Immunology is a branch of biomedical science that
covers the study of all aspects of the immune system in all organisms. It deals
with the physiological functioning of the immune system in states of both
health and diseases, malfunctions of the immune system in immunological
disorders (auto immune diseases, hypersensitivities, immune deficiency,
transplant rejection); the physical, chemical and physiological characteristics
of the components of the immune system in vitro, in-situ and in-vivo.
Immunology has applications in several disciplines of science.
Even before the
concept of immunity was developed numerous early physicians characterized organ
that would later prove to be part of the immune system. The key primary
lymphoid organs of the immune system are the thymus and bone marrow
and secondary lymphatic tissue such as spleen, tonsils, lymph vessels, lymph
nodes, adenoids, skin and liver. When health condition warrant,
immune system organs including the thymus, spleen, portions of bone marrow,
lymph nodes and secondary lymphatic tissue can be surgically excised for
examination while patients are still alive.
Many components of the immune system
are actually cellular in nature and not associated with any specific organ but
rather are embedded or circulating in various tissues located through out the
body.
CELLS AND ORGANS OF THE IMMUNE SYSTEM
The cell of the lymphoid system
respond specifically to immunological stimuli. During embryonic life primitive
lymphocytes are derived from haemopoietic stem cells in the yolk sac, liver and
bone marrow along with the cells of the myeloid and erythriod series.
The lymphoid stem cells which
originate from the bone. Marrow, differentiate to form two distinct lymphocyte
subpopulations. One set of populations called T-cells, require to be
differentiated in the thymus before they start functioning as immunologically
competent cells. The cells of the other subpopulation, B-cells are independent
of thymus and are differentiated in the bursa of fabricus, a lymphoid portion
of the hindgut birds, however, human B-cell are differentiated in the bone
marrow.
PRIMARY
LYMPHOID ORGANS of the human immune system are thymus and bone marrow, from
where lymphocytes are developed throughout the life span of an individual.
Secondary lymphoid organ, are tissues rich in lymphocytes through which mature
lymphocytes are constantly migrating and are being activated for a specific
immune response. These secondary lymphoid organ include lymph nodes, white pulp
of the spleen and miccosa associated lymphoid tissue (MALT) eg tonsils, peyers
patehes and appendix. T-cells and B-cells occupy area specific for their
development.
LYMPHOCYTE MARKERS: These are T-lymphocyte cells that are found to have
many surface receptors. They can be identified by the use of monoclonal
antibodies raised against the receptors. These cells are called CD (cluster of
differentiation) markers because one
marker may bind with several monoclonal antibodies forming a cluster. Thus, the
sheep red cell receptor marker is CD2 and is present in all T-cells. And CD4 is
expressed primarily by T-helper inducer cells and are thought to interact with
class II HLA molecules on the surface of antigen presenting cells. T-cells can
be divided into various subsets based on their functions and surface receptors.
The main subsets are:-
(i) T-helper cells (T4):- These T-cells are
responsible for producing lymphokines that positively regulate or help B-cells
in the process of antibody production especially against haptens. B.cells
cannot produce antibodies to haptens until the hapten carrier has interacted
with helper T-cells. T-cells also help effector T-cells in cell mediated immune
response. A large number of T-cells have CD4 receptors on their surface and
also CD3, TCR ∂ β
(ii) T-
suppressor cells (Ts) some T-lymphocytes are able to negatively regulate or
suppress the immune responses of both T-and B-lymphocytes. These cells have CD8
surface markers. Overactivity of Ts cells can result in the state of
immunodeficiency leading to many intracellular opportunistic infections such as
that by pneumocystis carinii.
(iii) T-
inducer cells. Generation of T-suppressor cells is induced by T-inducer cells.
They also have a CD4, CD3, TCRỵ8 marker on the surface.
(iv) T-cytoxic
cells (Tc) some T-cells are lethal or cytoxic for cells that carry specific
antigen on their surfaces eg vinus infected cells, tumor cells or grafted
(transplanted) cells. The Tc cells may also mediate the graft- versus host
(GVH) reaction. A large number of Tc-cells have CD8, CD3, TCR ∂ β
surface markers.
(v) T-
delayed hypersensitivity cells (TDH) these T-cells secrete lymphokines which
produce an inflammatory response in delayed allergic reaction or in defence
against infection caused by bacteria, fungi, and parasites all TDH have CD4
surface marker. They also have CD16, CD56 but not CD3.
MONOCLONAL ANTIBODIES
These are single specific antibodies that are the same
because they are made up by identical immune cells that are all clones of a
unique parent cell. Monoclonal antibodies have high affinity and avidity for
antigen to bind to the same epitope. Given almost any substance, it is possible
to produce monoclonal antibodies that specifically bind to that substance, they
can serve to defect or purify that substance.
PRODUCTION-HYBRIDOMA
CELL TECHNIQUE
Monoclonal antibodies are typically made by fusing
cells (myeloma and spleen) from mouse that has been immunized with the desired
antigen. A technique for the production of a pure, specific antibody by such a
clone of cells is called a hybridoma technique. The two types of cells used for
the hybrid are;-
1. Multiple
Myeloma Cells: multiple myeloma is a malignant tumour of antibody producing
plasma cells, usually of no determined specificity. This multiple myeloma cells
selected for this technique can multiply indefinite. They must lack the enzyme
hypoxanthine guanine phosphoribosyl transferase
(HGPRT) because myeloma cells does not synthesize this enzyme which is
necessary for the synthesis of nucleic acids. The absence of HGPRT is not a
problem for these cells unless the de novo purine synthesis pathway is
disputed. This pathway are by passed by exposing cells to aminopterian (a folic
acid analogue, which has the ability to inhibit dihydrogolate reductase, DHFR)
they become fully auxotrophic for nucleic acids requiring supplementation
(briclone) to survive.
2. Splenic
cells of a Mouse Immunized with the Specific Antigen
A mouse is immunized with the
antigen for which the antibody is to be produced, the mouse responds by
producing antibodies to many determinants or epitopes on the antigen injected.
The mouse spleen is removed, minced and prepared as a single cell suspension.
This should include the B-cell population, which produces antibodies to the
injected antigen. These B-cells cannot multiply indefinitely but posses the
enzyme hypoxanthine guanine phosphoribosy transferase (HGPRT) and can grow in
hypoxanthine, ammopterin and thymidine medium (HAT) this culture medium is
selective for fused hybridoma cells. Unfused mycloma cells cannot grow because
they lack HGPRT and this cannot replicate their DNA. Unfused spleen cells
cannot grow indefinitely because of their limited life span. Only fused hybrid
cells, referred to as hybridomas, are able to grow indefinitely in the media
because the spleen cell partner supplies HGPRT and the mycloma partner has
traits that make it dimilar to caner cell.
FUSION OF SPLENIC B-CELLS WITH MULTIPLE
MYELOMA CELLS
When these two types of cells are suspended in a HAT
medium in the presence of polyethelyne glycol (PEG) a spontaneous fusion occurs
between some cells resulting in a hybrid with the hybrid cells are capable of
growing indefinitely in HAT medium and produce antibodies to the desired
antigen. The hybrids formed by the
fusion will be the only cells surviving indefinitely. This is the hybridoma
technique.
FLOW CYTOMETRY
This is a laser based, biophysical
technology used in cell counting, sorting, biomarker defection and protein
engineering. It operates by suspending cells in a stream of fluid and passing
them by an electronic detection apparatus. It allows simultaneous
multiparametric analysis of physical and or chemical characteristics of up to
thousands of particles per second.
Flow cytometry in routinely used in
the diagnosis of health disorders, especially blood cancers but has many other
applications in basic research, clinical practice, and clinical trials. If
physically sort particles based on their particles, so as to purify population
of interest.
Modern flow cytometers are able to
analyze several thousand particles per second in real time. A flow cytometer is
similar to microscope except that instead of producing an image of the cell.
Flow cytometers offers an automated quantification of large numbers of cell
parameters. To analyze solid tissue, a single-cell suspension must first he
prepared.
COMPONENTS OF FLOW CYTOMETER
1. Flow
cell –Made up of liquid stream
(sheath fluid) this component carries and aligns the cells so that they pass in single file through the light beam
for sensing.
2. Measuring
System- Commonly used for measurement of impedance
(or conductivity) and optical systems such as lamps (mercury, xenon); high
power water-cooled lasers (argon, krypton, dye laser); low-power air-cooled
lasers (argon (488nm), red- HeNe (633nm) green- HeNe, HeCd (UV); diode laser
(blue, green red, violet) resulting on light signals.
3. Detector
and analogue –To-digital conversion
(ADC) system –which generates FSC and SSC as well as fluorescence signals from
light into electrical signals that can be processed by a computer.
4. An
Amplification System –Linear or logarithmic
5. A
Computer for analysis of the signals.
The process of collecting data from
samples using the flow cytometer is called acquisition. Acquisition is
mediated by a computer physically connected to the flow cytometer and the soft
were which handles the digital inferface with the cytometer. The software is
capable of adjusting parameters (ie voltage, compensation etc) for the sample
being tested and also assists in displaying initial sample information while
acquiring sample data to ensure that parameters are set correctly.