IMMUNOLOGY AND SEROLOGY
Chapter 1: Introduction to Immunology
HISTORY OF IMMUNOLOGY
 430 B.C – Thucydides noticed that smallpox survivors
did not get re-infected.
 1000 A.D – Chinese introduced variolation, the practice
of inhaling smallpox scabs to induce protection.
 Around 1700 – Lady Mary Wortley Montagu helped
spread variolation to Turkey and the rest of Europe.
 1798 – Edward Jenner discovered smallpox vaccination
and cross-immunity.
 1880-1881 – Louis Pasteur discovered the first
attenuated vaccine through his chickens (cholera) and
applied it to a rabies patient. Father of Immunology.
 1883 – 1905 – Elie Metchnikoff discovered cellular
theory of immunity through phagocytosis.
 1890 – Emil von Behring and Kitasata – Discovered the
theory of Humoral Immunity (noncellular elements)
 1891 – Koch – Demonstration of cutaneous (delayed
type) hypersensitivity.
 1903 – Almoth Wright linked the two theories and
observed that certain humoral factors called
“opsonins” acted to coat a bacteria so that it would be
more susceptible to phagocytosis.
 1938 – Marrack hypothesized about antigen-antibody
binding
 1949 – Salk and Sabin developed the polio vaccine
 1951 – Reed developed a vaccine against the yellow
fever
 1985 – 1987 – Identification of genes for T- cell
receptor
 2005 – Frazer developed the Human Papillomavirus
vaccine
DEFINITION OF TERMS
Immunology – Study of the response of a host when
foreign substances are introduced to it.
Antigen – Foreign substance that can induce an immune
reaction.
Antibody – A molecule produced by the body to neutralize
and eliminate antigens.
Immunity – Condition of being resistance to infection
Immune System – The whole system including the cells,
tissues and molecules that mediate immunity
Immune Response – The coordinated action of the
immune system
CELLS OF THE IMMUNE SYSTEM
Neutrophils
 50-70% of Total WBCs. 10-15um and has between two
to five lobes
 Contains granules
 Primary Granules – Myeloperoxidase, Elastase,
Proteinase 3, Lysozyme, Cathepsin G, and
Defensins
 Secondary Granules – Collagenase, Lactoferrin,
Lysozyme, NADPH Oxidase.
 Tertiary Granules – Gelatinase and Plasminogen
activator
 Half in Marginating pool, Half in Circulating pool
 Diapedesis – Movement through blood vessel walls
 Macrophages help induce endothelial cells to express
selectins.
 Chemotaxins - chemical messengers that cause cells to
migrate in a particular direction
 Once in tissues, neutrophils have lifespan of 5 days.
Basophils
 Less than 1% of all circulating WBCs
 Contain deep-bluish purple granules that often obscure
the nucleus
 Contains histamine, a small amount of heparin, and
eosinophil chemotactic factor-A
 IgE binds to Basophils
 The granules lack hydrolytic enzymes, although
peroxidase is present. Basophils exist for only a few
hours in the bloodstream.
Mast Cells
 Resemble basophils but are actually connective tissue
cells of mesenchymal origin.
 Long life span between 9 and 18 months
 Enzymes include - acid phosphatase, alkaline
phosphatase, and protease
 Also binds IgE
Eosinophils
 1-3% of Circulating WBCs
 Number increases in allergic reactions and parasitic
infections
 Nucleus is usually bilobed and ellipsoidal and is often
eccentric.
 Takes up the acid eosin dye
 Cytoplasm filled with large orange granules
 Primary Granules – Acid phosphatase, arylsulfatase
 Specific Granules - major basic protein, eosinophil
cationic protein, eosinophil peroxidase, and eosinophil-
derived neurotoxin.
Monocytes/Macrophage
 4-10% of Circulating WBCs. Stay in peripheral blood for
up to 70 hours.
 Largest cells in the peripheral blood. 12-22um. Average
of 18um
 Irregularly folded or horseshoe-shaped nucleus that
occupies almost one-half of the entire cell’s volume
 Abundant dull grayish blue cytoplasm with ground-
glass appearance
 Granules include peroxidase, acid phosphatase, and
arylsulfatase (similar to neutrophils) and B-
glucoronidase, lysozyme, and lipase.
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 Macrophages have specific names according to their Mechanism Antibody Cell mediated
particular tissue location. Mediated
 Alveolar Macrophages – Lungs Cells B Lymphocytes T Lymphocytes
 Kupffer Cells – Liver involved
 Microglial Cells – Brain Mode of Abs in Serum Direct cell-to-cell
 Histiocytes – Connective tissue action contact or soluble
 Functions include microbial killing, tumoricidal activity, products secreted by
intracellular parasite eradication, phagocytosis, cells
secretion of cell mediators, and antigen presentation Purpose Primary defense Viral and Fungal
against bacterial infections, Intracellular
Dendritic Cells antigen organisms, tumor
 Main function is to be an APC antigens, and graft
 Classified according to tissue location rejections
 Langerhans Cells – Skin and Mucous Membranes
 Interstitial Dendritic Cells – Populate major organs INNATE AND ADAPTIVE IMMUNITY
and G.I Tract Kind Active Passive
 Interdigitating dendritic cells – Present in Natural Artificial Natural Artificial
secondary lymphoid tissue and thymus Mode of Infection Vaccination Transfer Infusion
 Most potent phagocytic cell in tissue Acquisition in vivo or of Abs
colostrums
NK Cells Ig Yes Yes No No
 Large granular lymphocytes Produced
 Play a role in innate and adaptive immune response by the
host?
 Kills virally infected cells. No CD4 and CD8 markers.
Duration of Long Long Short Short
immune
Lymphocytes response
 Key Cell involved in the immune/specific response
 Represent 20-40% of circulating WBCs
Chapter 2: Natural Immunity
 Large rounded nucleus that is usually indented
FIRST LINE OF DEFENSE
 Separated into two main classes
 Unbroken skin and mucous membranes
 Recognize foreign antigens, directly destroy some cells,
 Lactic acid in sweat and fatty acids from sebaceous
or produce antibodies as plasma cells
glands maintain skin pH at 5.6
 Circulation is complex and is regulated by different cell
 Mucus and cilia adhering to the membranes of the
surface adhesion molecules and by chemical
nose and nasopharynx traps microorganisms. Earwax
messengers called cytokines
protects auditory canals
 Flushing action of liquid and solid wastes through
LINES OF DEFENSE
urinary and gastrointestinal processes
First Line of Defense
 Acidity (up to pH 1) and alkalinity of G.I tract and
 External. First barrier to infection is unbroken skin and
acidity of vagina (Through lactic acid in vagina that
mucosal membrane surfaces.
maintains pH at 5)
Second Line of Defense
 Tears and Saliva contain Lysozyme that attacks and
 Internal. How body resists infection after
destroys the cell wall of certain bacteria
microorganisms have penetrated the first line of
defense.
SECOND LINE OF DEFENSE
 Natural immunity (inborn or innate resistance)
Phagocytosis
Third Line of Defense
 Can be divided into six steps
 Internal.
1. Chemotaxis
 Adaptive/Acquired immunity and if the microorganism
 The physical occurrence of damage to tissues, by
overwhelms the natural immunity.
trauma or microbial multiplication, releases
substances such as activated complement
HUMORAL AND CELL-MEDIATED components and products of infection to initiate
IMMUNITY phagocytosis.
Humoral Cell-Mediated  Mediators produced include interleukin-1 (IL-1),
Type of Extracellular Intracellular which is released by macrophages in response to
Microbe/Ag

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 FAR EASTERN UNIVERSITY – MANILA
infection or tissue injury. Another is histamine.
Mediators cause capillary and venular dilation.
 Neutrophils can be found in the site within an
hour. They roll along the blood vessels and then
penetrate through to the tissue by means of
diapedesis.
 P-selectin on endothelial cells is the primary
adhesion molecule for capture and the initiation of
rolling. Functional E-selectin ligands include CD44
2. Adherence
 The receptors on phagocytes come into contact
with the foreign particle enhanced by opsonins
(greek: to prepare for eating)
 Opsonins include CRP, complement components,
and antibodies
 PPRR (Primitive pattern recognition receptors) –
Direct interaction
3. Engulfment
 The cellular cytoplasm flows around the particle
and eventually fuses with it.
 The principal factor in determining whether
phagocytosis can occur is the physical nature of the
surface of the bacteria and phagocytic cell.
 This invagination leads to the formation of an
isolated vacuole (phagosome) within the cell.
4. Phagosome Formation
5. Fusion
 Contact with cytoplasmic granules takes place, and
fusion between granules and the phagosome
occurs. At this point, the fused elements are known
as a phagolysosome.
 An increase in oxygen consumption, known as the
respiratory, or oxidative, burst, occurs within the
cell
 Patients with chronic granulomatous disease (CGD)
= NADPH mutation
6. Digestion and Destruction
 A radical known as O2 – (superoxide) is formed.
Superoxide is highly toxic but can be rapidly
converted to more lethal products. By adding
hydrogen ions, the enzyme superoxide dismutase
(SOD) converts superoxide to hydrogen peroxide or
the hydroxyl radical OH.
 The granules then release their contents, and
digestion occurs. Any undigested material is
excreted from the cells by exocytosis.
 Nitroblue tetrazolium dye test – Test for activation of
NADPH Oxidase. Colorless to deep blue
 Neutrophil-Extracellular Traps (NETs) - Composed of
chromatin components, including histones, and
neutrophil antimicrobial proteins. Microbes are
trapped in NETs, where they encounter high
concentrations of antimicrobial proteins
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Toll-like receptors (TLR)
 The highest concentration of these receptors occurs on
monocytes, macrophages, and neutrophils
 There are 11 TLRs in humans. Each of these receptors
recognizes a different microbial product.
 For ex. TLR2 recognizes teichoic acid and peptidoglycan
found in gram-positive bacteria, while TLR4 recognizes
lipopolysaccharide found in gram-negative bacteria
NK Cells
 Do not express T cell and B cell markers and are
generally larger than them.
 Make up 5-10% of circulating lymphoid pool.
 Ability to mediate cytolytic reactions and kill target
cells without prior exposure to them
 Arise from the CLP and differentiate into a T/NK Cell
 IL-15 plays a critical role in NK’s development
 CD16+ (receptor for Fc portion of IgG) CD56+
 NK Cell receptors
1. Inhibitory receptors – based on MHC Class I
protein (If NK cells react with MHC class I
proteins, then inhibition of natural killing
occurs.) (Diseased and cancerous cells tend to
lose their ability to produce MHC proteins. NK
cells are thus triggered by a lack of MHC
antigens, sometimes referred to as recognition
of “missing self)
2. Activatory receptors - These proteins are
named MICA and MICB. Receptors called
CD94/ NKG2C and CD94/NKG2D on NK cells
bind MICA or MICB.
3. CD16 receptor for IgG (FcyR) – For Antibody-
dependent cell cytotoxicity
 NK cells release substances called perforins and
granzymes
Interferons (Natural Humoral)
Interferon Origin Prominent Biological Activity
IFN-α Leukocytes Antiviral, increased MHC class I
expression
IFN-β Fibroblasts, Same ^
epithelial
cells
IFN-γ T cells, NK Major macrophage activator;
cells induces MHC class II molecules on
many cells and can synergize with
TNF; augments NK cell activity;
antagonist to IL-4.
Type I IFN = IFN-a and IFN-B
 mediate the early innate immune response to viral
infections
 structurally different but that bind to the same cell
surface receptor and induce similar biologic responses.
 FAR EASTERN UNIVERSITY – MANILA
Type II IFN = IFN-y
 Principal macrophage-activating cytokine and serves a
critical function in innate immunity and in specific cell-
mediated immunity
Inflammation
 Overall reaction of the body to injury or invasion by an
infectious agent
 Cardinal Signs: Tumor, Rubor, Dolor, Calor, Functio
Laesa
 Major events:
 Increased blood supply to the affected area
 Increased capillary permeability
 Migration of WBCs (mainly neutrophils, from the
capillaries to the surrounding tissue)
 Migration of Macrophages
 Neutrophils, which are mobilized within 30 to 60
minutes after the injury, are the major type of cell
present in acute inflammation. Neutrophil emigration
may last 24 to 48 hours.
 Migration of macrophages from surrounding tissue and
from blood monocytes occurs several hours later and
peaks at 16 to 48 hours
Fever and Sepsis
 If an inflammation overwhelms the whole body,
systemic inflammatory response syndrome (SIRS) is
diagnosed (alteration of body temp <36 or >38,
increased heart rate, respiratory rate, and a leukocyte
count of >12 x 109/L)
 Sepsis = SIRS + Infection
 Severe sepsis = Sepsis + Evidence of Organ dysfunction
 Fever can be induced by bacterial toxins, ag-ab
complexes, and IL-1 (along with IL-6 and TNF) which all
act as pyrogens. Pyrogens reset the body’s thermostat
and cause a rise in temperature.
ACUTE PHASE PROTEINS
 Innate body defense. Nonspecific indicator of
inflammatory process
 Normal serum constituents that increase rapidly by at
least 25 percent due to infection, injury, or trauma to
the tissues
 They are produced primarily by hepatocytes (liver
parenchymal cells) within 12 to 24 hours in response to
an increase in certain intercellular signaling
polypeptides called cytokines
 CRP
 originally thought to be an antibody to the c-
polysaccharide of pneumococci
 increases rapidly within 4 to 6 hours following
infection, surgery, or other trauma (Levels increase
dramatically as much as a hundredfold to a
thousandfold, reaching a peak value within 48
hours. They also decline rapidly with cessation of
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the stimulus. CRP has a plasma half-life of about 19
hours)
 acts somewhat like an antibody, as it is capable of
opsonization (the coating of foreign particles),
agglutination, precipitation, and activation of
complement by the classical pathway
 CRP is the most widely used indicator of acute
inflammation
 Recent research indicates that an increased level of
CRP is a significant risk factor for myocardial
infarction and ischemic stroke in men and women
who have no previous history of cardiovascular
disease. A concentration of more than 2 mg/L has
been defined as the threshold for high
cardiovascular risk
 Serum Amyloid A
 Apolipoprotein synthesized in liver
 associated with HDL cholesterol, and it is thought
to play a role in metabolism of cholesterol
 increase significantly more in bacterial infections
than in viral infections
 Complement
 Mannose-binding proteins (Mannose-binding lectin)
 trimer that acts as an opsonin and is calcium-
dependent
 recognize foreign carbohydrates such as mannose
and several other sugars found primarily on
bacteria, some yeasts, viruses, and several
parasites
 A1-Antitrypsin
 major component of the alpha band when serum is
electrophoresed
 general plasma inhibitor of proteases like trypsin
and elastase
 counteract the effects of neutrophil invasion
during an inflammatory response
 deficiency can result in premature emphysema,
especially in individuals who smoke
 Haptoglobin
 primary function is to bind irreversibly to free
hemoglobin
 Once bound, the complex is cleared rapidly by
Kupffer and parenchymal cells in the liver. Levels
decrease during hemolysis
 important role in protecting the kidney from
damage and in preventing the loss of iron by
urinary excretion
 Fibrinogen
 most abundant of the coagulation factors
 forms the fibrin clot through cleavage by thrombin
 increases the strength of a wound and stimulates
endothelial cell adhesion and proliferation
 Ceruloplasmin
 principal copper-transporting protein in human
plasma
 ceruloplasmin acts as a ferroxidase, oxidizing iron
from Fe2+ to Fe3+
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 depletion of ceruloplasmin is found in Wilson’s
disease
Chapter 3: Complement System
THE COMPLEMENT SYSTEM
 Complex series of more than 30 soluble and cell-bound
proteins that interact in a very specific way to enhance
host defense mechanisms against foreign cells.
 Jules Bordet elucidated the nature of complements
 Complement promotes opsonization and lysis of
foreign cells and immune complexes
 Most plasma complement proteins are synthesized in
the liver, with the exception of C1 components, which
are mainly produced by intestinal epithelial cells, and
factor D, which is made in adipose tissue
 There are three pathways: Classical pathway,
Alternative pathway, and the Lectin Pathway
CLASSICAL PATHWAY
 The complement proteins were named by the
sequence wherein the proteins were isolated
 Main antibody-directed mechanism for triggering
complement activation
 IgM, IgG1, IgG2, and IgG3
 IgG3>IgG1>IgG2
 May also be activated by CRP, several viruses,
mycoplasmas, some protozoa, and certain gram-
negative bacteria such as Escherichia coli
 Divided into three stages: Recognition unit, Activation
unit, and Membrane attack complex
Recognition Unit
 Consists of three subunits: C1q, C1r, and C1s which is
stabilized by calcium.
 Binding occurs at the CH2 region for IgG and at the CH3
region for IgM
 Once C1s is activated, the recognition stage ends.
Activation Unit
 Ultimately results in production of C5 convertase
 C1s cleaves C4 to split off C4a and leaves of C4b. C4b
must bind to protein or carbohydrate within a few
seconds, or it will react with water molecules to form
iC4b, which is rapidly degraded.
 C2 is the next component to be activated. When
combined with C4b in the presence of magnesium ions,
C2 is cleaved by C1s to form C2a and C2b.
 Combination of C4b and C2a is known as C3 convertase
(C4b2a)
 This cleaves C3 into two parts: C3a and C3b (C3 is the
major constituent of the complement system)
 C3b also serves as a powerful opsonin
 C4b2a3b = C5 convertase (last step in the formation of
the activation unit. The cleaving of C5 with deposition
of C5b at another site on the cell membrane
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constitutes the beginning of the membrane attack
complex (MAC)
Membrane Attack Complex
 C4b2a3b splits of C5 into C5a and C5b
 C5b is extremely labile, and it is rapidly inactivated
unless binding to C6 occurs
 Subsequent binding involves C6, C7, C8, and C9
 Lysis can already be observed at C5bC6C7C8 but this is
small and is capable of lysing erythrocytes but not
nucleated cells. For lysis of nucleated cells to be
achieved, C9 must bind to the complex.
ALTERNATIVE PATHWAY
 Part of innate or natural immunity
 Pathway was originally named for the protein
properdin
 In addition to properdin, the serum proteins that are
unique to this pathway include factor B and factor D
 Triggering substances include bacterial cell walls,
especially those containing lipopolysaccharide; fungal
cell walls; yeast; viruses; virally infected cells; tumor
cell lines; and some parasites, especially trypanosomes
 In plasma, native c3 is not stable. Water hydrolyzes C3
to form C3b (or iC3). This then binds to Factor B and
once bound, factor D cleaves B into Ba and Bb. C3Bb is
then formed. This enzyme is stabilized by properdin,
and it continues to cleave additional C3. If a molecule
of C3 remains attached to the C3bBbP enzyme, the
convertase now has the capability to cleave C5 (C5
convertase like) C5 is cleaved to produce C5b, the first
part of the membrane attack unit.
LECTIN PATHWAY
 Lectins are proteins that bind to carbohydrates
 Part of the innate immunity.
 involves nonspecific recognition of carbohydrates that
are common constituents of microbial cell walls and
that are distinct from those found on human cell
surfaces
 MBL (Mannose-binding Lectin) binds to mannose or
related sugars in a calcium-dependent manner to
initiate this pathway
 MBL is considered an acute phase protein
 MBL is similar to C1q. Activates MBL-serine proteases
(MASPs). Once MBL binds to a cellular surface, MASP-
2, which is homologous to C1s, autoactivates. MASP-2
thus takes the active role in cleaving C4 and C2.
SYSTEM CONTROLS
Regulation of Classical and Lectin Pathways
 C1 Inhibitor – main role is to inactivate C1. Also
inactivates MASP-2. (inhibits activation at the first
stages of both the classical and lectin pathways)
 Factor I - inactivates C3b and C4b when bound to one
of four main regulators:
 FAR EASTERN UNIVERSITY – MANILA

 C4b-binding protein (C4BP) – Soluble  CR3 (CD11b/CD18) - binds particles opsonized with
 capable of combining with either fluid-phase or iC3b, a C3b degradation product in a calcium-
bound C4b, so C4b cannot bind C2 and is made dependent fashion. Key role in mediating
available for degradation by factor I. phagocytosis of particles coated with these
 Complement receptor 1 (CR1) – Cell-bound complement fragments.
 CD35  CR4 (CD11c/CD18) - function appears to be similar to
 Found mainly on peripheral blood cells that of CR3
 Binds C3b and C4b but has the greatest affinity
for C3b MANIFESTATIONS OF ACTIVATION
 One of its main functions is to act as a receptor  Aside from cell lysis complements have other uses.
on platelets and red blood cells and to mediate Complement molecules’ effector functions can be
transport of C3b-coated immune complexes to classified into three main categories: anaphylatoxins,
the liver and spleen chemotaxins, and opsonins
 Immune Adherence - ability of cells to bind  Anaphylatoxin - small peptide that causes increased
complement-coated particles vascular permeability, contraction of smooth muscle,
 Membrane cofactor protein (MCP) – Cell-bound and release of histamine from basophils and mast cells
 CD46 (C3a, C4a, C5a)
 Found on virtually all epithelial and endothelial  C5a also serves as a chemotaxin
cells except erythrocytes  C3a, C4a, and C5a can be inactivated by
 MCP is the most efficient cofactor for factor I– Carboxypeptidase N
mediated cleavage of C3b  C4b, C3b and iC3b can act as opsonins
 Decay accelerating factor (DAF) – Cell-bound
 CD55 COMPLEMENT DISEASE STATES
 Wide tissue distribution  Complements can be harmful if:
 Capable of dissociating both classical and 1. Activated systemically on a large-scale such as
alternative pathway C3 convertases gram-negative septicemia
 The presence of DAF on host cells protects 2. Activated by tissue necrosis
them from bystander lysis and is one of the 3. Red Cell lysis (PNH)
main mechanisms used in discrimination of self Deficiency Associated Disease
from nonself, because foreign cells do not C1 Lupus-like; recurrent infections
possess this substance C2 Lupus-like; recurrent infections;
Atherosclerosis
Regulation of Alternative Pathways C3 Severe recurrent infections;
 Factor H - principal soluble regulator of the alternative glomerulonephritis
pathway C4 Lupus-like
 Binds to C3b C5-C8 Neisseria infections
 C3b in the fluid phase has a hundredfold greater C9 No known disease associated
affinity for factor H than for factor B
C1NH Hereditary Angioedema
 On cell surfaces, C3b preferentially binds factor B
DAF PNH
 Factor H can also act as a cofactor for factor I (to
MIRL PNH
breakdown C3b)
Factor H/Factor I Recurrent pyogenic infections
Regulation of Terminal components MBL Pneumococcal diseases, sepsis,
Neisseria infections
 S protein (Vitronectin) – Binds C5b67 complex
Properdin Neisseria infections
 Membrane inhibitor of reactive lysis (MIRL) (CD59) -
MASP-2 Pneumococcal diseases
acts to block formation of the membrane attack
complex.  Most common (major pathway component) deficiency
is C2.
 Second most common is MBL deficiency
COMPLEMENT RECEPTORS AND ROLES
 Most serious is C3 deficiency since it is the key
 CR1 (CD35) – Ligand is C3b and C4b. Cofactor for factor
mediator in all pathways.
I. Transport of immune complexes.
 PNH - These individuals appear to have a deficiency in
 CR2 (CD21) – Ligand is C3d (degradation products of
the glycophospholipid anchor of the DAF/MIRL
C3b). EBV entry to B cells. Found on mature B cells.
molecule that prevents its insertion into the cell
Important role as part of the B-cell coreceptor for
membrane
antigen.

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LABORATORY DETECTION
Individual Components (Quantitative)
 Most frequently used:
 RID - Sensitive Technique takes at least 24 hours.
Radius of Circle = Antigen concentration
 Nephelometry – Measures amount of light
scattered by reagent antibody and complement
antigen complexes. Results are available quickly.
Also sensitive.
Assays for Classical Pathway (Qualitative)
 CH50 Assay (Hemolytic titration) – Most commonly used
 Expressed in CH50 units which is the reciprocal of
the dilution that is able to lyse 50 percent of the
sensitized sheep erythrocytes.
 Another CH50 assay has been developed and is based
on lysis of liposomes that release an enzyme when
lysed.
 Lytic activity through radial hemolysis
 ELISA – Best screen for complement abnormalities.
Color change indicates a positive reaction.
Assays for Alternative Pathway (Qualitative)
 AH50 – Almost same as CH50 but magnesium chloride
and EGTA (ethylene glycol tetraacetic acid) are added
to chelate calcium
 ELISA – Detect C3bBbP or C3bP
A test system to detect all pathways:
Strip for classical pathway – Coated with IgM
Strip for alternative pathway – Coated with LPS
Strip for lectin pathway – Coated with Mannose
Decreased levels of complements/activity can be due to:
1. Decreased production
2. In vivo consumption
3. In vitro consumption
Complement inactivation in vitro
1. Heat
 56oC for 30 mins – Inactivates C1, C2 and C4
 50oC for 20 mins – Inactivates Factor B
2. Anticoagulants
 EDTA – Calcium and Magnesium
 Heparin – Inhibits cleavage of C4 by C1
3. Storage – Alters C4
Chapter 4: Adaptive Immunity
PRIMARY LYMPHOID ORGANS
 Bone Marrow
 Largest tissue of the body
 T, B, and NK cells arise from a common precursor
known as the common lymphoid precursor (CLP)
 Center for antigen-independent lymphopoiesis
 B-cell maturation takes place here
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 In the blood, B cells = 10-20%, T cells = 61-89%, and
NK Cells = up to 5-22%
 Thymus
 Small, flat, bilobed organ found in the thorax, or
chest cavity, right below the thyroid gland and
overlying the heart
 Thymus atrophies with age
 T cells develop here. They mature from the Cortex
to the Medulla in a span of 2-3 weeks.
SECONDARY LYMPHOID ORGANS
 After differentiation in Primary Lymphoid Organs, T
cells and B cells go the the Secondary Lymphoid
organs.
 Lymphocytes travel by way of the Thoracic Duct
 Lymphopoiesis occurs in secondary lymphoid organs.
 Most naïve or resting lymphocytes die within a few
days after leaving the primary lymphoid organs
Spleen
 Serves as a filtering mechanism for antigens in the
bloodstream
 Largest secondary lymphoid organ
 Divided into two: Red Pulp and White Pulp
 Red Pulp – Function is to destroy RBCs
 White Pulp – Contains the lymphoid tissue arranged
around arterioles
 Attached to the PALS are the Primary Follicles and a
Marginal Zone
Lymph Nodes
 Filters fluid from tissues
 Located along lymphatic ducts
 Numerous near joints and where the arms and legs join
the body
 Tissue is organized into: Outer cortex, Paracortex, Inner
medulla
 Enter via afferent lymphatic vessels and exit via
efferent lymph vessels
 In Outer cortex one can find the:
 Primary Follicles – Contains macrophages, follicular
dendritic cells and mature, resting B cells
 Secondary Follicles – Contains antigen-stimulated
proliferating B cells. The inside of a secondary
follicle is called a Germinal Center. Plasma cells
and Memory B cells are found here.
 Paracortex – T lymphocytes are found here
 Transit time through a lymph node is approximately 18
hours
 Contact with antigen causes lymph nodes to shut down
= Lymphadenopathy
Other Secondary Organs
 MALT (Mucosal associated lymphoid tissue) - found in
the gastrointestinal, respiratory, and urogenital tracts.
Peyer’s patches – lower ileum of intestinal tract
 FAR EASTERN UNIVERSITY – MANILA
 Tonsils - respond to pathogens entering the respiratory
and alimentary tracts
 Appendix
 CALT (Cutaneous associated lymphoid tissue)
SURFACE MEMBRANE MARKERS
Clusters of Differentiation (CD) - acts as a reference in
standardizing names of membrane proteins found on all
human white blood cells
CD2 – T cells (Also NK Cells)
CD3 – T cells (Associated with T cell antigen receptor)
CD4 – Helper T cells (Coreceptor for MHC Class II)
CD5 – Mature T cells; Subset of B cells (B1)
CD8 – Cytotoxic T cells (Coreceptor for MHC Class I)
CD10 – B and T cell precursor. BM Stromal Cells (marker for
pre-B CALLA)
CD16 – NK Cell (Low affinity Fc receptor)
CD19 – B cells (Part of B cell coreceptor)
CD21 – B cell and Immature thymocytes (Part of B cell
coreceptor) (Receptor for C3d)
CD23 – B cells (IgE synthesis) (triggers release of IL-1, IL-6,
and GM-CSF from monocytes)
CD25 – Activated T cells and B cells (Receptor for IL-2)
CD44 – Leukocytes (Adhesion molecule)
CD45R – All hematopoietic cells
CD56 – NK cells
CD94 – NK cells
T CELL DEVELOPMENT AND CELL-
MEDIATED IMMUNE RESPONSE
Thymocytes – Lymphocyte precursors
Maturation is from cortex to medulla and is driven by
chemokines
IL-7 – Critical for T cell growth and development
Early surface markers are CD44 and CD25
Double Negative Stage
 Lack CD4 and CD8 markers (hence they are known as
double-negative thymocytes (DN)
 Rearrangement of the genes that code for the antigen
receptor known as TCR begins (alpha chain and a beta
chain) (CD3 – main part of T cell receptor)
 B chain rearrangement occurs first (B chain + CD3 = pre
T cell receptor) (Leads to TCR2)
 Some thymocytes (10%) rearrange and express two
other chains, gamma (γ) and delta (δ). (TCR1)
Double Positive Stage
 Thymocytes express both CD4 and CD8
 Rearrangement for the alpha chain starts
 Positive Selection starts - allows only double-positive
cells with functional TCR receptors to survive.
Recognize MHC.
 After positive selection, a second process starts.
Negative Selection - Strong reactions with self-peptides
send a signal to delete the developing T cell
8|Page
Mature T cells
 Helper or Inducer T cells/CD4+ - recognize antigens
presented through MHC Class II
 Th1 – Produce IFN-γ and TNF-B. Protect cells
against intracellular pathogens.
 Th2 – Produce IL-4, IL-5, IL-10, and IL-13. Help
B cells produce antibody against extracellular
pathogens.
 Treg – CD4+ and CD25+ (These cells comprise
approximately 5 to 10 percent of all CD4-
positive T cells). Suppress immune response to
self antigens by producing IL-10 and
transforming growth factor B.
 Cytotoxic T cells/CD8+ - recognize antigens presented
through MHC Class I
Antigen Activation
 When antigen recognition occurs, T lymphocytes are
transformed into large activated cells
 Express receptors for IL-2
 T lymphoblasts differentiate into functionally active
small lymphocytes that produce
 This is called Cell-mediated immunity
 In addition to effector cells, T memory cells are also
generated
B CELL DEVELOPMENT AND HUMORAL
IMMUNE RESPONSE
Pro-B cells
 Earliest B cell precursor.
 pro-B cell has distinctive markers that include surface
antigens CD19, CD45R, CD43, CD24, and c-Kit
 Intracellular proteins found in this stage include
terminal deoxyribonucleotide transferase (TdT) and
recombination activating genes (RAG-1 and RAG-2)
 Gene rearrangement of the DNA that codes for
antibody production (Rearrangement of genes on
chromosome 14, heavy-chain, takes place first)
 C-kit interacts with stem cell factor
Pre-B cells
 First heavy chains synthesized are the μ chains. Pre-B
cells also lose the CD43 marker as well as c-Kit and TdT
 Express a surrogate light chain
 Mu chains + surrogate light chain on cell surface = pre
B-cell receptor
Immature B cells
 Distinguished by appearance of complete IgM
molecules on cell surface.
 Rearrangement for genetic sequence coding for light
chain starts (Chromosome 2 and 22)
 Other surface proteins: CD21, CD40, MHC Class II
 Immature B cells that tightly bind self-antigens through
cross-linking of surface IgM molecules receive a signal
 FAR EASTERN UNIVERSITY – MANILA

to halt development, and they are eliminated or  3 layers: Plasma at the top, a mononuclear layer
inactivated banding on top of the density gradient solution, and
RBC and granulocytes at the bottom
Mature B cells  Segregation into subsets is accomplished via flow
 In spleen become known as Marginal Zone B cells once cytometry.
B cell matures.  Rosette Technique - Sheep cells attach to the CD2
 In other secondary lymphoid organs become known as antigen, found only on T cells (200 cells are counted)
Follicular B cells once it matures. Not as precise as the previous technique due to factors
 Exhibit IgD as surface receptor in addition to IgM such as autoagglutination.
 Life span of a mature B cell is only a few days if not
activated. If activated, transforms to a blast stage that Chapter 5: Antigen and MHC System
transforms to memory cells or antibody-producing Immunogen – Macromolecules capable of eliciting the
plasma cells. formation of immunoglobulins or sensitized cells in an
immunocompetent host.
Activated B cells Antigen - substance that reacts with antibody or sensitized
 Exhibit CD25 T cells but may not be able to evoke an immune response
 Occurs when antigens cross-link several surface in the first place
immunoglobulins on select B cells Epitope – Portion of antigen recognized by antibody or by T
cells; also called determinant site
Plasma Cells Hapten – nonimmunologic materials that, when combined
 Characterized by presence of abundant cytoplasmic Igs with a carrier, create new antigenic determinants.
and little to no surface Igs  Several factors such as age, overall health, dose, route
 Nucleus is eccentric or oval of inoculation, and genetic capacity influence the
 Most fully differentiated lymphocyte and its main nature of this response
function is antibody production  Innate immune response takes care of small amounts
 Located in germinal centers of pathogens and leave the adaptive response for
 Memory cells (also found in germinal centers) have a pathogens that are present in large numbers
much longer life span.  Very large doses can result in T- and B-cell tolerance
 Actual amount of immunogen needed to generate an
LABORATORY IDENTIFICATION OF immune response differs with the route of inoculation
LYMPHOCYTES
T cells B cells FACTORS AFFECTING IMMUNOGENICITY
Macromolecular size
Develop in Thymus Develop in Bone Marrow
 Immunogen must have MW of at least 10,000 (With
Found in blood (60%–80% of Found in bone marrow,
exceptions)
circulating lymphocytes), spleen, lymph nodes
lymph nodes thoracic duct  Rule of thumb is that the greater the molecular weight,
fluid, lymph nodes the more potent the molecule is as an immunogen
Identified by rosette Identified by Surface
formation with SRBCs Immunoglobulins Chemical composition and complexity
End product of activation are End product of activation  Proteins and polysaccharides are the best immunogens
cytokines are antibodies  Carbohydrates are somewhat less immunogenic than
Antigens include CD2, CD3, Antigens include CD19, protein, because the units of sugars are more limited
CD4, CD8 CD20, CD21, CD40, MHC than the number of amino acids in protein
Class II  Pure nucleic acids and lipids are not immunogenic by
Located in paracortical region Located in cortical regions themselves
of lymph nodes of Lymph Nodes
 Most methods are based on separation of Foreignness
mononuclear cells from whole blood and detection of
 The more distant taxonomically the source of the
specific cell surface markers
immunogen is from the host, the better it is as a
 Density gradient centrifugation with Ficoll-Hypaque stimulus
(S.G that varies between 1.077 and 1.114)
 Diluted defibrinated or heparinized blood is carefully Ability to be processed and presented with MHC
layered on top of the solution, and the tube is molecules
centrifuged  Enzymatic digestion to create small peptides or pieces
that can be complexed to MHC molecules to present to
responsive lymphocytes

9|Page
 FAR EASTERN UNIVERSITY – MANILA
Conformation and accessibility
 Linear epitopes = Amino acids following one another
on a single chain
 Conformational epitopes = Folding of one chain or
multiple chains
 B cells recognize both linear and conformational
epitopes while T cells recognize linear epitopes only.
HAPTENS
 Once antibody production is initiated, the hapten is
capable of reaction with antibody even when the
hapten is not complexed to a carrier molecule
 Precipitation and agglutination do not occur with
haptens
 Poison ivy is an example (contains substances called
catechols with are haptens)
ANTIGEN:HOST RELATIONSHIPS
Autoantigens – Antigens that belong to the host
Alloantigens – Antigens from other members of the same
species
Heteroantigens – From other species
Heterophile Antigens - heteroantigens that exist in
unrelated plants or animals but are either identical or
closely related in structure so that antibody to one will
cross-react with antigen of the other
ADJUVANTS
 Defined as a substance administered with an
immunogen that increases the immune response
 Aluminum salts are the only ones approved for clinical
use in the U.S.
 Freund’s complete adjuvant consists of mineral oil,
emulsifier, and killed mycobacteria. Causes granulomas
so is not used in humans.
 Adjuvants enhance immune response by:
1. Prolonging existence of immunogen in the area
2. Increasing effective size of immunogen
3. Increasing the number of macrophages
involved in antigen processing
METHODS OF ANTIGEN RECOGNITION
1. Direct
 Cell interacts directly with receptor on antigen
 Phagocytes through PPRR (TLR) that attaches to
PAMPs (Pathogen-associated molecular patterns)
 NK Cells through KAR and KIR
2. Indirect
 Another molecule binds the antigen
 Phagocytosis through opsonins
3. Adaptive
 B cells recognize antigens via membrane Igs
 T cells recognize antigen fragments bound to MHCs
10 | P a g e
MHC
 Originally referred to as Human Leukocyte Antigens
 Main function is to bring antigen to the cell surface for
recognition by T cells
MHC Genes
 Most polymorphic system found in humans
 Found on the short arm of Chromosome 6
 Divided into three classes:
1. Class I Molecules – coded at 3 different loci: A,
B, and C
2. Class II Molecules – situated in the D region.
DR, DQ and DP
3. Class III Molecules – Between class I and II
region. Code for complement proteins and
cytokines such as tumor necrosis factor. Not
expressed on cell surfaces.
Alleles - alternate forms of a gene that code for slightly
different varieties of the same product.
Example: at least 580 different alleles of HLA-A, 921 alleles
of HLA-B, and 312 alleles of HLA-C have been identified at
this time.
 The probability that any two individuals will express
the same MHC molecules is very low
 These genes are described as codominant
 Since the MHC genes are closely linked, they are
inherited together as a package called a haplotype.
* Each MHC molecule can present only one peptide at a
time, because there is only one clef, but each MHC
molecule is capable of presenting many different peptides
(broad specificity)
MHC I Molecules
 Expressed on all nucleated cells. Highest levels on
lymphocytes and low or undetected on liver
hepatocytes, neural cells, muscle cells, and sperm
 HLA-C antigens are expressed at a lower level than
HLA-A and HLA-B antigens
 Made up of an alpha chain and B2-Microglobulin
 Mainly present peptides that have been synthesized
within the cell to CD8 (cytotoxic) T cells
 Another group of molecules called the nonclassical
class I antigens are designated E, F, and G.
 G antigens are expressed on trophoblast cells during
the first trimester of pregnancy
MHC I Antigen Processing
 Both class I and class II molecules are synthesized in
the rough endoplasmic reticulum
 Class I molecules, however, actually bind peptides
while still in the endoplasmic reticulum
 Peptides are derived from partial digestion of proteins
synthesized in the cytoplasm. These intracellular
peptides may include viral, tumor, or even bacterial
antigens.
 FAR EASTERN UNIVERSITY – MANILA
 Digestion of these defective or early proteins is carried
out by proteases (found in proteasomes)
 Transporter associated with antigen proteins (TAP1
and TAP2) bring the digested peptides in the ER.
MHC II Molecules
 Found on antigen-presenting cells (APCs), which
include B cells, monocytes, macrophages, dendritic
cells and endothelial cells
 Made up of an a chain and a B chain
 Mainly bind exogenous proteins—those taken into the
cell from the outside and degraded and present
antigen to CD4 (helper) T cells.
 At least three other class II genes have been
described—DM, DN, and DO, the so-called nonclassical
class II genes. Products of these genes play a regulatory
role in antigen processing
MHC II Antigen Processing
 Class II molecules must be transported from the
endoplasmic reticulum (ER) to an endosomal
compartment before they can bind peptides
 the invariant chain (Ii) is bound to the Class II molecule
and it prevents interaction of the binding site with any
endogenous peptides in the endoplasmic reticulum
 The invariant chain is degraded by a protease, leaving
just a small fragment called class II invariant chain
peptide (CLIP) attached to the peptide-binding cleft.
HLA-DM molecules help to mediate the reaction by
removing the CLIP fragment.
 If binding occurs with a T-cell receptor on a CD4 T cell,
the T helper cell recruits and triggers a B-cell response,
resulting in antibody formation
ANTIGENS THAT ACTIVATE LYMPHOCYTES
Monoclonal Activators – substances that stimulate cells
expressing antigen receptors specific for an epitope. After
contact with T or B cells, the antigen generates a
population of cell that forms one clone.
Oligoclonal Activators – trigger the production of
lymphocytes clones of T cells from different subsets
Polyclonal Activators – causes proliferation of many types
of cells
Mitogens – plant proteins that bind to molecules
present on virtually all T cells and/or B cells.
 Phytohemagglutinin and Concanavalin A –
activates T cells
 Pokeweed Mitogen – activates T and B
 Lipopolysaccharide – B cells
Chapter 6: Antibodies
Immunoglobulins – glycoproteins found in the serum
portion of blood. When subjected to electrophoresis at pH
8.6, appear in the gamma band. Humoral branch of
immune response.
11 | P a g e
ANTIBODY STRUCTURE
Basic four-chain polypeptide unit that consists of 2 heavy
chains and 2 light chains. Held together by noncovalent
forces and disulfide bonds.
Basic structure was elucidated by Edelman and Porter
Papain
 Cleaved IgG into 3 equal pieces
 2 Fab fragments and 1 Fc fragment
 Fab Fragment – Have antigen-binding capacity. Has the
amino terminal end
 Fc Fragment – Fragment crystallizable (spontaneously
crystallized at 4oC). Important in effector functions. Has
the carboxy terminal end
Pepsin
 Cleave IgG at the carboxy-terminal side of the hinge
region forming two parts.
 F(ab)2 and Fc’
Nature of Light Chains
 Bence Jones proteins – L chains secreted by malignant
plasma cells in multiple myeloma. When heated to
60ºC, they precipitate from urine, but on further
heating to 80ºC, they redissolve
 2 main types of L chains – Kappa and Lambda (Had a
constant region and a variable region
 Difference of the two lies in amino acid substitutions at
a few locations along the chain
Gen. structure
 Segment of H chain located between the CH1 and CH2
regions is known as the hinge region. Has high content
of Proline for flexibility.
 All types of immunoglobulins contain a carbohydrate
portion - between the CH2 domains of the two H
chains.
Functions include
(1) increasing the solubility of immunoglobulin
(2) providing protection against degradation
(3) enhancing functional activity of the FC domains.
 FAR EASTERN UNIVERSITY – MANILA

IG VARIANTS  The 5 monomeric units are held together by a J chain

Isotype - unique amino acid sequence that is common to (joining chain)
all immunoglobulin molecules of a given class in a given  Because of its large size, IgM is found mainly in the
species. Heavy chain constant region. intravascular pool and not in other body fluids or
Allotype - Minor variations of these sequences that are tissues
present in some individuals but not others. Occur in the  Primary response antibody
four IgG subclasses, in one IgA subclass, and in the kappa  Functions of IgM include:
light chain 1. Complement fixation
Idiotype - variable portions of each chain (antigen 2. Agglutination
recognition unit) 3. Opsonization
4. Toxin neutralization
IMMUNOGLOBULIN CLASSES 5. B-cell surface receptor for antigens
IgG IgM IgA IgD IgE
MW 150k 900k 160k 180k 190k IgA
Sedimentation 7S 19S 7S/11S 7-S 8S  Two subclasses. IgA1 – serum (monomer); IgA2 –
Coefficient
secretions at mucosal surfaces (dimer)
H chain γ1, γ2, None α1, α2 None None
 IgA2 is held together by a J chain
subclasses γ3, γ4
 IgA is synthesized at a much greater rate than that of
Constant 3 4 3 3 4
IgG
domains
% of Total Ig 70-75 10 10-15 <1 0.002  Combines with a Secretory Component (SC) that is
Serum half-life 23 6 5 1-3 2-3 derived from epithelial cells. Once binding takes place,
(days) IgA and SC precursor are taken inside the cell and then
Electrophoretic γ2–α1 γ 1– γ2–β2 γ1 γ1 released to the opposite surface by a process known as
migration β12 transcytosis
Complement Yes Yes No No No  The SC may thus act to facilitate transport of IgA to
fixation mucosal surfaces. It also makes the dimer more
Crosses Yes No No No No resistant to enzymatic digestion.
placenta  Aggregation of IgA immune complexes may activate
Breastmilk/ + --- +++ --- --- the alternative pathway
Colostrums  Capable of acting as opsonins

IgG IgD
 Major functions of IgG  Found on the surface of B lymphocytes. Second type of
1. Providing immunity for the newborn Ig to appear in B cell maturation.
2. Fixing complement  May play a role in regulating B-cell maturation and
3. Opsonization differentiation
4. Neutralizing toxins and viruses
5. Agglutination and Precipitation reactions IgE
IgG1 IgG2 IgG3 IgG4
 Least abundant Ig in serum
# of disulfide 2 4 5 2
bonds in hinge  Most heat-labile of all Igs. Heating to 56ºC for between
region 30 minutes and 3 hours results in conformational
% of total serum 70 20 6 4 changes and loss of ability to bind to target cells
IgG  Attaches to basophils and mast cells through high-
Half-life (days) 23 23 7 23 affinity FC ϵ RI receptors
Complement ++ + +++ ---  Help induce Type I hypersensitivity reactions
binding (3>1>2)
Placental transfer +++ + +++ +++ SYNTHESIS AND FUNCTION (IMMUNE
(1>3>4>2)
RESPONSES)
Binding to FcyR +++ +/- +++ +
Production of antibodies is induced when the host’s
 Subclasses differ mainly in the number and position of lymphocytes come into contact with a foreign antigenic
the disulfide bridges between the y chains substance that binds to its receptor
IgM Clonal Selection or Activation and Proliferation happens
 Known as macroglobulin
 Secretions = Pentamer form; B cells = Monomer form  Primary Antibody Response
1. Lag Phase – no antibody is detectable

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 FAR EASTERN UNIVERSITY – MANILA
2. Log Phase – the antibody titer increases
logarithmically
3. Plateau Phase – the antibody titer stabilizes
4. Decline Phase – the antibody is catabolized
 Secondary Antibody Response (Anamnestic)
An anamnestic response differs from a primary response as
follows:
1. Time – Shorter lag phase, longer plateau, more
gradual decline.
2. Type of Antibody – IgG class is the predominant form
3. Antibody Titer – Higher titer. 10 fold or greater than
primary response
Ehrlich’s side chain theory – cells had specific surface
receptors for antigen that were present before contact
with antigen occurred.
Clonal selection theory - Niels Jerne and Macfarlane
Burnet theorized that individual lymphocytes are
genetically preprogrammed to produce one type of
immunoglobulin and that a specific antigen finds or selects
those particular cells capable of responding to it.
AG:AB INTERACTIONS (MOLECULAR BASIS
CHAPTER SA TURGEON TOO)
Specificity - Ability of a particular antibody to combine with
a particular antigen
Cross-reactivity - Antibodies directed against one type of
antigen will also react with the other type of antigen
Affinity - Initial force of attraction that exists between a
single Fab site on an antibody molecule and a single
epitope or determinant site on the corresponding antigen
Avidity - functional combining strength of an antibody with
its antigen
Immune Complex - Noncovalent combination of antigen
with its respective specific antibody.
An immune complex may be of the small (soluble) or large
(precipitating) type, depending on the nature and
proportion of antigen and antibody. Under conditions of
antigen or antibody excess,soluble complexes tend to
predominate. If equivalent amounts of antigen and
antibody are present, a precipitate may form
Types of bonding in Ag:Ab reactions
1. Hydrophobic bonds (major bonds formed)
2. Hydrogen bonds
3. Van der Waals forces
4. Electrostatic forces
Class Switching - The variable and constant regions are
joined at the ribonucleic acid (RNA) level, thus conserving
the DNA of the constant regions
Allelic Exclusion - If a successful rearrangement of DNA on
one chromosome 14 occurs, then the genes on the second
chromosome are not rearranged
13 | P a g e
MONOCLONAL ANTIBODIES
- Purified antibodies cloned from a single cell
 Discovered by Kohler, Milstein, and Jerne
 To induce the cells to fuse, they used Sendai virus, an
influenza virus that characteristically causes cell fusion
Modern Method:
 Mice are immunized with a specific antigen
 Spleen cells are mixed with cultured mouse
myeloma cells
 Polyethylene glycol (PEG) rather than Sendai virus
is added to the cell mixture to promote cell
membrane fusion
 The fused cell mixture is placed in a medium
containing hypoxanthine, aminopterin, and
thymidine (HAT medium)
 The supernatant is tested for specific antibody
using methods such as ELISA
Chapter 7: Cytokines
INTRODUCTION
Cytokines - polypeptide products of activated cells that
control a variety of cellular responses and thereby regulate
the immune response
Autocrine – Affecting the same cell that secreted it
Paracrine – Affecting a target cell in close proximity
Endocrine – effect is sytematic
Cytokine Storm - massive overproduction and
dysregulation of cytokines that leads to shock, multiorgan
failure, or even death
Interleukins - Cytokines produced by leukocytes that act on
other leukocytes
Pleiotropy – having many different effects
Redundancy – different cytokines that have the same
effect
Synergy – act in networks; one cytokine may stimulate the
release of other cytokines that have similar or overlapping
activities
Antagonism – cytokine may produce effects that terminate
the activity of another cytokine
INNATE
IL-1
 Produced by monocyte and macrophages
 Induced by the presence of microbial pathogens,
bacterial lipopolysaccharides, or other cytokines
 Endogenous pyrogen and induces fever in the acute
phase response through its action on the
hypothalamus
 Also helpful in diapedesis
TNF-a
 Induce lysis of tumors
 Most prominent member of the TNFs
 FAR EASTERN UNIVERSITY – MANILA
 Main trigger for TNF- production is the presence of
lipopolysaccharide, found in gram-negative bacteria
IL-6
 Plays an important role in acute phase reactions
and the adaptive immune response
 IL-6 stimulates B cells to proliferate and
differentiate into plasma cells and induces CD4 T
cells to produce greater quantities of both pro- and
anti-inflammatory cytokines
Chemokines
 a family of cytokines that enhance motility and
promote migration of many types of white blood cells
toward the source of the chemokine (chemotaxis)
 The chemokine receptors CXCR4 and CCR5 are utilized
by HIV as co-receptors for infection of CD4+ T
lymphocytes and macrophages
TGF-B
 A factor that induces antiproliferative activity in a wide
variety of cell types
 TGF- blocks the production of IL-12 and strongly
inhibits the induction of IFN-
IFN-a and IFN-B
 Interferons were originally so named because they
interfere with viral replication
 Type I that function primarily in this manner (a and B)
 Produced by dendritic cells and induce production of
proteins and pathways that directly interfere with viral
replication and cell division (Enhances expression of
MHC Class I)
ADAPTIVE
Th1 lineage is driven by IL-12. Th2 lineage is driven by IL-4.
Treg cells are derived from IL-10-responsive naïve T cells
Th1 Cytokines
 IFN-y – Causes increased expression of MHC I and II.
IFN-y is a strong stimulator of macrophages and boosts
their tumoricidal activity
 IL-2 - Also known as the T-cell growth factor
 IL-2 and IFN-y induce the development of Th1 cells,
which, in turn, induces macrophage activation and
delayed type hypersensitivity
Th2 Cytokines
 IL-4 - Turns on the genes that generate Th2 cells and
turns off the genes that promote Th1 cells. Promotes
the production of IgG2a and IgE and, along with IL-5,
drives the differentiation and activation of eosinophils
in both allergic immune responses and the response to
parasitic infections
 IL-10 - Anti-inflammatory and suppressive effects on
Th1 cells. Suppression of IL-12 synthesis (Thus, in
14 | P a g e
contrast to most other cytokines, IL-10 serves as an
antagonist to IFN-—it is a down-regulator of the
immune response)
Treg Cytokines
 Tregs affect T cell activity primarily through the actions
of TGF-B
HEMATOPOIETIC STIMULATORS
IL-3 – For lymphocytes
Erythropoietin – RBC Production
G-CSF – For neutrophils
M-CSF – For macrophages
GM-CSF – Other cell types besides lymphocytes
IL-3 + GM-CSF – Basophils and Mast Cells
IL-5 + IL-3 + GM-CSF – Eosinophils
Chapter 8: Precipitation Reactions
Precipitation - combining soluble antigen with soluble
antibody to produce insoluble complexes that are visible
Agglutination - process by which particulate antigens such
as cells aggregate to form larger complexes when a specific
antibody is present
 Precipitation first noted by Kraus
ANTIGEN-ANTIBODY BINDING
Affinity - initial force of attraction between a single Fab site
and a single epitope or determinant site
 Ionic bonds
 Hydrogen bonds
 Hydrophobic bonds
 Van der Waals forces
Cross-reactivity - reacting with antigens that are
structurally similar to the original antigen that induced
antibody production
Avidity - sum of all the attractive forces between an
antigen and an antibody
 strength with which a multivalent antibody binds a
multivalent antigen
 High avidity can actually compensate for a low affinity
 All antigen–antibody binding is reversible and is
governed by the law of mass action. Free reactants are
in equilibrium with bound reactants
PRECIPITATION CURVE
Zone of Equivalence - number of multivalent sites of
antigen and antibody are approximately equal
 The lattice hypothesis, as formulated by Marrack, is
based on the assumptions that each antibody molecule
 FAR EASTERN UNIVERSITY – MANILA
must have at least two binding sites, and antigen must
be multivalent
Prozone – Antibody excess (diluting out antibody and
performing the test again may produce a positive result)
Postzone – Antigen excess (repeated with an additional
patient specimen taken about a week later)
MEASUREMENT OF PRECIPITATION BY
LIGHT SCATTERING
Turbidimetry - measure of the turbidity or cloudiness of a
solution. Measures the reduction in light intensity due to
reflection, absorption, or scatter
Nephelometry - measures the light that is scattered at a
particular angle from the incident beam as it passes
through a suspension
 Although the sensitivity of turbidity has increased,
nephelometry is more sensitive, with a lower limit of
detection of 1 to 10 mg/L
 End Point Nephelometry - reaction is allowed to run
essentially to completion
 Kinetic or Rate Nephelometry - rate of scattering
increase is measured immediately after the reagent is
added
PASSIVE IMMUNODIFFUSION TECHNIQUES
Passive Immunodiffusion - determined in a support
medium such as a gel. Reactants are added to the gel, and
antigen–antibody combination occurs by means of
diffusion. No electrical current is used.
 The rate of diffusion is affected by the size of the
particles, the temperature, the gel viscosity, and the
amount of hydration. An agar concentration ranging
from 0.3 percent to 1.5 percent allows for diffusion of
most reactants. Agarose is preferred to agar, because
agar has a strong negative charge, while agarose has
almost none
Radial Immunodiffusion – Modification of single
immunodiffusion. Antibody is uniformly distributed in the
support gel, and antigen is applied to a well cut into the
gel.
 Mancini (Endpoint method) – Takes between 24-72
hours. Square of the diameter is then directly
proportional to the concentration of the antigen
 Fahey and Mckelvey (Kinetic Method) – Takes 18
hours. Diameter is proportional to the log of the
concentration
Ouchterlony Double Diffusion - both antigen and antibody
diffuse independently through a semisolid medium in two
dimensions, horizontally and vertically
15 | P a g e
ELECTROPHORETIC TECHNIQUES
Rocket Immunoelectrophoresis
 One-dimension immmunoelectrophoresis
 Developed by Laurell
 Electrophoresis is used to facilitate migration of the
antigen
 Antibody is distributed in the gel, and antigen is placed
in wells cut in the gel
 End result is a precipitin line that is conical in shape,
resembling a rocket
Immunelectrophoresis
 Double-diffusion technique
 Introduced by Grabar and Williams
 Serum is electrophoresed then a trough is cut in the gel
parallel to the line of separation. Antiserum is placed in
the trough, and the gel is incubated for 18 to 24 hours
Immunofixation Electrophoresis
 Described by Alper and Johnson
 Similar to immunoelectrophoresis except that after
electrophoresis takes place, antiserum is applied
directly to the gel’s surface rather than placed in a
trough
 Typically, patient serum is applied to six lanes of the
gel, and after electrophoresis, five lanes are overlaid
with one each of the following antibodies: antibody to
gamma, alpha, or mu heavy chains and to kappa or
lambda light chains. The sixth lane is overlaid with
antibody to all serum proteins and serves as the
reference lane. Reactions in each of the five lanes are
compared to the reference lane.
Chapter 9: Agglutination Reactions
Agglutinins – Antibodies that produce aggregation
reactions
 Is a two-step process involving Sensitization and Lattice
Formation.
 Gruber and Durham first reported agglutination
reactions
 Widal and Sicard developed tests for Typhoid Fever,
Brucellosis, and Tularemia
 FAR EASTERN UNIVERSITY – MANILA
STEPS IN AGGLUTINATION
Sensitization - Antigen–antibody combination through
single antigenic determinants on the particle surface
 Affected by nature of antibody molecules (Affinity and
Avidity) and the nature of the antigen-bearing surface
(Are they sparse? Are they obscured?)
Lattice Formation - formation of cross-links that form the
visible aggregates
 Governed by physicochemical factors such as the
milieu’s ionic strength, pH, and temperature
Enhancement of lattice formation
• 1) Decreasing ionic strength (LISS or Albumin)
• 2) Increasing viscosity (Dextran or Polyethylene
glycol)
• 3) Enzymes (Bromelin, Papain, Trypsin, Ficin)
• 4) Agitation and Centrifugation
• 5) Alterating Temp or pH (pH 6.5-7.5)
• 6.) Use of Anti-human globulin
TYPES OF AGGLUTINATION REACTIONS
Direct Agglutination
 antigens are found naturally on a particle
 One such example is the Widal test
 The antigens used in this procedure include Salmonella
O (somatic) and H (flagellar) antigens
 significant finding is a fourfold increase in antibody
titer over time
 Hemagglutination – Agglutination reaction that
involves RBCs
Passive Agglutination
 Indirect agglutination
 particles that are coated with antigens not normally
found on their surfaces
 Problems encountered with the use of erythrocytes as
carrier particles include the possibility of
crossreactivity, especially with heterophile antibody
 IgG was naturally adsorbed to the surface of
polystyrene latex particles
Reverse Passive Agglutination
 antibody rather than antigen is attached to a carrier
particle
 Use of monoclonal antibodies has greatly cut down on
cross-reactivity
 rheumatoid factor will cause a false positive as it reacts
with any IgG antibody, so this must be taken into
account
Agglutination Inhibition
 competition between particulate and soluble antigens
for limited antibody-combining sites
 lack of agglutination is an indicator of a positive
reaction
16 | P a g e
 Hemagglutination inhibition - reactions use the same
principle, except red blood cells are the indicator
particles (Detects antibodies)
Coagglutination
 bacteria as the inert particles to which antibody is
attached
 Staphylococcus aureus and Protein A
ANTIGLOBULIN-MEDIATED
AGGLUTINATION (COOMB’S TEST)
Direct Antiglobulin Test
 used to demonstrate in vivo attachment of antibody or
complement to an individual’s red blood cells
 hemolytic anemia, hemolytic disease of the newborn,
sensitization of red blood cells caused by the presence
of drugs, or a transfusion reaction
 A positive test indicates that an immune reaction is
taking place in that individual
 One-step process
Indirect Antiglobulin Test
 used to determine the presence of a particular
antibody in a patient, or it can be used to type patient
red blood cells for specific blood group antigens
 Two-step process
 washed red blood cells and antibody are allowed to
combine at 37°C, and the cells are then carefully
washed again to remove any unbound antibody
 Failure to wash cells = FALSE NEGATIVE
INSTRUMENTATION
 Turbidimetry
 Nephelometry
 Particle-Counting Immunoassay (PACIA)
 measuring the number of residual
nonagglutinating particles in a specimen
 counted by means of a laser beam
 RATE ASSAY
 END-POINT ASSAY
Chapter 10: Labeled Immunoassays
CHARACTERISTICS
• Competitive vs. Noncompetitive Assays
 Competitive – Inversely proportional
 Noncompetitive – Directly proportional
• Antibodies – More affinity, more specific/sensitive
• Standards of Calibrators - Differing amounts of
standards are added to antibody–antigen mixtures to
ascertain their effect on binding of the labeled reagent
• Separation Methods – Need a partitioning step
• Detection of the Label – A system for counting
 FAR EASTERN UNIVERSITY – MANILA
• Control – Blank tube (usually Phosphate-buffered
saline) (check for background). Negative control + High
and Low positive control. Tests usually run in duplicate
RADIOIMMUNOASSAYS
• Pioneered by Yalow and Berson
• I125 is the most popular radioisotope. Emits gamma
radiation
• COMPETITIVE BINDING ASSAYS
• Difficult and expensive to maintain a license, disposal
problems, short half-life, and expensive equipment are
difficulties usually encountered
ENZYME IMMUNOASSAYS
 Naturally occurring molecules that catalyze
biochemical reactions
 Cheap, readily available, long shelf-life
 Labels used: horseradish peroxidase, glucose-6-
phosphate dehydrogenase, alkaline phosphatase, and
β-D-galactosidase
Heterogenous Enzyme Immunoassays
1. Competitive EIA
2. Noncompetitive EIA – Higher sensitivity. ELISA
(Enzyme-linked immunosorbent assay)
3. Capture Assays (sandwich immunoassays) – subject to
hook effect
4. Rapid Immunoassays: Membrane-Based Cassette
Assays
 Single-use disposable assays in a plastic cartridge.
Membrane usually nitrocellulose. Rapid flow and
high surface area increasing speed and reactivity of
test.
 IMMUNOCHROMATOGRAPHY – combine addition
of px sample, wash reagent, labeled antibodies and
substrate in ONE STEP.
Homogenous Enzyme Immunoassays
 Enzyme Multiplied Immunoassay Technique (EMIT)
 Developed by Syva corporation
 Directly proportional
 Serum Ag, Rgt Ab, Rgt Ag+Enzyme
FLUORESCENT IMMUNOASSAYS
• Albert Coons
• These fluorescent compounds are called fluorophores
or fluorochromes
• Absorb energy and convert that energy into light of a
longer wavelength and lower energy
• The two compounds most often used are fluorescein
and rhodamine, usually in the form of isothiocyanates.
Other compounds used are phycoerythrin, europium
(β-naphthyl trifluoroacetone), and lucifer yellow VS
1. Direct Immunofluorescent assays – ONE STEP
2. Indirect Immunofluorescent assays – TWO STEPS
17 | P a g e
3. Other Fluorescent immunoassays:
Heterogenous/Homogenous (FPIA) (Fluorescence
polarization immunoassay)
 Inversely proportional
 Rgt antigen (unpolarized), Rgt antibody, Serum
antigen
CHEMILUMINESCENT IMMUNOASSAYS
 Emission of light caused by a chemical reaction
 most common substances used are luminol, acridinium
esters, ruthenium derivatives, and nitrophenyl oxalates
 Acridinium esters - quick burst or flash of light
 Luminol and dioxetane – light remains for a longer time
 Excellent sensitivity. Up to Attamoles and Zeptomoles.
Detection = Photomultiplier tubes.
Chapter 11: Molecular Diagnostic Techniques
CHARACTERISTICS OF NUCLEIC ACIDS
• Two Main Types: DNA and RNA
• Each nucleotide consists of a cyclic, 5-carbon sugar,
with a phosphate group at the 5′ C and one of four
nitrogenous bases at the 1′ C. The sugar deoxyribose is
present in DNA, while ribose is the primary sugar found
in RNA
• PURINES: Adenine and Guanine
• PYRIMIDINES: Cytosine, Guanine, and Uracil
• Transcription – generation of mRNA that codes for the
gene for the protiein
• Translation – mRNA is translated in cytoplasm to
proteins
• Types of RNA:
• mRNA – translate DNA to proteins
• tRNA – transports amino acids to make
proteins
• rRNA - acts as the site for protein synthesis
directed by the mRNA
HYBRIDIZATION TECHNIQUES
 Nucleic acid probe – Short strand of DNA/RNA of a
known sequence
 The two strands should share at least 16 to 20
consecutive bases of perfect complementarity to form
a stable hybrid
 Probes are labeled with a marker (such as a
radioisotope, a fluorochrome, an enzyme, or a
chemiluminescent substrate
1. Solid Support Hybridization
 Dot-blot - samples added to membrane. Heated and
separated then labeled probes added.
 Sandwich Hybridization Assays – modification of dot-
blot. Use TWO probes.
 To characterize DNA, we can use Restriction
endonucleases (cleave, separated, then stained with
Ethidium Bromide). Results in RFLPs (Restriction
Fragment Length Polymorphisms)
 FAR EASTERN UNIVERSITY – MANILA
 These enzymes cleave both strands of a double-
stranded DNA at specific recognition sites that are
approximately 4 to 6 base pairs long
 Southern Blot – DNA
 Northern Blot - RNA
2. Solution Hybridization
 Free to interact in mixture
 S1 Nuclease digest unannealed DNA then hybrids are
recovered through precipitation or column binding
 Hybridization protect assay (HPA), a chemiluminescent
acridinium ester is attached to a probe. Solution is
subject to alkaline hydrolysis after hybridization
(hydrolyzes the ester if the probe is not attached to
target) Probes that remain attached gives off light.
3. In Situ Hybridization
 Target is in intact cells
 For probes to reach the nucleic acid, they must be
small enough, usually limited to 500 bases or less, to
penetrate the cells in question
 Endogenous control probe must be used.
 Fluorescent in situ hybridization (FISH)
4. DNA Chip Technology
 Uses Biochips (microarrays)
 After amplification, the sample, labeled with a
fluorescent tag, is loaded onto the chip.
MISMATCH IS A PROBLEM
To ensure that there is stringency (or correct pairing):
1. Decrease salt conc.
2. Increase temp
3. Increase concentration of formamide or urea
DNA Sequencing – GOLD STANDARD for many molecular
applications
AMPLIFICATION METHODS
Target Amplification
1. Polymerase Chain Reaction (PCR)
 Best known and most widely used amplification
method
 Developed by Kary Mullis
3 STEPS
1. Denaturation – 95oC (Separate)
2. Annealing - 52oC (Primers attach)
3. Polymerization – 72oC (Polymerase binds to 3’ end)
 One cycle = 60-90 secs
 DNA Fragments = Amplicons
 Real-time PCR – reduces time it takes
 RT-PCR – PCR with RT to convert RNA to DNA first
 Used for HLA identification also
 Carryover causes false (+)
2. Transcription-based Amplification: TMA and NASBA
18 | P a g e
 TMA (Transcription-mediated amplification) – Uses two
enzymes (RNA Polymerase and RT)
 NASBA (Nucleic acid sequence-based amplification) –
Uses three enzymes (RT, RNase H, and bacteriophage
T7 DNA-dependent RNA polymerase)
3. Strand Displacement Amplification (SDA)
Probe Amplification
 Ligase Chain Reaction (DNA Ligase amplification)
Signal Amplification
 Branched DNA (bDNA) signal amplification
 Because the patient nucleic acid itself is not
replicated or amplified, this type of technique is
less prone to contamination problems
 Less sensitive than enzyme amplification
Chapter 12: Immunodeficiencies
• Defects in Humoral Immunity = Pyogenic Bacterial
Infection (particularly Respiratory Tract)
• Defects in T cell-mediated immunity = Intracellular
pathogens (Virus, fungi, intracellular bacteria) Almost
always develop mucocutaneous candidiasis
DEFICIENCIES OF B CELL SYSTEM
1. Transient Hypogammaglobulinemia of Infancy
 Babies still have low levels of immunoglobulins
past 5-6 months of age
2. X-Linked Bruton’s agammaglobulinemia – deficiency of
Bruton Tyrosine Kinase
 Lack mature CD19 cells. Differentiation stops at
pre-B cell stage
3. IgA Deficiency
 Most common congenital immunodeficiency
 IgA <5 mg/mL considered SEVERE
4. Common Variable Immunodeficiency - most common
primary immune deficiency with a severe clinical
syndrome
 Heterogenous group of disorders that cause
hypogammaglobulinemia
 Natural agglutinins are low or absent
 Have mature B cells (do not differentiate)
5. Isolated IgG Subclass deficiency
 level of one or more of the IgG subclasses is more
than two standard deviations below the mean age-
appropriate level
 Mostly against protein – IgG1 and IgG3
 Mostly against carbohydrates – IgG2 and IgG4
DEFICIENCIES OF CELLULAR IMMUNITY
1. Digeorge Anomaly - abnormality of the third and fourth
pharyngeal pouches that affects thymic development
 Decrease in T cell numbers
 Can develop tetany, mental retardation, absence of
ossification of the hyoid bone, cardiac anomalies,
 FAR EASTERN UNIVERSITY – MANILA
abnormal facial development, and thymic
hypoplasia
2. Purine Nucleoside Phosphorylase Deficiency
 Rare autosomal recessive trait
 number of T cells progressively decreases because
of the accumulation of deoxyguanosine
triphosphate, a toxic purine metabolite
COMBINED DEFICIENCES OF CELLULAR
AND HUMORAL
1. Severe Combined Immunodeficiency - group of related
diseases that all affect T- and B-cell function but with
differing causes. X-linked SCID is the most common.
Abnormal gene is common gamma chain.
 JAK3 gene defect (T- B+ NK-)
 X-linked SCID (T- B+ NK+)
 Adenosine deaminase deficiency (T- B- NK-)
 RAG1/RAG2 mutation (T- B- but NK is functioning)
 Present early in infancy. Oral candidal infections,
pneumonia, and diarrhea are the most common
manifestations.
2. Wiscott-Aldrich Syndrome
 X-linked. Triad of Eczema, Immunodeficiency,
Thrombocytopenia.
 Low IgM, High IgE
 WASp gene defect – Abnormal CD43
3. Ataxia Telangiectasia
 Cerebellar ataxia + Telangiectasia. Gene defect in
chromosome 11 that leads to a defective kinase
involved in DNA repair and in cell cycle control
 Increased AFP. Low IgG2, IgA and IgE
DEFECTS OF NEUTROPHIL FUNCTION
1. Chronic Granulomatous Disease
 Most common neutrophil abnormality
 Defect in NADPH oxidase system leading to
incapacity to perform respiratory burst
 Originally diagnosed through Nitroblue Tetrazolium
Dye Test
 Now, neutrophils are labeled with
dihydrorhodamine (DHR)
2. Leukocyte Adhesion Deficiency
 CD18 deficiency. Cannot transmigrate
LABORATORY EVALUATION OF IMMUNE
DYSFUNCTION
Screening Tests
1. All immunodeficiencies - CBC, WBC Differential
2. Humoral Immunity - Ig levels, Isohemagglutinin titers,
IgG response to protein and carbs
3. Cellular Immunity – Delayed hypersensitivity skin tests,
Chest Xray (For thymus)
4. Phagocyte defect – NBT Test, IgE level (Hyper-IgE
syndrome)
5. Complement – CH50, Serum C3 level
Confirmatory Tests
19 | P a g e
1. Humoral immunity – B cell counts, B cell proliferation in
vitro, Histology of lymphoid tissues
2. Cellular immunity – T cell counts and function in vitro,
Enzyme assays (ADA, PNP)
3. Phagocyte defect – LAD molecule analysis (CD11a, 11b,
11c, 18), Phagocytosis and bacterial killing assays,
Chemotaxis assay, Enzyme assays (NADPH oxidase etc.)
4. Complement – other specific component assays
Chapter 13: Hypersensitivity Reactions
TYPE I HYPERSENSITIVITY
(ANAPHYLACTIC HYPERSENSITIVITY)
• Short Time Lag. Key reactant is IgE
• Atopic antigens or Allergens
• ATOPY - inherited tendency to respond to naturally
occurring inhaled and ingested allergens with
continued production of IgE
• Passive Cutaneous Anaphylaxis – Serum transferred,
nonallergic individual will show allergy
• IL-5 and IL-9 are involved in the development of
eosinophils,
• IL-4 and IL-9 promote development of mast cells.
• IL-4, IL-9, and IL-13 all act to stimulate overproduction
of mucus, a characteristic of most allergic reactions.
• Tendency for allergy – Linked to MHC
• Mast cells – Principle effector cells
• Histamine is higher in mast cells than basophils
Mediators/Granules
1. Histamine – vasoactive amine. Responsible for local
erythema/redness and wheal and flare formation.
2. Heparin
3. Eosinophil chemotactic factor of Anaphylaxis
4. Neutrophil chemotactic factor
5. Proteases - Tryptase (generates Bradykinin which
induces prolonged smooth muscle contraction and
increases vascular permeability and secretory activity)
Clinical Manifestations and Treatment of Type I
Hypersensitivity
Anaphylaxis – Systemic response to allergens. Most severe
type of allergic reaction. Involves multiple organs.
 Typical agents that induce anaphylaxis include
venom from bees, wasps, and hornets; drugs such
as penicillin; and foods such as shellfish, peanuts,
or dairy products
 latex sensitivity is now a significant cause of
anaphylaxis among health-care workers and in
patients who have had multiple surgery procedures
 Treatment – avoidance, antihistamines,
decongestants and bronchodilators
 Hyposensitization – Building up IgG antibodies
Rhinitis – most common form of Atopy
Asthma - defined clinically as recurrent airflow obstruction
Testing for Immediate Hypersensitivity
IN VIVO Skin Tests
 FAR EASTERN UNIVERSITY – MANILA
1. Cutaneous (Prick test) – (+) is wheal that is 3mm
greater than negative control (Higher concentration)
2. Intradermal - more sensitive than cutaneous testing
(same positive). <3 years not recommended. (Higher
amount of Antigen)
IN VITRO Tests
1. Total IgE (Screening) – RIST (Radioimmunosorbent test)
2. Allergen-specific IgE – RAST (Radioallergosorbent test)
TYPE II HYPERSENSTIVITY (CYTOTOXIC
HYPERSENSITIVITY)
• Reactants are IgG and IgM
• Triggered by antigen on cells
• Antibody coats cellular surfaces and promotes
phagocytosis by both opsonization and activation of
the complement cascade
• Isohemagglutinins – naturally occurring antibodies
1. TRANSFUSION REACTIONS
2. HEMOLYTIC DISEASE OF THE NEWBORN – Severe HDN
is called Erythoblastosis fetalis
 For babies, bilirubin levels >20mg/dL may lead to
Kernicterus (deposition in brain)
 Treatment involves exchange transfusion or
intrauterine transfusion
3. AUTOIMMUNE HEMOLYTIC ANEMIA (Warm and Cold)
4. REACTIONS INVOLVING TISSUE ANTIGENS –
Goodpasture’s syndrome, Hashimoto’s disease,
Myasthenia Gravis, Type 1 DM
Testing for Type II Hypersensitivity
DAT
IAT
TYPE III HYPERSENSITIVITY (IMMUNE
COMPLEX HYPERSENSITIVITY)
• Antibody involved is also IgG and IgM
• Antigen here is SOLUBLE
• Immune complexes in tissue bind complement, causing
damage to the particular tissue
• Sites in which this typically occurs include the
glomerular basement membrane, vascular
endothelium, joint linings, and pulmonary alveolar
membranes.
ARTHUS REACTION – Localized Type III reaction described
by Maurice Arthus. (Rabbits immunized then intradermal
injection of antigen)
Serum Sickness - passive immunization with animal serum,
usually horse or bovine, used to treat such infections as
diphtheria, tetanus, and gangrene
Systemic Lupus Erythematosus – Autologous antigens are
involved. Antibodies are directed against constituents such
as DNA and nucleohistones
Rheumatoid Arthritis – Autologous antigens are also
involved. Presence of rheumatoid factor (Against IgG)
TYPE IV HYPERSENSITIVITY
20 | P a g e
• First described by Robert Koch through observation of
Mycobacterium tuberculosis
• Major cells - sensitized T cells, primarily Th1.
• The reaction cannot be transferred from one animal to
another by means of serum
• Langerhans cells in skin and macrophages in tissue
present antigen to Th1 cells.
• CONTACT DERMATITIS – due to LMW compounds that
touch the skin. Most common is poison ivy, poison oak
and poison sumac. Give off urushiol. Can also be
caused by nickel, rubber, formalin, cosmetics, latex,
medications, antiseptics, antibiotics, and anesthetics.
• Hypersensitivity Pneumonitis – inhaled allergens
(Farmer’s lung, Breeder’s disease, Humidifier lung
disease)
• Tuberculin-type Hypersensitivity – MTB (Tuberculin
skin test)
• Patch Test – gold standard for contact dermatitis
• Mantoux Method – test for Type IV
Chapter 14: Autoimmune Diseases
• Central Tolerance - Destruction of potentially self-
reactive lymphocytes in the primary lymphoid organs
• Peripheral Tolerance – Secondary lymphoid organs
• Molecular Mimicry – many individual viral or bacterial
agents contain antigens that closely resemble self-
antigens (Poliovirus VP2 and Acetylcholine receptors;
measles virus P3 and myelin basic protein; and
papilloma virus E2 and insulin receptors)
• Sequestered Antigens – Antigens that are protected
from encountering the circulation are not exposed to
potentially reactive lymphocytes
• Polyclonal B cell activation - One defect in particular,
FC receptor polymorphisms, the receptor for antibody
that normally down-regulates antibody production,
may cause continual B-cell stimulation
• HLA B27 – Ankylosing Spondylitis
SYSTEMIC LUPUS ERYTHEMATOSUS
• Prototype of human autoimmune diseases
• Peak age: 20-40 years. Women more likely than men
• Typical patient average of 3 circulating autoantibodies
• CLINICAL SIGNS: Joint Involvement. Butterfly rash (and
other erythematous rashes on exposure to UV) Renal
Involvement. Cardiac involvement. Neuropsychiatric
manifestations. Hematologic abnormalities.
 FAR EASTERN UNIVERSITY – MANILA
• In order for a clinical diagnosis of lupus to be made,
four of eleven specific criteria must be present
• LE Cell – Neutrophil that engulfed antibody-coated
nucleus of another neutrophil
• First test for SLE – test for ANA (Anti-nuclear
antibodies). FANA is the most widely used and
accepted test.
• Mouse kidney or human epithelial HEp-2 cells are fixed
to a slide and allowed to react with patient serum
• Ds-DNA = Most specific for SLE
• Crithidia luciliae, a hemoflagellate – Test for anti
dsDNA
• Anti-histone = Drug-induced lupus
• Development of Antiphospholipid antibodies – Cause
prolonged PT and APTT
RHEUMATOID ARTHRITIS
• Women more likely than men
• RA characterized as chronic, symmetric, and erosive
arthritis of the peripheral joints that can also affect
multiple organs such as the heart and the lungs
• strongest association with certain DR4 alleles
• At least 4 of 7 symptoms must be present for 6 weeks
or more for the diagnosis to be made
• Felty’s Syndrome = Chronic RA with neutropenia,
splenomegaly, and possibly thrombocytopenia
• Macrophages and neutrophils attracted to area -
results in formation of organized mass of cells
(PANNUS)
• 75% of RA patients have RF (Ig against IgG)
• Anti-CCP is now the lead marker for detection of
RA (much more specific than RF)
AUTOIMMUNE THYROID DISEASES
• Hashimoto’s and Grave’s Diseases
• The Thyroid consists of units called follicles that are
lined with cuboidal epithelial cells. Follicles filled with
colloid. Colloid – mostly made of Thyroglobulin
• Thyrotropin-releasing hormone (TRH) is secreted by
the hypothalamus to initiate relase of TSH by the
pituitary. TSH then acts on the thyroid gland.
• Hashimoto’s – One trigger is high iodine intake
• HLA-DR3 – Grave’s disease
• HLA-DR3,4,5 and DQw7 – Hashimoto’s
• Hashimoto’s – Chronic autoimmune thyroiditis.
Combination of goiter, hypothyroidism, and thyroid
21 | P a g e
autoantibodies. The goiter is irregular and rubbery. Abs
to thyroglobulin PREDOMINATE.
• Grave’s – Hyperthyroidism. Manifested as
thyrotoxicosis. Diffusely enlarged goiter that is soft
instead of rubbery. EXOPHTHALMUS
• Major Abs in Graves’ disease - thyroid-stimulating
hormone receptor antibody (TSHRab) and antibodies
to thyroid peroxidase
TYPE 1A DIABETES MELLITUS
• Insufficient insulin production caused by selective
destruction of the beta cells of the pancreas.
• HLA DR3 and DR4; HLA-DQ
• Antibodies that can be found – Ab to insulinoma
antigen 2 (IA-2 or ICA 512) and IA-2βA (phogrin); anti-
insulin antibodies; antibodies to the enzyme GAD; and
antibodies to various other islet cell proteins, called
islet cell antibodies (ICAs)
• Combined screening for IA-2A, ICA, and GAD antibodies
appears to have the most sensitivity and best positive
predictive value
MULTIPLE SCLEROSIS
• characterized by formation of lesions (plaques) in
white matter of brain and spinal cord = destruction of
myelin sheath.
• Most closely associated with DRB1*1501
• Theory = triggered by molecular mimicry
• 2 most common tests for diagnosis = oligoclonal
banding and CSF IgG index
• Antimyelin antibody
MYASTHENIA GRAVIS
• Affects neuromuscular junction
• Anti-acetylcholine receptors - leads to progressive
muscle weakness
• Might be linked to A1, B8, and DR3
• RIA procedures are used to detect antibody. Radio-
labeled snake venom (α-bungarotoxin) irreversibly
binds to ACHRs; precipitation of receptors caused by
combination with antibody is then measured
GOODPASTURE’s SYNDROME
• Autoantibody to basement membranes (specificity for
the noncollagenous region of the alpha 3 chain of type
IV collagen)
• More likely to occur in young males between the ages
of 18 and 35. Often follows a viral infection
• Western blot – Confirmatory Test
Chapter 15: Transplantation Immunology
HISTOCOMPATIBILITY SYSTEMS
1. Major Histocompatibility System
 Class I and Class II
 HLA Genes are inherited as haplotypes
 FAR EASTERN UNIVERSITY – MANILA
 While this has successfully enabled populations to
survive infectious challenges, it severely restricts
the ability to transplant foreign tissues or cells
between any two individuals
2. Minor Histocompatibility Antigens - mHAs are non-HLA
proteins that demonstrate polymorphism in amino acid
sequence within a species (CD4 and CD8 mediated)
3. MHC class I–related chain A (MICA Antigens) - encodes
a cell surface protein that is involved in gamma/delta
T-cell responses
4. ABO Bloodgroup Antigens - the only blood group
system that impacts clinical transplantation
5. Killer Immunoglobulin-like receptors – Killer Activating
and Inhibitory Receptors (Connected to MHC)
ALLORECOGNITION
Different types of Graft
• Autograft - self
• Syngraft – identical twins
• Allograft – two individuals of same species
• Xenograft – different species
Types of allorecognition
• Direct Allorecognition - recipient T cells bind and
respond directly to foreign (allo) HLA proteins on graft
cells
 The mixed lymphocyte response (MLR) is an in
vitro correlate of direct allorecognition (CD8)
• Indirect Allorecognition - second pathway by which the
immune system recognizes foreign HLA proteins
X`
TRANSPLANT REJECTION
• Hyperacute – minutes to hours. Mediated by
preformed antibodies (ABO,HLA). Thrombus formation
leading to ischemia and necrosis of transplanted tissue
• Accelerated - individuals possess very low levels of
donor-specific antibody in pre-transplant period
• Acute – days to weeks. Mediated by CD8+ and CD4+
and humoral immunity. Parenchymal and Vascular
injury.
• Chronic – Months to years. CD4+ and B cells.
Progressive fibrosis and scarring with narrowing of the
vessel lumen due to proliferation of smooth muscle
cells.
GRAFT-VERSUS-HOST DISEASE
• Usually happens to stem cell transplants
• Infused products often contain Mature T cells
• These cells have several beneficial effects, including
promotion of engraftment, reconstitution of immunity,
and mediation of a graft-versus-leukemia effect.
However, these mature T cells may also mediate GVHD
• Acute GVHD – within 100 days. Targets skin, G.I tract,
and Liver
• If mismatched – Target is HLA. If matched –
Minor histocompatibility antigens
22 | P a g e
• Chronic GVHD – after 100 days. Fibrosis of skin, eyes,
mouth, and other mucosal surfaces
IMMUNOSUPPRESIVE AGENTS
• Corticosteroids
• Antimetabolic agents
• Calcineurin inhibitors – Cyclosporine, Tacrolimus,
Rapamycin (Sirolimus)
• Monoclonal antibodies
• Polyclonal antibodies
CLINICAL HISTOCOMPATIBILITY TESTING
• HLA Typing - phenotypic or genotypic definition of HLA
 HLA Phenotyping – Complement-dependent
cytotoxicity (CDC). Antisera + Lymphocytes. T for
Class I, B for Class II. After incubation, complement
is added.
 HLA Genotyping - use polymerase chain reaction
(PCR)–based amplification of HLA genes followed
by analysis of the amplified DNA to identify the
specific HLA allele or group of alleles.
• HLA Antibody Screening and Identification - donors
possessing those HLA antigens can be eliminated. CDC
can also be used. AHG can be added to detect low
levels of Ab.
• Percent panel reactive antibody (%PRA) - proportion of
lymphocytes in the panel (usually 30 to 60 unique
lymphocyte preparations are included in the panel)
that are killed by the patient’s serum
• Enzyme-linked immunosorbent assay (ELISA) has been
developed in recent years as a substitute for CDC-
based HLA antibody testing
• Another approach for antibody detection and
identification is flow cytometry – most sensitive
• Once a donor has been identified for a particular
patient, a donor–recipient crossmatch test is
performed to confirm the absence of donor-specific
antibody. Donor lymphocytes are incubated with
recipient serum in a CDC assay to verify a lack of
binding as detected by microscopic analysis after
addition of a vital dye.
Chapter 16: Tumor Immunology
• Proto-oncogenes = Regulatory genes that promote cell
division
• Tumor-suppressor genes = Produces growth-inhibitory
signals
• Benign = a tumor that does not invade surrounding
tissue and where normal body function is largely
preserved
• Malignant = can invade surrounding tissues
• Metastasis = malignant cells travel through the body
• Anaplastic = tumors more similar to fetal or embryonic
tissue
• TNM System = Classification of tumors (T=size,
N=lymph nodes, M=metastasis)
 FAR EASTERN UNIVERSITY – MANILA

• IMMUNOSURVEILLANCE – process where immune • Outermost cell wall component = two major proteins
system eradicates cancer cells as they form has long (M and T) – can determine serogroup or serotype
been postulated. • Group-specific carbohydrate underneath the protein
layer, divides into 20 groups called lancefield groups
Phases • Major virulent factor = M protein
1. Induction phase = cells are exposed to a variety of • Additional virulence factors = exoantigens/exotoxins
environmental insults (Exhibit dysplasia then neoplasia) Pyrogenic exotoxins (A,B,C) responsible for rash in
2. In situ phase =when neoplastic cells have formed but scarlet fever
are confined to the tissue of origin • Antibodies are produced to the following exoantigens:
3. Invasion phase = If cells become malignant and cause enzymes streptolysin O, deoxyribonuclease B (DNase
dissemination B), hyaluronidase, nicotinamide adenine dinucleotidase
(NADase), and streptokinase
• Tumor-Associated antigens (TAAs) - antigens present in • Two main damaging sequelae = ARF (Acute Rheumatic
the tumor tissue in higher amounts than in normal Fever) and Glomerulonephritis
tissue • Acute rheumatic fever - sequela to pharyngitis or
• Screening tests – used in ostensibly normal people to tonsillitis (NO SKIN INFECTION CAUSE)
detect occult cancer • Glomerulonephritis - deposition of immune complexes
• Diagnostic tests - those that help determine differential in glomeruli (Skin or Pharynx infection-caused)
diagnosis, tumor stage, prognosis, and therapy
selection DETECTION METHODS
• Cytogenetic studies, Nucleic acid amplification ASO Testing
techniques, and FISH have been used for tumor marker • Streptolysin O able to lyse cells
detection • Indicates recent streptococcal infection in patients
TUMOR MARKERS CANCERS suspected of having acute rheumatic fever or
AFP Nonseminomatous testicular, Liver, poststreptococcal glomerulonephritis following a
Germ Cell throat infection
B-2-Microglobulin Lymphocyte Malignancies • Ability of antibodies in the patient’s serum to
Calcitonin and Ca+ Familial medullary thyroid carcinoma neutralize the hemolytic activity of streptolysin O
• Expressed in Todd units
CD Markers White Blood Cell Malignancies • Range of expected normal values is variable and
CEA Colorectal; Breast depends on the (1) patient’s age, (2) the geographic
CA125 Ovarian adenocarcinoma location, and the (3) season of the year
CA15-3 Breast • A single ASO titer is considered to be moderately
CA19-9 Pancreatic elevated if the titer is at least 240 Todd units in an
ER/PR Breast adenocarcinoma adult and 320 Todd units in a child
Fecal occult blood Colorectal cancer
Anti-DNAse
hCG Nonseminomatous testicular; germ • useful in patients suspected of having
cell; trophoblastic glomerulonephritis preceded by streptococcal skin
HER2/neu Breast infections
Monoclonal free Ig Plasma Cell/B lymphocytes • antibodies to DNase B may be detected in patients
light chains with acute rheumatic fever who have a negative ASO
PSA Prostate
test result
PTH and Ca++ Parathyroid Carcinoma
• NEUTRALIZATION METHODOLOGY
Thyroglobulin Thyroid
• 4+ indicating that the intensity of color is unchanged,
and a 0 indicating a total loss of color
Chapter 17: Serology of Bacterial Infections • Normal titers for children between the ages of 2 and
BACTERIAL INFECTIONS 12 years range from 240 to 640 unit
• Bacteria have developed several ways to inhibit the
immune response or make it more difficult for the Streptozyme testing
immune response to occur. • Slide agglutination screening test
• Three main mechanisms are (1) avoiding antibody, (2) • Sheep red blood cells are coated with streptolysin,
blocking phagocytosis, and (3) inactivating the streptokinase, hyaluronidase, DNase, and NADase
complement cascade • Hemagglutination is a positive test

GROUP A STREPTOCOCCI HELICOBACTER PYLORI

23 | P a g e
 FAR EASTERN UNIVERSITY – MANILA

• Major cause of both gastric and duodenal ulcers. Chapter 18: Spirochetes
• If untreated, H. pylori infection will last for the TREPONEMA PALLIDUM AND SYPHILIS
patient’s life and may lead to gastric carcinoma • Gram negative, corkscrew motility
• Culturing for H. pylori, histologically examining gastric • T. pallidum subsp. pertenue – Yaws
biopsy tissue, and performing a urease biopsy test are • T. pallidum subsp. endemicum – Nonvenereal endemic
techniques that can be used to detect H. pylori syphilis
infections • T. pallidum subsp. carateum – Pinta
• Rapidly destroyed by heat, cold, and drying out
MYCOPLASMA PNEUMONIAE • Dies after an hour outside the host
• Lack cell walls and have a small genome, sterols in their • T. pallidum lipoprotein induces the expression of CCR5
cell membrane, and complex growth requirements in macrophages – therefore people who’ve had syphilis
• Leading cause of respiratory infections is susceptible to HIV infection
• Laboratory diagnosis of Mycoplasma pneumoniae STAGES OF DISEASE
involved testing for cold agglutinins, because these are • Primary – painless, hard chancre. Harbors T. pallidum
present in about 50 percent of patients with the and is very infectious. Chancre spontaneously heals in a
infection month
• Molecular techniques provide a more rapid means of • Secondary – multiple widespread lesions on mucous
detecting Mycoplasma infection membranes and skin. (disseminated rash) Fever and
malaise. 40% have neurologic signs. HIGHLY
RICKETTSIAL INFECTIONS INFECTIOUS.
 Short rods, or coccobacilli, that are obligate, • Latent Stage – No symptoms. Noninfectious except for
intracellular, gram-negative bacteria preggies.
Spotted Fever Group • Tertiary – Months to years after secondary infection.
R. rickettsii – Rocky Mountain Spotted Fever (Tick) 3 Major manifestations
R. japonica – Japanese Spotted Fever (Tick) 1. Gummatous/Late benign syphilis – Granulomatous
R. felis – Flea-borne spotted fever lesions. Not contagious
R. akari – Rickettsial pox (Mite) 2. Cardiovascular syphilis – aortitis or aortic aneurysm
Typhus Group 3. Neurosyphilis – Tabes dorsalis (degeneration of lower
R. typhi – Endemic Typhus/Murine Typhus (Flea Feces) spinal cord and general paresis)
R. prowazekii – Epidemic Typhus/Brill’s dse (Louse Feces) • HUTCHINSON’s TRIAD – DEAFNESS, PEG SHAPED
- Recrudescent Typhus (Years after Epidemic TEETH, INTERSTITIAL KERATITIS
Typhus)
• WEIL-FELIX TEST DETECTION OF SYPHILIS
• OX-19 and OX-2 strains of Proteus vulgaris and the OX- Direct Detection
K strain of Proteus mirabilis A. Direct Microscopic Darkfield
• Detection of rickettsial antibodies • Primary and Secondary
• Exudates
Disease OX-2 OX-19 OX-K
• Corkscrew motility
• Need experience
R. prowazekii Epidemic + 4+ --- B. Fluorescent Antibody Testing
typhus/Brill’s • Use of fluorescent labeled antibody
dse
• Direct
R. typhi Murine Typhus + 4+ --- • Indirect
Nontreponemal Tests
R. ricketsii RMSF + 4+ --- • Focus is detection of reagin (Anticardiolipin)
• Cardiolipin – Chemically known as diphosphatidyl
R. akari, Ricketssial pox --- --- --- glycerol; usually from beef
R. burnetii, Q fever • Expected result = Flocculation (precipitation over a
B. quintana Trench Fever narrow range of Ag conc.)
• WASSERMANN – First serological test. Nontreponemal.
O. Scrub Typhus --- --- 4+
Uses complement fixation technique
tsutsugamushi
1. Venereal Disease Research Laboratory (VDRL)
2. Rapid Plasma Reagin (RPR)
• IFA test and the micro-IF are currently considered the 3. Toluidine Red Unheated Serum Test (TRUST)
gold standard for detecting rickettsial antibodies 4. Unheated Serum Reagin (USR)
5. Reagin Screen Test (RST)

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 FAR EASTERN UNIVERSITY – MANILA
VDRL
• Specimen: serum (inactivated or heated at 56oC for 30
minutes)
• Antigen:
• 0.03% Cardiolipin (beef heart)
• 0.9% Cholesterol (carrier)
• 0.21% Lecithin (carrier)
• 1% NSS at pH 6.0
• USE 0.05mL PATIENT SERUM
• Hamilton syringe (18g) is used to dispense. 60 drops.
14mm diameter rings. Rotate for 4 minutes at 180 rpm.
If reactive, Quantitative by serial dilution.
• QUANTITATIVE: 19G – 75 Drops or 23G – 100 drops
• CSF: 21/22G – USE BOERNER AGGLUTINATION SLIDES
100drops (8 mins 180rpm) If (+) – Neurosyphilis
RPR
• Antigen suspension contains charcoal (For reading)
• Stable up to 3 months
• Contains EDTA, thimerosal, choline chloride to
inactivate complement in serum
• 18 mm diameter rings. 20G 60drops/mL
• 8 minutes at 100RPM
• More sensitive than VDRL in primary syphilis
Treponemal
1. Fluoresence treponemal antibody-Absorption Test
(FTA-ABS) (Detects IgG)
• Use reiter strain (nonpathogenic) to remove
crossreactivity in serum inactivated with heat then
add to slide with fixed Nichols strain T. pallidum
(dead)
• 100% in secondary and latent stage
• 20% in primary stage are nonreactive
• Reactive for life
• Most widely used confirmatory test
2. MHA-TP (Microhemagglutination T. pallidum)/TPHA (T.
pallidum hemagglutination test)
3. HATTS (Hemagglutinaton treponemal test for Syphilis)
4. TPI (Treponema pallidum immobilization test)
5. TP-PA (Treponema pallidum particle agglutination test)
OTHER TESTS:
 Congenital syphilis – Western blot is recommended
test.
 EIA – detects IgM in baby
 VDRL – neurosyphilis (lacks sensitivity but highly
specific) (Increased lymphocyte count and elevated
total protein (>45mg/dL)
LYME DISEASE
Borrelia burgdorferi
• Spirochete
• OSP-A to OSP-F = lipoprotein antigens
• Responsible for crossreaction with B. recurrentis and T.
pallidum
• Grows in culture using liquid medium Barbour-
Stoenner-Kelly at 33oC for 6 weeks or longer
25 | P a g e
• Reservoir is Peromyscus leucopus (white-footed
mouse) or White-tailed deer
• Vector is Ixodes (mostly active in summer)
Stages of disease
• 1) Localized Rash/Cutaneous = Erythema chronicum
migrans (Bull’s eye rash)
• 2) Blood stream dissemination = Multiple lesions; Pain
in joints, tendons, muscles and bone. Neurologic and
Cardiac involvement. Facial palsy. Neuroborreliosis.
• 3) Late Disseminated stage – Arthritis and late
neuroborreliosis (peripheral neuropathy and
encephalomyelitis)
LABORATORY TESTING OF LYME DISEASE
• Rash – Presumptive finding
• History of tick bite, clinical symptoms, confirmation
using serological tests
1) IFA – first test to evaluate Ab response. Positive if
1:256. False (+) – Syphilis, Recurrentis, RA
2) EIA – Good screening test
3) Western Blot – Confirmatory test
 IGM (+) if bands in 2 of the following are present:
23 (Osp C), 39, and 41 (flagellin)
 IgG (+) if 5 out of ten bands are present
4) PCR – Most sensitive`
Chapter 19: Parasitic Immunology
POSSIBLE RESULTS OF PARASITIC INFECTIONS
1) No infection
2) Parasite becomes established but is the killed and
eliminated by the host defense mechanisms
3) Host is killed
4) Long lasting infection
5) Host’s immune system attacks the parasite and in doing
so also causes damage to host tissues.
EVASION OF HOST DEFENSES
1) Antigenically complex with immunologically different
stages in different body locations during their life cycle
2) Hiding inside host cells
3) Acquiring host antigens
4) Changing the surface protein
5) Shedding of antigen
6) Redirecting the immune response from a vulnerable
target to a dominant but irrelevant antigen
7) Cannot be eliminated leading to immune suppression,
autoimmune disorders and hypersensitivity
HOST RESPONSE
1) Interferon gamma activated macrophages
2) Antibodies – IgM, IgG, and IgA
3) Release of IL-3 and GM-CSF to induce the BM to produce
more Leukocytes
4) IgE production
5) Release of eosinophil chemotactic factor
TOXOPLASMOSIS
 FAR EASTERN UNIVERSITY – MANILA
• Causative agent: Toxoplasma gondii (Humans:
Intermediate Cats: Definitive
• Infective stage for humans: oocyst
• This disease is nearly always asymptomatic. Rarely, the
parasite reaches the central nervous system (CNS) or
the eye.
• Can survive indefinitely in macrophages
Modes of transmission
1) Ingestion of tissue cysts in raw or uncooked meat
2) Fecal-oral contamination from infected cats
3) Transplacental transmission from infected mother
4) Blood transfusion or organ transplant from infected
individual
CLINICAL MANIFESTATIONS
• Immunocompetent – Asymptomatic. Acute infection
(resemble IM)
• Immunocompromised – Severe infection manifesting
as disseminated disease
• Associated with stillbirth and abortion especially during
1st trimester
• ToRCHeS panel – Detection of IgM against toxoplasma,
rubella, CMV, and HSV, and Syphilis
LAB TESTING
• Bioassay – “Gold standard” – Neutralization procedure
using patient’s blood or other body fluids with parasite
inoculated into a mouse or culture eclls
• IFA – widely used. Detects antibody
• Sabin-Feldman Dye Test – Patient serum (Abs) + live
T.gondii + methylene blue = Dead parasites will not
stain blue
• Indirect EIA – method of choice. Most sensitive. Uses
ALP as enzyme
Chapter 20: Fungal Immunology
Fungal Infections Increases with
1) Increased use of broad spectrum antibiotics
2) Use of immunosuppressive drugs
3) Organ transplants
4) Immunodeficiency
• Usually introduced in the body traumatically or through
inhalation.
• Four-fold increase in titer is significant in making a
diagnosis
• One of the most common opportunistic diseases in
patients with AIDS is pneumonia caused by
Pneumocystis carinii, also known as Pneumocystis
jiroveci
Immune response to fungi
• Factors that affect virulence of fungus – Ability to
survive at 37oC at a neutral pH and production of toxins
• Opsonization is not necessary for phagocytosis of most
fungi, but it is required for phagocytosis of
Cryptococcus neoformans
26 | P a g e
• Cellular immunity is the most important defense
against fungal infection
FUNGAL INFECTIONS
• Candidiasis – Most common cause of serious fungal
disease
• Opportunistic pathogen. Normal inhabitant of
alimentary canal.
• Aspergillosis – Found worldwide in soil, air, decaying
vegetables, and stored grains
• Ouchterlony immunodiffusion (ID) or
Counterimmunoelectrophoresis (CIE) – Positive only
for immunocompetent host
• EIA – Detects serum galactomannan antigen. For
immunocompromised
• Coccidioidomycosis – C. immitis. Pulmonary infection.
Ag is Coccidioidin
• Complement fixation – most widely used. 1:2-1:4 –
Presumptive evidence of early infection. 1:16 –
active infection.
• Greater than 1:16 = disseminated. 1:2 or higher titer
in CSF = Coccidioidal meningitis
• Immunodiffusion – Most commonly used screening
test
• Cryptococcosis – C. neoformans. Chief vector is
pigeons. Pulmonary inf.
• Latex Particle Agglutination (LPA) Antigen Test –
Detect capsule polysaccharide ag
• India Ink
Chapter 21: Viral Immunology
Hepatitis
• Primary – Hepa A, B, C, D, E, G
• Secondary – EBV, CMV, HSV
• Labs: Increased bilirubin, ALT, and AST
• Hepa A and E – Mostly fecal route
• Hepa B, C, D – Mostly parenteral
HEPATITIS A
• Picornaviridae. Single Stranded RNA Virus. Average
incubation of 28 days
• Does not progress to a chronic state and is usually self-
limiting
• Antigens are shed in feces.
HEPATITIS B
 FAR EASTERN UNIVERSITY – MANILA
• Hepadnaviridae. DNA Virus. Incubation period of 45-90
days.
• Most HBV-infected adults recover within 6 months and
develop immunity to the virus, but about 1 percent
develop fulminant liver disease with hepatic necrosis,
which has a high rate of fatality
• Transmitted through Parenteral, Sexual, Perinatal
routes
HEPATITIS C
• Flaviviridae. Single stranded RNA. Average incubation
of 7 weeks (2 to 30 weeks)
• Although the majority of infections are asymptomatic,
the infection is problematic, because about 85 percent
of persons develop chronic infection, which leads to
cirrhosis in about 20 percent of these individuals
• Hepatocellular Carcinoma
HEPATITIS D, E, G
Hepatitis D - Delta Hepatitis. Parenterally transmitted. HDV
is a defective virus that requires the help of HBV. Largely
confined to IV Drug Users.
• Coinfection – HDV and HBV occur simultaneously
• Superinfection – HDV infects individuals who are
already chronic HBV carriers
• HDV RNA, a marker of active viral replication that is
present in all types of active hepatitis D infection
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Hepatitis E - Hepeviridae. Single Stranded RNA
• causes an acute, self-limiting hepatitis that lasts 1 to 4
weeks in most people who become infected.
Fulminant hepatitis, associated with rapidly
progressing disease and a high mortality rate, occurs
more commonly in pregnant women
Hepatitis G - Flaviviridae. Transmitted by the bloodborne
route, perinatal route, and possibly through sexual contact.
High Risk: Dialysis and renal transplant recipient
Transfusion-Transmitted Virus - Annellovirus. Parenteral
transmission of the virus through contaminated blood has
been clearly evidenced, and presence of the virus in stool,
bile, and saliva, suggest transmission by the fecal-oral and
respiratory routes as well
HERPES VIRUS INFECTIONS
INCLUDES 8 VIRUSES THAT CAN CAUSES DISEASE IN
HUMANS - herpes simplex viruses (HSV-1 and HSV-2);
varicella-zoster (also known as human herpes virus-3 or
HHV-3); the Epstein-Barr virus (HHV-4); cytomegalovirus
(HHV-5); and the human herpes viruses 6, 7, and 8 (HHV-6,
HHV-7, and HHV-8), the latter of which has been associated
with Kaposi’s sarcoma
• All can cause latent infection. Complex DNA Virus.
EPSTEIN-BARR VIRUS
• Causes a wide spectrum of diseases
• Most common – intimate contact with salivary
secretions (KISSING DISEASE) (also, blood transfusions,
bone marrow and solid organ transplants, sexual
contact, and perinatal transmission (less frequent)
• Poor Hygiene – During childhood. Higher standard of
hygiene – Adolescence/adulthood
• EBV infects CD21+ cells
• Primary infection in Adults/Adolescents – IM (Fever,
Lymphadenopathy, Sore Throat)
ANTIGENS
EARLY ACUTE LATE ACUTE LATENT
EA – D (Early VCA (Viral Capsid EBNA (EBV Nuclear
Antigen – Diffuse) Antigen) Antigens)
EA – R (Early MA (Membrane LMP (Latent
Antigen – Antigen) Membrane
Restricted) Proteins)
Laboratory Testing
• Absolute lymphocytosis = >50% lymphocytes (W/ 10%
atypical)
• Presence of Heterophile Abs and antibodies to EBV
antigens.
• Heterophile antibodies are IgM – React with Horse,
Ox, Sheep (Adsorbed by Beef). (Replaced by rapid latex
agglutination or solid phase immunoassays (Bovine
RBCs are the antigens)
 FAR EASTERN UNIVERSITY – MANILA

• Monospot test – Differential slide agglutination test • Definitive diagnosis – Based on identifying VZV
using Horse RBCs antigens in skin lesions, tissue, or vesicular fluids.
• Paul-Bunnell – Hemagglutination test using inactivated • Rapid - multinucleated giant cells called Tzanck cell
patient serum and sheep RBCs through examination of smears
ANTI-VCA ANTI-EA • Most accurate and sensitive – PCR
• Serology – Most useful in determining immunity and in
CONDITION IgM IgG EA-D EA-R ANTI- HETERO identifying VZV-susceptible who may benefit from
EBNA PHILE
AB (IgM) prophylactic treatment
• Most sensitive and reliable serological test for VZV –
UNINFECTED - - - - - - FAMA (fluorescent antibody to membrane antigen)

ACUTE IM + ++ + - - + RUBELLA
CONVALESCE • Single stranded RNA. Togaviridae. German Measles
- + - +/- + +/-
NT (12-23 days incubation)
PAST • Transmitted through: Respiratory droplets and
- + - - + -
INFECTION Transplacental
CHRONIC • Produces a characteristic erythematous,
- +++ + ++ +/- -
ACTIVE maculopapular rash
INFECTION
• Dangerous during pregnancy (miscarriage, stillbirth or
Congenital Rubella Syndrome (CRS)
CYTOMEGALOVIRUS
• Ubiquitous Laboratory Testing
• Spread through: Close contact with secretions, • Viral culture not routinely used (Too time-consuming)
Intimate sexual contact, Blood transfusions, Solid organ • Serological tests – Method of choice
transplants, and perinatal • ELISA – most commonly used (IgM)
• Primary infections usually asymptomatic • Primary Rubella Infection: IgM or 4-fold titer in IgG
• Latent in monocytes, dendritic cells, myeloid • IgG indicates Immunity (10-15 IU/mL considered
progenitor cells, and peripheral blood leukocytes protective)
• Most important infectious agent associated with organ • RT-PCR - highly sensitive and specific aid in prenatal or
transplantation, and most common cause of congenital postnatal diagnosis and can be used to detect rubella
infections. RN

Laboratory Testing RUBEOLA

• Preferred diagnostic methods – Culture, CMV antigens,
or CMV DNA
• For culture, shell vial method is preferred (Reduces
time to 16-72 hours) - Detection of the CMV lower
matrix protein pp65 in CMV-infected leukocytes =
MORE RAPID DIAGNOSIS
• Quantitative PCR - monitor effectiveness of antiviral
treatment and identify patients at risk for developing
disseminated CMV disease
• Serology tests – most useful in documenting past
infection
VARICELLA-ZOSTER VIRUS
• Causes Varicella (Chicken pox) and Herpes Zoster
(Shingles) – Most risky for neonates, pregnant women,
and immunocompromised patients
• Transmitted though inhalation and transplacental
transmission
• Can lead to congenital malformations
• Establishes latency in Dorsal Ganglion cells
• Most common complication in Shingles: Postherpetic
neuralgia
Laboratory Testing
• Single stranded RNA. Paramyxoviridae. Measles (10-12
days incubation)
• Transmitted through: Direct contact with droplets
• Koplik spots appear in prodromal period
• Produces erythematous, maculopapular eruption
• Adults, Children <5 years old, and
Immunocompromised are prone to complications
(diarrhea, otitis media, croup, bronchitis, pneumonia,
and encephalitis)
• Subacute Sclerosing Panencephalitis (SSPE) may occur
• Dangerous during pregnancy (higher risk of premature
labor, spontaneous abortion, or low birth weight)
Laboratory Testing
• Culture – not generally performed
• Optimal time to recover the virus
• Nasopharyngeal aspirate, Throat Swab, Blood – up
to 3 days after rash onset
• Urine – 1 week after appearance of rashes
• Diagnosis of Measles (acute) – IgM or 4-fold rise in IgG
• IgM detectable 3-4 days after rashes up to 8-12 weeks
• IgG detectable 7-10 days after onset of symptoms
(Persists for life)

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 FAR EASTERN UNIVERSITY – MANILA

• Most commonly used – ELISA CHARACTERISTICS AND VIRAL

• Molecular methods – If serology is inconclusive or REPLICATION
inconsistent • Etiologic agent of AIDS (Acquired Immunodeficiency
Syndrome)
MUMPS • Luc Montagnier, Robert Gallo and Jay Levy identified
• Single stranded RNA. Paramyxoviridae. 14-18 days HIV-1
incubation • HIV-1 = Formerly called HTLV-III, Lymphadenopathy-
• Transmitted through: Respiratory droplets and possibly associated virus (LAV), and AIDS-associated retrovirus
fomites (ARV).
• Most common clinical manifestation: Inflammation of • HIV-2 = Primarily in West Africa. Less pathogenic and
the parotid glands lower transmission
• Complications include Meningitis, testicular • Transmission: Sexual contact, contact with blood/body
inflammation (Orchitis), ovarian inflammation, and fluids, perinatal.
deafness • 0.3% - percutaneous 0.09% - mucous membrane
• Dangerous in pregnant women (increased risk of fetal • Genus Lentivirinae, Family Retroviridae. RNA virus with
death at first trimester) Reverse Transcriptase.
• Structural Genes: gag, pol, env
Laboratory Testing GAG p55 (precursor)
• First few days – Saliva, Urine, CSF, excretory duct of Core structural proteins: p6, p9, p17, p24 (located in
parotid gland nucleocapsid)
• Culture – shell vial cultures of rhesus monkey kidney POL gp160 (precursor)
cells or human embryonic lung fibroblasts Cleaves into gp120 (protruding) and gp41
• RT-PCR – if culture is not successful (transmembrane)
• Serology – most simple and practical for mumps
• Most commonly used: ELISA and IFA ENV For HIV Replication
p31 – Reverse transcriptase (integrase) p10 – protease
• Recent Infection: IgM or fourfold increase in IgG Their subunits: p66 and p51 p66 (degradation of RNA)
• IgG - immunity
1. Attachment – Binding of Gp120. Can attach to T helper,
HUMAN T-CELL LYMPHOTROPIC VIRUS macrophage/monocyte, dendritic cells, Langerhans,
• RNA Viruses that have Reverse Transcriptase (HTLV 1 and microglial cells
and 2) • T-tropic or X4 strains
• 3 structural genes: gag (Viral core proteins), pol (RT), • M-tropic or R5 strains
env (envelope glycoproteins) 2. Penetration/Fusion – CXCR4 and CCR5
• 2 major regulatory genes: tax and rev 3. Uncoating
• Transmitted through: Bloodborne, Sexual contact, and 4. Eclipse - Provirus
Mother to child. 5. Synthesis – DNA to mRNA to viral proteins to viral
• HTLV 1 – associated with T-cell leukemia/lymphoma, particles to virions
HTLV-associated myelopathy/tropical spastic 6. Release – acquire envelope
paraparesis (HAM/TSP), HTLV uveitis, sicca syndrome
and infective dermatitis. MANIFESTATIONS, SYMPTOMS, and
• HTLV 2 – less severe TREATMENT OF HIV
• Initial viral replication – Increased levels of p24 antigen
Laboratory Testing and Viral RNA
• Culture: Hard to perform • First antibodies – against gag. Most immunogenic –
• ELISA tests used initially to screen (use gag and env as viral envelope
antigens) • Not completely eliminate virus (B cells and CTL) due to:
• Confirmatory is WESTERN BLOT. • Rapid genetic mutations, Altered antigens,
• Specimens are considered positive for HTLV-I or HTLV-II Downregulation of MHC I, and Eclipse
by this test if bands representing antibodies to the gag • Hallmark: Decreasing CD4+ (decreased humoral and
protein p24 and the env glycoproteins of either virus cell-mediated immunity)
are present STAGES OF PROGRESSION
• More sensitive – Radioimmunoprecipitation assay 1. Primary Acute
(RIPA), PCR • Viremia, Acute retroviral syndrome, IM-like
2. Latency
• Median length of 10 years in untreated px
Chapter 22: Human Immunodeficiency Virus • LNTP – Long-term non-progressors (Normal or
mildly decreased CD4+, low viral load and

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asymptomatic even in absence of antiretroviral 4th EIA/IFA HIV-1, 2, and

therapy) Gen p24
3. AIDS
• <200/uL CD4+ or certain opportunistic • Detuned assays – differentiate recent from established
infections/malignancies associated with AIDS infection
• Immunosuppression, viremia, life-threatening • Rapid Tests = Lateral flow/Flow-through immunoassays
infections, malignancies, neurological symptoms (Colorimetric)
TREATMENT FALSE Neg.
• 3 main types of drugs: 1. Spx collection prior to antibody formation (no
1. Nucleoside analogue reverse transcriptase seroconversion yet)
inhibitors – Inhibit further action of RT when they 2. Immunosuppressive therapy/replacement therapy
incorporate themselves into viral DNA (bind to viral 3. Defective Ab synthesis (Hypogamma)
DNA) 4. Technical errors
2. Non-nucleoside reverse transcriptase inhibitors – FALSE Pos.
stops RT from transcribing RNA to DNA (bind 1. Heat inactivation of serum
directly to enzyme) 2. Repeated freeze and thaw
3. Protease inhibitors – prevent cleavage of precursor 3. Autoreactive antibodies
proteins 4. Multiple pregnancies
• HAART – Highly Active Anti-Retroviral Therapy 5. Severe hepatic disease
• THERE IS NO CURE 6. Passive Igs
• Ultimate means of prevention – Formulation of a 7. Recent vaccination
vaccine and community-based education 8. Some malignancies

LABORATORY TESTING FOR HIV Antibody Detection – Confirmatory

INFECTIONS • Western Blot (99% PPV)
CD4+ T Cell Enumeration • More technically demanding. Nitrocellulose strip w/
• Hallmark = Decreased CD4+ (<200/uL) HIV proteins separated by PAGE then reacted with
• Monitor effectiveness of therapy (Every 3-6 months) - serum + AHG w/ enzyme label then washed
<350/uL • Early HIV antibodies are p24 and p55 followed by gp41,
• If CD4+ still declines by more than 25%, 120 and 160
therapy is changed • Positive = At least 2 of p24, gp41 and gp120/160 are
• Gold standard in CD4+ T cell Enum = present
Immunophenotyping by FLOW CYTOMETRY • NEG = No bands present or no corresponding bands
• % CD4+ T cells = (# of CD4+ / # of Total Lymphocytes) x • Specimen that have some bands but do not meet
100 criteria = Indeterminate
• Absolute = WBC Ct. x %Lymphocytes x %CD4+ T • Other confirmatory tests: IFA, RIPA, Line
cells Immunoassays, Rapid confirmatory tests
• Can also use ratio of CD4 to CD8. If with AIDS, usually
<1:1 Antigen Detection
• p24 testing
Antibody Detection – Screening • Correlate with amount of HIV replication
• Screening = Standard is ELISA – goal is to detect ALL • Undetectable as anti-p24 develops
INFECTED PERSONS • Appears 1 week earlier than Ab
• First implemented 1985 • All (+) results should be confirmed by a neutralization
• Cornerstone of screening procedures (easy to perform, assay
large num. of samples, high sensitivity and specificity) • Good for early diagnosis, newborns, and to monitor
METHOD ANTIGEN ANTIBODY LIMITATION therapy

1st Indirect Viral Lysate HIV-1 Abs Prone to false Viral Nucleic Acid Detection
Gen ELISA (+) and can’t • Viral Load tests = quantitative tests for HIV nucleic acid
detect HIV-2 • Becomes detectable 11 days after infection
2nd Indirect Recombinant HIV-1 and 2 Decreased
and rise to very high levels
Gen ELISA HIV Abs sensitivity
against certain • “Set point” – Stable level of HIV RNA
HIV subtypes • USES – disease progression, patient management,
3rd Sandwich Recombinant HIV-1 and 2 response to therapy (prediction and monitoring),
Gen ELISA HIV Abs

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detection of HIV in seronegative patients or thise with

confusing results
• Change in Viral Load considered significant if there is at
least a three-fold or 0.5log increase or decrease in
number of copies/mL
• Viral Load assays: RT-PCR, Branched chain DNA assay
amplification, NASBA (nucleic acid sequence-based
amplification)
• Drug Resistance Testing:
• Genotypic Resistance Assays – Detects
mutations in RT and Protease
• Phenotypic Resistance Assays – Determines
ability to grow in presence of drugs

Culture
• Virus isolation – DEFINITIVE
• Best sample is peripheral blood although CSF,
saliva, cervical secretion, semen, tears, and
organ biopsies can be used
• Detects RT activity or p24 ag in culture
• NOT USUALLY DONE

• Neonatal testing – Difficult to test for antibodies. BEST

diagnosed using molecular methods. (IgG passed from
mother. Takes >15 months)

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