IMMUNOLOGY AND SEROLOGY, LEC 2.

The organism must be isolated from a host

Prof. Ruby Garcia-Meim, RMT, MSPH containing the disease and grown in pure
culture
UNIT 1: HISTORY OF IMMUNOLOGY 3. Samples of the organism taken from pure
culture must cause the same disease when
inoculated into a healthy, susceptible animal
INTRODUCTION
in the lab
➢ Experiential Immunology Period
4. The organism must be isolated from the
➢ Experimental Immunology Period
inoculated animal and must be identified as
➢ Modern immunology Period – molecular
the same original organism first isolated from
biology
the originally diseased host
Immunology act as an independent subject: (In 1971,
International Conference of Immunology, in USA)
Pasteurization
In 1880, Pasteur
I. Experiential Immunology Period
(The 17th century – the middle of 19th century) • Anticholera live attenuated vaccine
• Old culture of chicken V. cholera
In ancient times, many serious infection diseases, o Artificial active immunity
such as smallpox, plague and cholera etc. caused - The body is the one that
innumerable people dead) synthesizes the antibody.

In 1670, Chinese medical practitioners: Active Immunity:

variolation -> process of collecting pustules from : form of adaptive immunity that is induced by
small pox lesions -> healthy individuals inhale -> exposure to a foreign antigen and in which the
exhibit immunity against small pox. immunized individual plays an active role in
responding to the antigen.
• Edward Jenner – An English physician
Discovered that cowpox vaccination protected 2. Passive Immunity: Ab is transferred
against smallpox in 1796
In the late eighties of 19th century
• Collected cowpox virus – vaccinia virus ->
➢ Roux and Yersin --- exotoxin in C.
injected to man -> immunity against smallpox
diphtheria
• Cross-reaction/cross reactivity – 2 Ag’s
• The discovery of diphtheriae antitoxin and
share the same glucolipid antigenic sites
bactericidins
o Ag1 = Ab2 = Ag2
Antitoxin --- Antibody (Ab)
Vacca: cow
Exotoxin --- Antigen (Ag)
Vaccine: A preparation of microbial antigen, often
combined with adjuvants, that is administered to • Yersin – discovered Y. Pestis – plague
individuals to induce protective immunity against ➢ Study on reaction of Ag and Ab in vitro
microbial infections o Serology – study of serum
Antigen : weakened/attenuated (old/aging/heat) ➢ In 1980, Von Behring and Kitasato
: killed, whole organism/subunits o diphtheriae antitoxin was applied
in treatment of Diphtheria
• Prophylactic (prevention) /experimental
o Artificial passive immunity
(cancer vaccines) /therapeutic
Vaccination: prophylaxis
Passive immunity:
• Immunization against infectious diseases,
• The form of immunity to an antigen that is
originally derived from immunization against
established in one individual by transfer of
smallpox
antibody or lymphocytes from another
individual who is immune to that antigen.
II. Experimental Immunology Period
(Middle of 19th century-the middle of 20th century)

1. Active Immunity

In the middle of 19th century

➢ Robert Koch --- isolated culture bacteria
successfully, TB Bacilli
➢ Louis Pasteur --- Infectious diseases were
caused by pathogens, anti-cholera vaccine

Koch’s Postulates ---- infectious diseases

Active – body is the one that synthesizes the Ab
1. The organism must always be present, in
every case of the disease • With memory T/B cells
Passive – you already have the Ab producing cells
(lymphocytes)
• Without memory

3. Study on antigen

In the early of 20th century, Karl Landsteiner

• study on antigenic determinant
(epitope)
• ABO blood type: Chimerism – one organism with two genetically
A, B, AB, O distinct cells
• Transfusion became safer with his discovery. e.g., Lady Fairchild

4. Study of immunochemistry

In 1938, Tiselius and Kabat

• Ab is y (gamma) globulin /
immunoglobulin

In the fifties of 20th century, Porter and Edelmen

• Molecular structure of Ab:
4 peptides
2 types of chain: heavy chain, light chain
• Antibody – gamma globulin.
Immunoglobulin

5. Study on immune tolerance

• No immune responsiveness
• No positive response to specific ag

In 1945, Owen found natural immune tolerance

6. Hypothesis for Ab formation
In 1953, Medawar set up animal model of
acquired immune tolerance in newborn period 1930, Breinl and Haurowitz
• Self Ag – naturally tolerance or else, it will • Templates postulates
produce autoimmune disorders
1940, Pauling
• Variable folding postulate

1955, Jerne
• Natural selection postulate

1959, Burnet
• Clonal selection theory
Clone: a group cells that stem from identical
cell
Variable folding theory
➢ 1st pregnancy: skin graft was get from twin 1
and transferred to twin 2 : didn’t reject the skin
graft; it tolerates
➢ 2nd pregnancy: got skin graft from 1st
pregnancy cattle and transplanted to 2 twins
on 2nd pregnancy. The 2 twins rejected
➢ Therefore; it tolerates only on the same
batch of pregnancy.

Clonal Selection Theory

 Pirquet and Shick – Hypersensitivity
(exaggerated immune response)

1903, Arthus – Arthus phenomenon

1907, Donath and Landsteiner – autoantibody

cause autoimmune disease

III. Modern Immunology Period

(Middle of 20th century – the 21st century)

Before birth, various clones of lymphocytes during 1. Study of Immune System

prenatal hematopoiesis will react to self-antigens.
Those that reacts are naturally deleted. In 1957, Glick Fabricius and Xianguang Zhang
• Chicken without bursa can not produce Ab
After birth, those that were not deleted • No B cell
They are the one responsible for reacting foreign • B cell deficiency – called Bruton’s
antigens and undergo proliferation. agammaglobulinemia

Clonal Selection Theory In 1961, Good and Miller

(1) There are various lymphocyte clones in our • Cell mediated immunity of new born mice
body, each of them bears a unique type of Ag whose thymus were taken away are
receptor which can recognize Ag specifically defective
(2) The clones of lymphocytes that can recognize • T cell deficiency – Digeorge syndrome
self-Ags will be destroyed or learn to tolerance
to self Ags (forbidden clones) at the early
stage of their development.
--- clone deletion
(3) The clones of lymphocytes that can be
interacted with corresponding Ag will be
selected and lead to activation, proliferation,
produce Ab and specific memory cells .
--- clone selection
(4) Forbidden clones can be revived and cause
autoimmunity

7. Mechanism of protective immunity 2. Study on monoclonal antibody

1883-1884, Metchnikoff In 1975, Kohler and Milstein

• Cell mediated immunity (CMI) • They performed hybridoma technology
o Microorganisms were engulfed and • Used plasma cell and myeloma cell together
destroyed by phagocytic cells • Antisera and test kits has monoclonal
antibodies
1897, Ehrlich
• Humoral Immunity (HI) 3. Study on immune genetics
o Ab in serum played important roled in
protective immunity In 1987, genetic control of antibody diversity
• Discovert of accurate mechanism of imune
1903, Wright & Dauglas response on gene level (MHC, TCR, BCR)
- Both HI and CMI were very important for
protective immunity.
- Ab in serum could promote the phagocytosis
of phagocytic cells

Humor – means secretions

8. Study on immune-pathology & immune

disease

1902, Richet and Portier – Anaphylaxis (severe Jean Dauset 1916 – Novel Prize 1980 for study on
allergic reaction) immunogenetics
MHC – single gene that controls rejection of transplant
4. Study on molecular mechanism of T/B
lymphocyte activation and signal
transduction

5. Study on effecttive mechanism of immune

cells

6. Study on clinical immunology

➢ Organ transplantation
➢ Autoimmune disease
➢ Tumor immunology
➢ Infectious diseases

7. Study on applied immunology

➢ Preparation of monoclonal antibody and

genetic engineering antibody
➢ Preparation of recombinant cytokines e.g.,
EPO
➢ Study on DNa vaccine
➢ Study on treatment with immune cells (stem
cell treatment)

8. New techniques of modern immunology

and application

➢ Separation of immune cells (B cells, T cells)

➢ Protein analysis technique
➢ Phage display technique
➢ Preparation of new animal model
UNIT 2: THE LYMPHOID SYSTEM Primary – production and maturation of T & B cells
Mature = immunocompetent cells
LYMPHATIC SYSTEMS / immune system Secondary – site of activation, proliferation, and
differentiation
Consists of:
1. Lymphatic vessels Primary Lymphoid Organs
2. Lymphoid tissues and lymphoid organs
BONE MARROW
Lymphatic system - Hematopoietic tissues
• One way system: to - Largest tissue of the body
the heart - Total weight: 1300-1500g (adult)
• Return of collected - Center for Ag-independent lymphopoiesis
excess tissue fluid - Lymphocyte stem cells from BM travel to other
• Return of leaked primary lymphoid organs where further
protein maturation takes place (T cells)
• “Lymph” is this fluid
• Edema results if
system blocked or
surgically removed

Lymph capillaries
• Have one-way minivalves allowing excess
fluid to enter but not leave
• Picks up bacteria and viruses as well as
protein, electrolytes and fluid (lymph nodes
destroy most pathogens)

Multipotential hematopoietic stem cell → Common

lymphoid progenitor → lymphocytes → B lymphocyte,
T lymphocyte, Natural Killer cells(granulated)

- Same with blood vessel but thinner and more

delicate
• Absent from bone, bone marrow, teeth, CNS
• Enter lymphatic collecting vessels

Lymphatic collecting vessels

• Similar to blood vessels (3 layers), but thin &
delicate
• Superficial ones in skin travel with superficial
veins
B cell – bone marrow – cell-mediated immunity
• Deep ones of trunk and digestive viscera T cell – thymus – antibody-mediated immunity
travel with deep arteries NK cells – immunological surveillance (cancerous
• Very low pressure and virally infected cells)
• Distinctive appearance of lymphangiography
• Drain into lymph nodes THYMUS
- Where T cells develop their identity
Lymphoid Organs - Small, flat, bilobed organ found in the thorax,
Primary right below the thyroid gland and overlying the
• Bone marrow heart
• Thymus - About 30g at birth, 35g at puberty, gradually
Secondary atrophies
• MALT (mucosa-associated lymphoid tissues) - Cortex: where surface AGs are acquired
• Tonsils - Medulla: where T lympho are released
• Appendix
• CALT (cutaneous-associated lymphoid tissues
Mature T cells
T helper – CD4
T cytotoxic cell – CD8

Pulp

Secondary Lymphoid Organs

• Spleen, lymph node, tonsils, appendix,

Peyer’s patches, MALTs
• Where reproduction of lymphocytes occurs
(Ag dependent lymphopoiesis)
• Naïve or resting lymphocytes die w/in a few
days after leaving the primary lymphoid
organs unless activated by foreign Ag
(mature lympho w/o Ag encounter)

SPLEEN
- Largest secondary
lymphoid organ
- 12cm length, 150g
weight (adult)
- Upper-left quadrant
of the abdomen
- Large
discriminating filter
- Pitting and culling
- Sequester 1/3
platelet mass

Splenectomy – platelet count increase

Splenomegaly – platelet count decrease
Pitting – plucking out of inclusion bodies
Culling – killing of old mature RBC LYMPH NODES
- Central collecting points for lymph fluid from
- Splenic tissue can be divided: adjacent tissues
o Red pulp – destroy old RBCs - Filtration and generation of B memory cells:
o White pulp – contains the lymphoid main function of this organ
tissue - w/ sinuses: macrophages
PALS (Periarteriolar Lymphoid Sheat) : T cells Cortex: macrophage, B cells
Primary follicles: B cells o primary follicle: resting B cells
Marginal zones: dendritic cells o secondary/germinal center: Ag
stimulated B-cells
Paracortex: T cells, interdigitating cells
Medulla: T cells, B cells, Macrophage, plasma
cells
Afferent lymphatic vessel – where lymph enters

Other Secondary Organs

• T and B cells are segregated and perform

specialized function

MALT: GIT, Respiratory, Urogenital

: Macrophages and lymphocytes
Parts of the intestine are so densely packed with MALT
that they are considered lymphoid organs

Peyer’s patches: specialized MALT

- Aggregated lymphoid nodules
o About 40 follicles, 1 cm wide
o Distal small intestine (ileum)
Appendix – vestigial organ

Tonsils: pathogens entering the respiratory and

alimentary tracts
- Simplest lymphoid tissue: swelling of mucosa
form a circle
- Crypts get infected in childhood
Palatine – usual tonsillitis
Lingual – tongue
Pharyngeal – “adenoids”
Tubal

CALT: epidermis plus lymphocytes

UNIT 3: INNATE IMMUNITY You might receive a gamma globulin shot if you travel
outside the country.
The Immune System
- Includes all parts of the body that help in the
recognition and destruction of foreign
materials.
- WBCs, phagocytes and lymphocytes, bone
marrow, lymph nodes, tonsils, thymus, and
spleen are all part of immune system

What is immunity?
• Immunity is the body’s ability to fight off
harmful microorganisms – pathogens – that
invade it.
• The immune system produces antibodies or
cells that can deactivate pathogens
• Fungi, protozoans, bacteria, and viruses are
all potential pathogens

2 types
1. Innate/ natural/ Non-specific – upon birth
2. Adaptive/ specific/ acquired – developed

NATURAL IMMUNITY
- Also called innate immunity
- Nonspecific/non-adaptive
- Ability to resist infection by means of normally
present body functions
- Same response for all pathogens
- No prior exposure is required
- Response does not change upon repeated
exposure
- Influenced by factors like nutrients, age,
fatigue, stress and genetic determinants

2 components:
• External defense mechanism
o keep microorganisms from entering
Natural Active Immunity – the body synthesizes the ab the body
Natural Passive – ab is transferred o First line of defense
o Physical barriers
ACTIVE IMMUNITY • Internal defense mechanism
- Occurs when one makes his/here own o both cellular and humoral factors
antibodies. o 2nd line of defense
- Long term immunity
• Acquired immunity
o 3rd line of defense
Getting the disease:
o Cell mediated immunity
if you get an infectious disease, often times, that
o Humoral immunity
stimulates the production of MEMORY CELLS which
▪ Lymphocytes (T,B cells)
are then stored to prevent the infection in the future
Innate Immune System
Gamma gobulin: a y globulin shot is purely an
- Includes physical, chemical, and cellular
injection of antibodies to provide temporary immunity.
barriers
 - Physical barriers eg. Skin and mucous o MBP
membranes o Fibrinogen
- Chemical barriers eg. Stomach acidity, o Haptoglobin
secreted antimicrobial peptides o Ceruloplasmin
- Cellular barriers eg. Macrophages, • Produced by hepatocytes/liver cells
neutrophils • 12-24 hours in response to cytokines
- Innate immune response activation occurs (intracellular signaling proteins)
within minutes of pathogen recognition
C-Reactive Protein
External Defense System - Non-specific inflammatory marker
- Structural barriers that prevent - Trace constituent of serum
microorganisms from entering the body - Thought to be an antibody to the c-
• Intact skin polysaccharide or pneumococci
• Mucous membranes - Increases w/in 4-6 hours following infection,
• Secretions surgery, trauma
o Lactic acid (sweat) - Peaks w/in 48
o Fatty acids (sebaceous glands) hours
o Lysosome (saliva, tears): attacks - Declines rapidly
gram+ bacteria w/cessation of
o Acid pH (stomach) stimulus
o Anti-fungal peptides called alpha- - Tillet & Francis
defensins (intestinal tract)
o Anti-microbial peptides called beta- • Half-life = 19 hours
defensins (respiratory, urogenital • MW = 118000 Daltons
tract) • Belongs to the family of pentraxins
o Surfactant-A and -D proteins • Acts like an antibody (primitive ab)
opsonize pathogens for enhanced • Capable of opsonization, agglutination,
phagocytosis (lung0 precipitation, C’ activation
o Lactic acid (vagina) • Ca dependent, non-specific
• Substrate is phosphocholine
Epithelial Defense Mechanisms
• Flushing action of urine (also acid pH) Uses:
• Motion of cilia o Indicator of acute inflammation
• Presence of normal flora (competitive o Non-specific indicator of disease or trauma
exclusion) o Following course of malignancy and organ
transplantation
First-line defenses – the body’s first line defense o Significant risk factor for Myocardial Infarction
against pathogens uses mostly physical and chemical
barriers (sneezing) Serum Amyloid A
• Apolipoprotein
Second-line Defenses – if a pathogen is able to get • MW = 11,685 Daltons
past the body’s first line of defense, and an infection • Normal circulating levels = 30ug/ml
starts, the body can rely on its second line of defense. • Associated w/ HDL-chole
(WBCs except lymphocytes)

Internal Defense System

• Cells and soluble factors play essential parts
• Recognize molecules
o CHO mannos
• WBCs, phagocytosis, inflammation
• Acute phase reactants: enhances
Functions:
phagocytosis
o Cleaning up chole from macrophages at the
site of tissue injuries
Acute Phase Reactants
o Recycles cell membrane cholesterol
• Normal serum constituents
• Increase rapidly due to infection, trauma, Complement
injury
• Series of serum proteins that are normally
• Eg. present
o C-RP Major Functions:
o Serum amyloid A o Opsonization – facilitated phagocytosis
o Complement components o Chemotaxis – chemical messenger
o Alpha1-antitrypsin
 o Cytolysis – cell lysis • Complex is cleared by the liver

Mannose-Binding Protein • Levels may drop because of intravascular

• Aka mannose-binding lectin hemolysis
• Trimer • Normal plasma conc = 40 to 290 mg/dl
• Acts as an opsonin Function:
• Ca-dependent o Plays a role in protecting kidneys from
Function: damage
o Recognize foreign CHO (mannose) found on o Prevents loss of iron
bacteria, yeasts, viruses, parasites o Prevents oxidative damage of free hgb
o Activates C’, helps promote phagocytosis o Binds unfolded CHONs
o Normal concentration = 10ug/mL
Fibrinogen
• Most abundant coagulation factors
• Factor I
• Forms fibrin clot
• Dimer
• MW = 340,000 Daltons
• Normal levels = 100 to 400 mg/dl

Alpha1-antitrypsin
• Major component of the alpha band in SPE
• Plasma inhibitor of proteases released from
WBC (eg. Elastase)
• Elastase: endogenous ENZ that degrade Functions:
elastin and collagen o Increases the strength of the wound
Function: o Stimulates endothelial cell adhesion and
o Acts a “mop-up” or counteracts the effect of proliferation
neutrophil invasion during inflammation o Forms a clot; a barrier to prevent spread of
o Regulates expression of pro-inflammatory microbes
cytokines o Promotes aggregation of RBCs
o Can also react w/ serine proteases triggering
C’ cascade or fibrinolysis Ceruloplasmin
• Consists of single polypeptide chain
• Copper containing protein
• MW = 132,000 Daltons
• Binds 90-95% copper found in the plasma
• Acts as ferroxidase
• Wilson’s disease: autosomal recessive
genetic disorder. Deficiency in ceruloplasmin

Haptoglobin
• Alpha2-globulin
• MW = 100,000 daltons
• Binds free hgb (released by intravascular Cellular defense mechanism
hemolysis) irreversibly • Myeloid cells: participate in phagocytosis
 • Granulocytes and monocytes - excessive ag-ab complex
• Lymphocytes: acquired immune response - neutralizing basophils and mast cells
- killing parasites
Leukocyte Players of Innate Immune responses
Basophils
- less than 1%
- rarest. Blue-black granules
- 10-15um (smallest granulocyte)
- w/ histamine (vasoactive amine that contracts
smooth muscle
- w/ heparin (anticoag)
- w. eosinophil chemotactic factor A
- IgE binds to surface of basophil
- Exist only for a few hours in the blood
- Allergic reaction

Mast cells
- Similar to basophils
- Basophils that migrated to tissues

Monocytes
- Largest in the peripheral blood
- 12-22um in diameter
- w/ peroxidase, acid phos, arylsulfatase
(similar lysosomes of neutrophil)
- other type of granules (beta-glucuronidase,
lysozyme, lipase)
- stay in peripheral blood up to 70 hours
- long lived but slow in motility – opsonin
(enhance phagocytosis)
Neutrophils function:
- Approx. 50-70% of the total peripheral WBCs o microbial killing, tumoricidal, intracellular
- 10-15um in diameter parasites eradication, phagocytosis, secretion
- 3-5 lobes (nucleus) hypersegmented if (4-5) of cytokines, Ag presentation
- w/primary, secondary, and tertiary granules
- azurophilic granules: antibacterial Tissue Macrophages
- w/ progressive cellular enlargement to b/w
• half is marginating, the other 50% is 25-80um
circulating for 6 to 10 hours - increase in ER, lysosomes, and mitochondria
• capable of diapedesis: movement thru blood - lungs: alveolar MO
vessel walls - liver: Kupffer cells
• Selectins: help make neutrophils sticky and - brain: microglial cells
enhance adherence to endothelial cells - connective tissue: histiocytes
• Form pseudopods, which squeeze thru - bone: osteoclasts
junctions
• Attracted to chemotactic factors Dendritic cells
- covered by long membranous extensions
• Chemotaxins: chemical messenger that
- phagocytose Ag and present in T helper cells
cause cells to migrate in a particular direction
- Langerhans cells: skin//mucous membrane
• Life span of neutrophil in the tx: 5 days
- Interstitial cell: organs like heart
• Frustrated phagocytes
- Interdigitating cell: T lympho areas in
• Pus cells – dead neutrophils secondary lymphoid organs

Eosinophils Innate Immune Receptors

- Approx.. 12-15um in diameter - Not clonally distributed
- 1-3% of the circulating WBCs in non-allergic - Binding of receptors results in rapid response
person
- Increases in allergic or parasitic infections 3 functions:
- Nucleus – usually bilobed, eccentrical o phagocytic receptors to stimulate pathogen
- Acidophile uptake
- w/ primary and specific granules o chemotactic receptors that guide phagocytes
- capable of phagocytosis but not as efficient as to site of infection
neutrophil
 o stimulate production of effector molecules and - promiscuous in receptor usage, each can bind
cytokines that induce innate responses and more than one receptor
also influence downstream adaptive immune - likewise, receptors are promiscuous
responses
e.g., • infection induces the release of various
Toll-Like Receptors (TLRs) chemokines
• these substances bind specific and
sometimes shared receptors to recruit various
types of immune cells to the site of infection

Cellular Localization:
- Lysosomal localization (i.e. subcellular) of
TLR-3 and TLR7-9
- TLR-3 and 7-9 recognize viral/bacterial
nucleic acids
- lysosomal expression isolates pathogen
nucleic acid recognition away from potential
cross-reaction with host mammalian nucleic
acid motifs

Inflammatory Cytokines
These cytokines are critical for host defense.
Egs:
o TNFa activates macrophage and PMN
phagocytosis and killing
o IFNab activates anti-viral mechanisms
o IL-1 stimulates inflammation and feve

Phagocytosis
Activated macrophages secrete proteins that drive
- physical contact b/w WBC and foreign particle
innate response
- formation of phagosomes
- Fusion = phagolysosome
Cytokines
- Digestion and release of debris
- induce response by binding to specific
receptors
- can function in autocrine or paracrine manner
- cytokines (and their receptors) are clustered
according to structural similarities
- critical cytokines secreted by macrophages
following activation include TNFa, IL-1, IL-6,
IL-12 to stimulate inflammation and
phagocytosis/killing

Chemokines
- diverse family of chemotactic cytokines,
induce directed chemotaxis of cells
- all related in amino acid structure
- certain chemokines induce cell activation in
addition to cell recruitment
Macrophage Microbial Killing
- Phagocytosis is initiated

Phagocytosis is active process:

- Internalization of pathogen into phagosome
- Acidification of phagosome
- Fusion of phagosome with lysosomes that
contain anti-microbial compounds
(phagolysosome)
- This may be sufficient to kill the pathogen
- If not, reactive oxygen and nitrogen species
may need to be generated

Cardinal Signs
➢ Rubor – redness
➢ Tumor – swelling
➢ Calor – heat
➢ Dolor – pain
➢ Function laesa – loss of function

Clinical symptoms of inflammation:

1. Increased vascular diameter, increased blood
Inflammation flow (heat, redness)
- Reaction to an injury 2. Activation of vascular endothelium to express
- Both cellular and humoral mechanisms adhesion molecules, increases leukocyte
- Increased blood supply in the area binding
- Increased capillary permeability 3. PMNs are first cell type recruited to site,
- Migration of WBCs followed later by monocytes
- Migration of macrophages 4. Increased vascular permeability results in
local swelling and pain

Microvascular coagulation helps prevent pathogen

spread into bloodstream (physical barrier)