BABS 2433 IMMUNOLOGY

Assignment 2
Name: Chong Khai En
Student ID: 15WAU08702
Group: RBS 2- Group 1
The early steps of rabiesvirus (CVS) infection in vitro were studied in
chicken embryo cells (CER) cells. The infection was monitored by
looking for specific viral inclusions using anti-rabies fluorescein
isothiocynanate at 24 hours after addition of virus. Cells were
incubated for various lengths of time after virus attachment with
ammonium chloride and chloroquine. The results are show in Figure 1.

20 mM NH4Cl
0.1mM
chloroquine

Figure 1

Question 1:
(i) What is the effect of ammonium chloride on the infection of CER
cells? Explain why there are such effects. (4 marks)
The results in Figure 1 show that inhibition of rabies virus infection by
ammonium chloride only occurred in the first hours. The addition of NH 4Cl one
hour after the binding step inhibited 50% of the infection only as determined by
fluorescence. If the NH4Cl was added 6 hour after the binding step, this
inhibition did not occur. The inhibitory effect of NH 4Cl is only restricted to early
events in infection in CER cells. The major effect of NH 4Cl is to raise the pH, thus
it affects a lysosome-dependent stage in the infectious process of rabies virus.
Rabies virus infection can occur at low pH, but the infection could be inhibited

by NH4Cl under these conditions. NH4Cl raises the intra-lysosomal pH in order to

prevent lysosomal fusion in the entry of rabies virus into CER cells.

(ii) What is the effect of treatment of CER cells with chloroquine?

Explain. (4 marks)
When chloroquine was added after the binding step, inhibition still occurred 5
hours after the binding step. Chloroquine has an inhibitory effect on the later
stages of rabies virus infection.
Ammonium chloride and chloroquine were used to prevent the virus fusion step
thus preventing infection. Both drugs were shown to inhibit the early steps of
infection, NH4Cl having a much earlier effect than chloroquine. The two drugs
had no effect on the attachment step nor did NH4Cl inhibit virus multiplication.

Question 2:
(a) What is the major role of peripheral self tolerance?
Peripheral self tolerance refers to mechanism acting on mature lymphocytes
after they have left the primary lymphoid organs. Peripheral tolerance is
induced when mature T cells recognize self antigens in peripheral tissues,
causing to functional inactivation (anergy) or death, or when the self-reactive
lymphocytes are suppressed by T cells. Antigen recognition without enough
costimulation results in T cell anergy or death, or makes T cells sensitive to
suppresses by regulatory T cells. Nave T lymphocytes require at least 2 signals
to induce their proliferation and differentiation. Signal 1 is antigen, signal 2 is
provided by costimulators that are expressed on antigen-presenting cells.

(b) Distinguish between clonal deletion and anergy.

Recognition of self antigens may trigger pathways of apotosis that result in
elimination or deletion of the self-reactive lymphocytes. T cells respond to
antigen presented by normal antigen-presenting cells (APCs) by secreting IL-2.
This expressing anti-apotosis proteins and undergo proliferation and
differentiation. The anti-apototic proteins prevent the release of mediators of
apotosis from mitochondria. Self antigen recognition by T cells without
costimulation leads to deficiency of intracellular anti-apototic proteins and
excess of pro-apototic proteins causes cell death by release of mediators from
mitochondria. Alternatively, self antigen may lead to expression of death
receptors and their ligands such as Fas and FasL on lymphocytes.

Anergy in T cells refers to long-lived functional inactivation that occurs when

these cells recognize antigens without adequate levels of the costimulators that
are needed for full T cell activation. On recognition of self antigens, T cells
engage one of inhibitory receptors of CD28 family, CTLA-4 (cytotoxic T
lymphocyte-associated antigen 4) or programmed death protein 1 (PD-1). They
terminate T cell activation. Thus, this results in long-lasting T cell anergy.
Clonal deletion
The physical loss of self-reactive
clones.
Self-reactive
lymphoid
cells
are
destroyed during the development of
the immune system.

Anergy
The functional inactivation of selfreactive clones induced by antigen.
Self-reactive T- or B-cells become
inactivated in the normal individual
and cannot amplify the immune
response.
Central tolerance by clonal deletion.
Peripheral tolerance by anergy.
B cells that have high affinity for B cells can come out from the bone
binding to normal cellular molecule are marrow but become inactivated and
deleted in bone marrow.
cannot proliferate.
(c) Central T cell tolerance is established by thymus selection, which
includes the process of non-selection (or neglect), positive selection
and negative selection. Explain the differences between these
selections. (5marks x 3)
Bone-marrow-derived pre-T-cells, which bear neither CD4 nor CD8, enter the
thymus from the bloodstream, develop into immature T-cells which are both CD4
and CD8 positive, then lose one or the other marker to become mature CD4 +
(helper) or CD8+ (cytotoxic) effector cells.
If the cell experiences a MHC interaction which is at some intermediate level,
the T-cell will eventually emigrate from the thymus as a mature CD8 + T-cell. This
process is termed positive selection. It is among this successful population
of T-cells, selected to recognize self-MHC at some intermediate level, which cells
may recognize a foreign antigen peptide in self-MHC strongly to proliferate to
generate an immune response when triggered to do so.
If an immature lymphocyte in thymus interacts strongly with a self antigen,
displayed as a peptide bound to a self MHC molecule, that lymphocyte receives
a signal that trigger apotosis, the process is negative selection. Antigens that
induce negative selection include proteins that are abundant in the body, such
as plasma proteins and cellular proteins.

Question 3:

What genes are related to X-linked hyper IgM syndrome? (5marks)

Mutations in the CD40LG gene cause X-linked hyper IgM syndrome. This gene
gives instructions for making protein called CD40 ligand, which can found on the
surface of immune system cells, T cells. CD40 ligand attaches to its receptor
protein, which is located on the surface of immune system cells called B cells. It
involved in antibodies production, and initially they are able to make only IgM
antibodies. When CD40 ligand and its receptor protein are connected, they
trigger chemical signals that instruct B cell to make IgG, IgA or IgE anibodies.
CD40 ligand also involved in T cell-macrophage interaction and plays a role in T
cell differentiation.
Mutations in the CD40LG gene cause an abnormal production of CD40 ligand or
prevent this protein production. If CD40 ligand is not attach to its receptor on B
cells, these cells cannot produce IgG, IgA or IgE antibodies. The T-lymphocytes
in patients with X-linked Hyper IgM are unable to instruct B-lymphocytes to
switch their production of immunoglobulins from IgM to IgG, IgA and IgE. As a
result, there are raised levels of IgM and reduced levels of IgG and IgA, an
absence of germinal centres and somatic hypermutation, and sometimes
defects in cell-mediated immunity.
X-linked hyper IgM syndrome is also caused by a mutation in an enzyme,
activation-induced cytidine deaminase (AID). The function of the enzyme is
limited to antibody switching, so the other T-lymphocyte functions of CD40
ligand are not affected, and these patients are less likely to have infections or
cancer. Mutation of AID will lead to giant germinal centres.

Question 4:
(i) There are four types of hypersensitivity reactions. Describe the
mechanisms involved in Type I hypersensitivity reactions. (6 marks)
Immediate or Type I hypersensitivity is caused by release of mediators from
mast cells. It is an IgE antibody- and mast cell-mediated reaction to particular
antigens that cause rapid vascular leakage and mucosal secretions, usually
followed by inflammation. IgE-mediated immediate hypersensitivity reactions
are also known as allergy or atopy.
Immediate hypersensitivity reactions are initiated by the introduction of antigen,
which stimulates TH2 cell responses to secrete IL-4, IL-5 and IL-13 which in turn
favor IgE class switch. IgE is produced in susceptible individuals, and then IgE
binds to Fc receptors (FcRI) on mast cells. A subsequent exposure to the same
antigen cross links the cell-bound IgE and triggers the release of various
pharmacologically active substances. Cross-linking of IgE Fc-receptor is

important in mast cell activation to release mediators that are important in

immediate hypersensitivity.

Induction and effector mechanisms in type I hypersensitivity.

Some mast cell mediators cause a rapid increase in vascular permeability and
smooth muscle contraction. This occurs within minutes of reintroduction of
antigen into previously sensitized individual, so it is called immediate
hypersensitivity. Other mast cell mediators are cytokines that recruits
neutrophils and eosinophils to the site of the reaction over several hours. This
inflammatory component is known as late-phase reaction. The most important
mediators produced by mast cells are vasoactive amines and proteases stored
in and released from granules, new generated products of arachidonic acid
metabolism, and cytokines. The cytokines produced by mast cells stimulate the
recruitment of leukocytes which can cause late-phase reaction.
Mast cells release cytokines, including TNF and IL-4 that promote neutrophil and
eosinophil-rich inflammation. Neutrophils and eosinophils liberate proteases
which causes tissue damage. Eosinophils are activated by IL-5 which is
produced by TH2 cells and mast cells.

(ii) Contact dermatitis is observed in response to the wearing of

disposable gloves. What type of hypersensitivity is associated with this
clinical condition? Explain. (4 marks)
Contact dermatitis can develop from frequent use of hand hygiene products,
exposure to chemicals and glove use. Contact dermatitis can be classifies to
irritant or allergic. Irritant contact dermatitis is non-allergic and develops as
itchy, irritated areas on the skin contacted. On the other hand, allergic contact

dermatitis is associated with type IV hypersensitivity, which can result from

exposure to disposable gloves.
Unlike classic type IV reactions which are mediated by CD4 + T cells and occur in
dermis, allergic contact dermatitis is mediated by CD8 + cells with a TH1-type
cytokine profile and occurs in the epidermis.
Type IV hypersensitivity reactions are caused by activated T H1 cells that are
activated by intracellular pathogens. This causes clonal expansion and
differentiation of antigen-specific cells into T H1 clones. This corresponds to the
sensitization phase of delayed-type hypersensitivity (DTH). The delay occurs
because it takes several hours for circulating effector T lymphocytes to the site
of antigen challenge, responds to the antigen at this site and induces a reaction.
If re-encounter with antigen, it is called effector phase. The antigen-specific T H1
clones undergo further clonal expansion and secretion of a variety of effector
molecules which include cytokines and chemokines. Examples of cytokines are
IFN- , TNF-, IL-2 and IL-3, while the example of chemokines are IL-8, monocyte
chemotactic and activating factor (MCAF) and migration inhibiting factor (MIF).