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A231 Immunology: Revision Package (Problems One to Three)

Additional resources

• Glossary of terms used in immunology: http://www.microbiologybytes.com/iandi/ImmGloss.html

• Overall picture of the immune response (animation): http://highered.mcgraw-


hill.com/sites/0072495855/student_view0/chapter24/animation__the_immune_response.html

• Innate vs. adaptive immunity: http://www.biology.arizona.edu/immunology/tutorials/immunology/page3.html

• E-book chapter on innate and adaptive immunity: http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=imm&part=A53#A60

Sequence of events in after tissue injury (Taken from http://legacy.owensboro.kctcs.edu/gcaplan/anat2/notes/Notes7%20Nonspepecific%20Defenses.htm)

Five hallmarks of inflammation (Taken from Sell, 2001)

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(Diagrams taken from Mak & Saunders, 2006)

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Practice Questions

1. Which of the following does not apply to "innate" immune mechanisms?


A) absence of specificity
B) activation by a stimulus
C) involvement of multiple cell types
D) a memory component

2. Which of the following is not an example of barriers?


A) Phagocytosis
B) Respiratory tract
C) Flushing action of tears
D) Lactic acid and fatty acid secretion

3. Out of the four cell types, which one of them does not belong to the immune system?
A) Basophils
B) Endothelial cells
C) Mast cells
D) Red blood cells

4. The first immunoglobulin synthesized by the fetus is


A) IgA.
B) IgE.
C) IgG.
D) IgM.
E) None; the fetus does not synthesize immunoglobulins.

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5. The primary and secondary antibody responses differ in

A) the predominant isotype generated.


B) the number of lymphocytes responding to antigen.
C) the speed at which antibodies appear in the serum.
D) the biologic functions manifested by the Ig isotypes produced.
E) All of the above.

Short Answer Question

6. Danny went on a hiking trip with his friends when he accidentally slipped and fell. He suffered a deep cut at his elbow. Swelling, pain, heat
and redness were observed. A few days later, pus started to form at the wounded site.

a. What was the protective response initiated?


b. Account for the observed symptoms.
c. Why was there pus formation at the site of Hubert’s injury?
d. Suggest possible treatment for Hubert’s injury.

True or False

1) The Fc receptor on mast cells binds IgE very strongly and cross-linking by antigen leads to mast cell activation and initiation of an
inflammatory reaction

2) IgE coating mast cells is a second line of defense of external body surfaces when they have been penetrated by an infectious agent

3) The first immunoglobulin heavy chain class to be expressed on the surface of a newly produced B-cell is IgD

4) There is a total of 4 polypeptide chains in a basic immunoglobulin molecule

A231 Immunology
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P4 (Clonal Expansion) – Practice Question

The graph below shows a patient’s immune response to Antigen A and B.

a. In the case of Antigen A, which antibody class is dominant in Response X and Y respectively?
Response X – IgM; Response Y – IgG\

b. Why is there a higher level of antibodies produced against A as compared to B?


This is due to the fact that during re-exposure to Antigen A in the secondary response, the specific memory B cells which were
generated after primary response were triggered to work against the antigen.

On the other hand, Antigen B is exposed to the immune system for the first time; hence, there is an absence of memory B cells in order
for an increased production of antibodies.

A231 Immunology: Revision Package (Problems 5 to 8)

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Additional resources

COMPLEMENT SYSTEM:

• Fc and complement receptors on phagocytes trigger the uptake and degradation of antibody-coated bacteria (Taken from Janeway, 2001 -
http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=imm&part=A1233&rendertype=figure&id=A1238)

Many bacteria resist


phagocytosis by macrophages
and neutrophils. Antibodies
bound to these bacteria,
however, enable them to be
ingested and degraded through
interaction of the multiple Fc
domains arrayed on the
bacterial surface with Fc
receptors on the phagocyte
surface.

Antibody coating also induces activation of the complement system and the binding of complement components to the bacterial surface.
These can interact with complement receptors (for example CR1) on the phagocyte. Fc receptors and complement receptors synergize in
inducing phagocytosis.

Bacteria coated with IgG antibody and complement are therefore more readily ingested than those coated with IgG alone. Binding of Fc and
complement receptors signals the phagocyte to increase the rate of phagocytosis, fuse lysosomes with phagosomes, and increase its
bactericidal activity.

• Details and summary about the complement system: http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=imm&part=A161#A192

• http://pathmicro.med.sc.edu/2006-immpdf/Complement06.pdf

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ANTIGEN PROCESSING AND PRESENTATION:

MHC Class II pathway MHC Class I pathway

Diagrams adapted from Benjamini,


Coico & Sunshine, 2000 –
‘Immunology: A Short Course’

Antigen Recognition by B and T


Cells:

Characteristic B cells T cells

Antigen interaction B-cell receptor (membrane Ig) binds antigen T-cell receptor binds antigen on MHC
Nature of antigens Protein, polysaccharide, lipid Peptide
Binding soluble antigens Yes No
Epitopes recognized Accessible Internal linear peptides produced by antigen
processing (proteolytic degradation)

*NOTE - Common misconceptions:

Misconception #1: ‘Antibiotics and antibodies are the same.’

Antibiotics are medicines that eliminate/ kill bacteria. Antibodies are proteins generated by the cells in the body to help eliminate pathogens
(e.g. bacteria, viruses etc).

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Misconception #2: ‘Vaccination is the same as immunisation.’

Immunisation - process of rendering a subject immune, or of becoming immune. Immunization can occur naturally as your own body creates
immunities to fight off a disease, or through the administration of a vaccine.

Vaccination - is the use of vaccines to help prevent certain diseases. The vaccine is a suspension (in liquid form given orally or by injection)
consisting of weakened or dead pathogens.

Practice Questions

Multiple Choice Questions

1. All the following are characteristics of both MHC class I and class II molecules except:

A) They are expressed codominantly.


B) They are expressed constitutively on all nucleated cells.
C) They are glycosylated polypeptides with domain structure.
D) They are involved in presentation of antigen fragments to T cells.
E) They are expressed on the surface membrane of B cells.

B MHC class I molecules are expressed on nearly all nucleated cells, but the constitutive expression of MHC class II molecules is more
limited (B cells, dendritic cells, and thymic epithelial cells). MHC class II expression can be induced on other cell types (such as
macrophages, endothelial cells, and human T cells) by cytokines.

2. A patient is admitted with multiple bacterial infections and is found to have a complete absence of C3. Which complement-mediated
function would remain intact in such a patient?

A) lysis of bacteria
B) opsonization of bacteria
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C) generation of anaphylatoxins
D) generation of neutrophil chemotactic factors
E) None of the above.

E All these functions are mediated by complement components that come after C3 and in its absence cannot be activated.

3. Immunodeficiency disease can result from

A) a developmental defect of T lymphocytes.


B) a developmental defect of bone marrow stem cells.
C) a defect in phagocyte function.
D) a defect in complement function.
E) All of the above.

E All are correct. Immunodeficiency disorders may result from defects in the development of bone marrow stem cells into lymphocytes
and other cells that participate in the immune response. They can also result from defects in phagocyte functions, which are important in
phagocytosis and presentation of antigen. Immunodeficiency disorders may also result from defects in complement function — an
absence or malfunction of one or more of the complement components, their activators, or regulators.

4. Products of TAP-1 and -2 genes

A) bind b2-microglobulin.
B) prevent peptide binding to MHC molecules.
C) are part of the proteasome.
D) transport peptides into the endoplasmic reticulum for binding to MHC class I.
E) transport peptides into the endoplasmic reticulum for binding to MHC class II.

D The products of the TAP-1 and -2 genes selectively transport peptides 8—9 amino acids in length from the cytoplasm into the ER
where they bind to MHC class I molecules.

5. Which of the following is incorrect concerning the processing of an antigen, such as a bacterial protein, in the acid compartments of the
cell?
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A) It results in production of potentially immunogenic peptides that associate with MHC class II molecules.
B) Predominantly exogenous antigens are processed by this pathway.
C) It may lead to activation of CD4+ T cells.
D) It may lead to the activation of CD8+ T cells.
E) Bacterially derived peptides displace a fragment of the invariant chain from the MHC class II binding groove.

D CD8+ T cells are generally not activated by processing in acid compartments; exogenous antigen processing in acid compartments
results in the generation of peptides, some of which can displace the CLIP fragment of the invariant chain from the MHC class II binding
groove. The peptide—MHC class II complexes move to the cell surface and can interact with a CD4+ T cell with the appropriate
receptor.

6. Which component(s) of complement could be missing and still leave the remainder of the complement system capable of activation by
the alternative pathway?

A) C1, C2, and C3


B) C3 only
C) C2, C3, and C4
D) C1, C2, and C4
E) C1, C3, and C4

D C3 is required for the alternative pathway of complement activation, while C1, C2, or C4 are not required.

7. An antigen—antibody immune complex in a C3-deficient individual will still result in

A) anaphylatoxin production.
B) depression of factor B.
C) production of chemotactic factors.
D) activation of C
E) activation of C5.
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D The immune complex will activate C2 (and C4) but will not activate C3 or any other components. Since the alternative pathway of
complement activation also requires C3, this pathway will not be activated. Anaphylatoxins and chemotactic factor generation require
C3, while the synthesis of factor B is not related to C

Short Answer Question

8. Summarise the advantage of having two distinct antigen processing pathways.

• T cell receptor (TCR) is unable to discern between extracellular/endogenous and intracellular/exogenous pathogens, and yet the
immune system needs to involve CD4+ T cells and CD8+ in reactions against the respective antigens (derived from the pathogens).

• To sove this problem, the peptides generated from extracellular proteins and the peptides generated from intracellular proteins are
segregated in the cell and trafficked to different classes of MHC molecules. These different classes in turn involve different T-cells:
CD4+ T cells – MHC Class II; CD8+ - MHC Class I.

9. After being injected with a vaccine for a microbe, both T and B-cell responses were observed. Identify the respective responses from
the graph and give an explanation for the responses.

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Response X – T cells response. T cells will be stimulated to proliferate but these are short lived and only seen during acute phase after
vaccination.

Response Y – B cells response. The vaccination induces prolonged serum antibody responses that can persist for months or years
after the vaccination.

10. The table below shows a comparison between two types of vaccines.

Pathogen A B

Type of vaccine Attenuated virus Toxoid

Vaccination Annually Every 10 years

Effectiveness Variable 0-85% 100%

a. The antibodies against toxoid ‘B’ disappear from the blood circulation within a year. Yet why is it only necessary to vaccinate against B
every 10 years?

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Memory is due to long-lived memory lymphocytes, which persist in the lymphoid tissues for many years. They will be reactivated if the
individual encounters the pathogen next time.

b. Why is the vaccine against ‘B’ always effective, whereas the vaccine against ‘A’ does not offer protection on all occasions?
The toxoid is a stable molecule (does not mutate or change in structure). Hence antibodies and lymphocytes which recognise it will still
be effective later on. Pathogen A may mutate frequently. The previous year’s antibodies or the antibodies generated by the current
vaccine may not be effective against new strain of pathogen.

A231 Immunology: Revision Package (Problems 9 to 15) – with Answers to practice questions

Additional resources

SDS-PAGE and Western Blot:

• http://www.chem.uky.edu/courses/che554/Lectures/CHE554Lect9_WesternBlot_30March2010.pdf

• http://www.abdserotec.com/uploads/WesternBlottingBrochure.pdf

Transplantation:

• Immunology of Transplant Rejection: http://emedicine.medscape.com/article/432209-overview

• Immunologic Aspects of Organ Transplantation: The Acquired Immune System: http://www.medscape.com/viewarticle/436533_10

• Basic concepts in Transplantation Immunology: http://www.aasld.org/conferences/Documents/CAQ%20Corner


%20Documents/Martinez.pdf

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• Transplant rejection (human allografts):

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Hypersensitivity reactions:

• The Modern Treatment of Allergies: http://chem4513.pbworks.com/The-Modern-Treatment-of-Allergies

• Allergy and Hypersensitivity (Janeway – online book): http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=imm&part=A1719

• Anaphylactic reactions:

• Differences between the four types of hypersensitivity


reactions (refer to diagrams):

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Taken from:

http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=imm&part=A1719&rendertype=figure&id=A1721

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• Allergic Reaction:

Taken from:
http://extension.entm.purdue.edu/publichealth/print/insects/stinging.html

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• Autoimmunity:

o http://www.theintellectualdevotional.com/blog/2010/01/25/autoimmune-diseases-basically-an-epic-body-fail/

o http://www.telepathology.com/courses/immune/auto.htm

o http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=imm&part=A1906#A1918

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Practice Questions

1. Figure A and B below shows the Western Blot results detecting presence of complement components C1q and C4 present in the urine
sample of a patient suffering from bacterial infection. The human serum serves as a positive control for the study.

Figure A Figure B
C4
C1q
Human serum Urine sample
Human serum Urine sample

Based on the results shown above, it was postulated that the classical complement pathway has been activated in this patient. Explain
why. (5 marks)

• Protein bands detected in the urine sample are more intense compared to the positive control - increase in production of complement
component C1q and maybe C4.

• C1q is higher than normal - classical complement pathway was activated.

• Increased level of antibodies binding to the pathogen (present in urine sample)  C1q bind to the antibodies  activation of the classical
complement cascade  pathogen removal

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2. Peter went on a service learning trip organised by his school and took part in a ceremony organised by the villagers to celebrate a couple’s
wedding. To join in the fun, he allowed the villagers to cover his arms with patterns drawn using variety of colours extracted from local
plants. When he returned to Singapore 3 weeks later, his arms started to itch and were red.

a. Explain what could be the cause of Peter’s condition. (3 marks)


• Contact sensitivity - T cell mediated (Type IV) delayed type hypersensitivity
• One of the dyes used to paint his arms could be the sensitive.

b. How could you confirm the diagnosis?


• Patch test using samples of the dyes to healthy areas of skin.
• Skin should show localised contact reaction 24-48 hours later at the site which the causative dye was applied.

c. If a biopsy is taken, what will one see? (1 mark)


• Biopsy should show high number of immune/mononuclear cells (monocytes).

3. The graph below shows the survival percentages of two groups of liver graft patients. One group received blood transfusions from their
donors before transplantation surgery. The other group did not receive any prior blood transfusions from their donors.

a. Based on the above results, which group of patients received blood transfusions prior to transplantation surgery? Justify your answer. (5
marks)
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• Group X
• Survival rate of liver grafts is higher for transfused patients - chance exposure to antigens which happen to be on their transplant.
• This blood transfusion effect will lower chances of organ rejection.

b. What are some of the possible pre-transplantation tests that can be conducted to increase the chances of graft survival? (3 marks)
HLA typing tests, ABO compatibility tests, DNA compatibility tests etc.

4. The presence or absence of Protein X (a 70 kDa protein) in patient’s serum is routinely established through the use of ELISA techniques
followed by a confirmatory test (if ELISA test is positive) using Western-blotting techniques.

a. Discuss why 2 tests are used to establish the presence of Protein X in patient’s serum. (Hint: Think of the overwhelming number of
patient’s samples to process in a clinical laboratory)

Any 4

 ELISA is a rapid method to establish presence or absence of Protein X

 Western blottings expts are slow and hence cannot process samples fast enough

 However, unspecific bindings of antibodies are not detected in ELISA

 Western blotting expts are able to pick up false positive ELISA results due to unspecific bindings of antibodies used.

 Size of proteins reacting with antibodies are revealed in Western-blotting expts; this allow us to realize unspecific binding of
antibodies.

b. In the above 2 tests mentioned, ELISA and Western-blotting experiments, the same pair of antibodies were used.
Primary antibody is rabbit anti-Protein X IgG; while secondary antibody used was enzyme-conjugated horse anti-rabbit
IgG. To reduce economic cost and time of testings, Mr. Chin Kay Kiang decided to carry out both ELISA and Western
blotting experiments using only enzyme-conjugated primary antibody. The antibody used here were identical (apart being
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conjugated to an enzyme) and of the same final concentration as the primary antibody used previously. Results of Mr.
Chin’s new test method (using only enzyme-conjugated primary antibody) on 5 clinical samples were compared to those
old method (using both primary and secondary antibodies) as shown in the table below.

New Method Old Method


(Enzyme-conjugated (Primary antibody and
primary antibody only) Enzyme-conjugated
Secondary antibody)
ELISA Western-blot ELISA Western-blot
Sample A + 70 kDa band + 70 kDa band
Sample B - No band + 70 kDa band
Sample C + 70 kDa band + 70 kDa band
Sample D - No band + 70 kDa band
Sample E - No band + 40 kDa band

i. Assuming the conditions used for the new method and old method were the same, discuss why the new method fails to detect Protein X
from Sample B and D; but was able to detect Protein X from Sample A and C?

Any 3

 Secondary antibodies allows amplification of signals

 Primary antibody is bounded by many Secondary antibodies

 Low amount of Protein X in Sample B and D

 High amount of Protein X in Sample A and C

ii. Looking at the results obtained for Sample E from Table 1, discuss why the new method was able to prevent a FALSE POSITIVE ELISA
results as compared to the old method.

Any 3

 Secondary antibodies bind non-specifically to a 40 kDa protein

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 This cause old method to produce false positive results

 This non-specific binding is revealed by Western blotting expts

 Secondary antibody NOT used in new method, hence no false positive results.

5. In an attempt to reduce graft’s rejection, the lymphocytes of Michael (patient), was isolated and mixed individually with fixed lymphocytes of
potential donors, Mr. A, Mr. B and Mr. C, in culture vessels; to observe for lymphocyte proliferation after a period of time. Fixed lymphocytes
are lymphocytes that had been subjected to treatment and cannot proliferate. Hence any proliferation of lymphocytes observed can be
assumed to be due to that of Michael’s. Controls were also set up where the lymphocytes involved in the tests are placed in the culture
vessels. These 4 individuals have the same O blood group. The results are as shown below.

Content of culture vessel Rate of lymphocyte


proliferation observed

Michael’s lymphocytes with Mr. A’s fixed lymphocytes. High

Michael’s lymphocyte with Mr. B’s fixed lymphocytes. Very High

Michael’s lymphocyte with Mr. C’s fixed lymphocytes. Very Low Rate

Michael’s lymphocytes Nil

Mr. A’s fixed lymphocytes Nil

Mr. B’s fixed lymphocytes Nil

Mr. C’s fixed lymphocytes Nil

a. Discuss if the results of this test is can be used to reduce the chances of graft’s rejection in Michael.

− Graft’s rejection is due to host’s immune response. (1 mark)

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− Proliferation of lymphocytes is a measure of immune response. (1 mark)
− This is because there is always proliferation of lymphocytes before an effective immune response can be effected; be it T-h cells, T-c
cells or B-cells. (2 marks)
− Strong immunogenic antigens that causes lymphocytes proliferation/graft’s rejections includes foreign MHC antigens (1 mark)
− MHC antigens are found on the surface of lymphocytes. (1 mark)
− Hence if the donor MHC molecules (found on the surface of fixed lymphocytes) can elicit rapid proliferation of Michael’s lymphocytes, it
is an indication that it will cause a strong immune response. (2 marks).
− Hence this test is a good indicator of suitability of organ transplants to Michael based on MHC antigens suitability.

b. Do you think there are any suitable donor(s) identified here? Justify your answer.
Mr. C fixed lymphocytes causes a very mild response from Michael’s lymphocytes (1mark).
Hence, Mr. C is a suitable donor. (1 mark)

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