone carrying a DNA fragment that contains the gene that encodes the
binding protein.
A. To select a clone carrying a DNA fragment that contains the gene encoding the
binding protein from a mouse genomic library, you would need to perform a screening
process using a technique called in situ hybridization.

First, the mouse genomic library, which consists of cloned bacterial colonies containing
fragments of the mouse genome, is transferred onto a nitrocellulose or nylon filter. The
filter is then treated with NaOH to lyse the bacteria and denature the DNA, allowing
single-stranded DNA to bind to the filter.

Next, a radioactive probe specific for the gene encoding the binding protein is incubated
with the filter. The probe will hybridize with DNA in the colonies on the filter that contain
the desired gene. The position of the positive colonies can be identified by
autoradiography.

By analyzing the position of the positive colonies on the filter, you can determine which
colonies contain the clone carrying the DNA fragment that contains the gene encoding
the binding protein. These colonies can then be isolated and further analyzed.

12. What are the major differences between transgenic mice and knockout mice and in
the procedures for producing them?
A. Transgenic mice are produced by injecting foreign cloned DNA (transgene) into a
fertilized mouse egg. The transgene integrates into the chromosomal DNA of the egg
and is passed on to the offspring. Transgenic mice express the transgene in all their
somatic cells. On the other hand, knockout mice are produced by introducing a mutant
or disrupted gene into embryonic stem (ES) cells. The ES cells are then injected into a
recipient mouse blastocyst and implanted into a pseudo-pregnant mouse. The resulting
chimeric offspring are mated to produce homozygous knockout mice. Knockout mice
lack the expression of a particular gene product. In summary, transgenic mice involve
the integration of a transgene into the genome, while knockout mice involve the
replacement of a normal gene with a mutant or disrupted allele.

13. How does a knock-in mouse differ from a knockout mouse?

A. A knock-in mouse is a genetically modified mouse in which a specific gene is
replaced or "knocked-in" with a mutated or altered form of the gene. This allows
researchers to study the effects of the altered gene on the mouse's phenotype and
function.

On the other hand, a knockout mouse is a genetically modified mouse in which a

specific gene is completely deleted or "knocked out". This results in the loss of function
of the gene, allowing researchers to study the effects of the gene's absence on the
mouse's phenotype and function.

14. How does the Cre/lox technology enhance knockout and knock-in strategies?
A. Cre/lox technology enhances immune system studies via tissue-specific gene
deletion/expression. Using Cre recombinase, targeting DNA loxP sites, researchers
control gene modulation.

For knockouts, genes flanked by loxP are deleted in specific tissues by mating modified
mice with Cre-carrying counterparts, clarifying tissue-specific gene impact.

In knock-ins, loxP-flanked stop codons in a gene's intron, alongside Cre under tissue-
specific promoters, enable controlled toxic gene expression, dissecting tissue roles.

Cre/lox aids immune research via precise gene control, advancing comprehension of
immune function and responses.

15. For each term related to recombinant DNA technology (a–i), select the most
appropriate description (1–10) listed below. Each description may be used once, more
than once, or not at all.

Terms

a. Yeast artificial chromosome

b. Restriction endonuclease
c. cDNA
d. COS sites
e. Retrovirus
f. Plasmid
g. cDNA library
h. Sticky ends
i. Genomic library

Descriptions

(1) Cleaves mRNA at specific sites.

(2) Cleaves double-stranded DNA at specific sites.
 (3) Circular genetic element that can replicate in E. coli cells.
(4) Used to clone DNA in mammalian cells.
(5) Formed from action of reverse transcriptase.
(6) Collection of DNA sequences within plasmid vectors representing all of the
mRNA sequences derived from a cell.
(7) Produced by action of certain DNA-cleaving enzymes.
(8) Used to clone very large DNA sequences.
(9) Used to introduce larger-than-normal DNA fragments in λ-phage vectors.
(10) Collection of λ clones that includes all the DNA sequences of a given
species.

A.

a. Yeast artificial chromosome - (8) Used to clone very large DNA sequences.
b. Restriction endonuclease - (2) Cleaves double-stranded DNA at specific sites.
c. cDNA - (5) Formed from the action of reverse transcriptase.
d. COS sites - (4) Used to clone DNA in mammalian cells.
e. Retrovirus - (5) Formed from the action of reverse transcriptase.
f. Plasmid - (3) Circular genetic element that can replicate in E. coli cells.
g. cDNA library - (6) Collection of DNA sequences within plasmid vectors representing
all of the mRNA sequences derived from a cell.
h. Sticky ends - (7) Produced by the action of certain DNA-cleaving enzymes.
i. Genomic library - (10) Collection of λ clones that includes all the DNA sequences of a
given species.