Molecular Theory Modules 1-6

Immunology

● Study of resistance to disease, specifically infectious disease

● Study of molecules, cells, organs and systems responsible for recognition and disposal of foreign
(nonself) materials
● How body components respond and interact
● There are desirable and undesirable consequences of immune interactions
● There are ways the immune system can be advantageously manipulated to protect against or treat
disease

Antigen and Antibody: Defined

● Antigen: Substance that can stimulate the production of antibodies

● Antibody: Protein produced by the immune response in response to exposure of an antigen. Also
called immunoglobulin

Role of the Immune System

1. Defending the body against infections

2. Recognizing and responding to foreign antigens
3. Defending the body against the development of tumors

The immune system accomplishes this by a series of interactions that result in:

● Specific recognition of antigens

● Capture of antigens for display to lymphocytes
● Elimination of antigens

Immune System Characteristics

● Specificity: distinguish among distinct antigens

● Memory: recognize and respond to antigens encountered previously
● Mobility: move quickly to specific body areas
● Replicability: produce exact copies of specific antibodies
● Cooperation between different cells or cellular products: produce a coordinated immune response

Immune System Functions

The function of the immune system is:

1. To recognize self from nonself

2. To defend the body against nonself

Desirable consequences of immunity:

● natural resistance
 ● recovery
● acquired resistance to infectious diseases

Undesirable consequences of immunity:

● allergies
● rejection of a transplanted organ
● an autoimmune disorder

1st Line of Defense

● Skin & mucosal membranes

○ The normal flora of the skin deter penetration or facilitate foreign microorganisms from
the body
○ Keratinization of the upper layer of the skin and the constant renewal of the skin’s
epithelial cells assist in the protective function of skin and mucosal membranes
● Secretions
○ Mucus adhering to the membranes of the nose and nasopharynx trap microorganisms,
which can be expelled by coughing or sneezing
○ Sebum produced by sebaceous glands of the skin and lactic acid in sweat both have
antimicrobial properties
○ Secretions produced in the elimination of liquid and solid wastes (urine and feces)
● Chemical properties

2nd Line of Defense

● Natural immunity (innate resistance)

○ One of the ways the body protects against infection after the first line of defense has been
penetrated
○ The elements of natural resistance include phagocytic cells, complement and acute
inflammatory reaction
● Nonspecific mechanism
○ Detection of microbial pathogens is carried out by sentinel cells of the innate immune
system (macrophages and dendritic cells) located in tissues in close contact with the
environment or that rapidly reunited to the site of infection (neutrophils)
○ Despite their lack of specificity, they are essential because they are responsible for
natural immunity to environmental organisms
● Involves cellular and humoral responses
○ phagocytic cells engulf invading foreign material
○ Inflammation causes changes that facilitate phagocytosis (engulfment and destruction)
○ If the degree of inflammation is sufficiently extensive, it is accompanied by an increase
in acute phase proteins or reactants (a group of glycoproteins)
○ Acute phase proteins are sensitive indicators of the presence of inflammatory disease and
useful in monitoring such conditions
○ Complement proteins are the major humoral (fluid) component of natural
immunity
○ Other substances of the humoral component include lysozymes and interferon
○ Interferon is a family of proteins produced rapidly by many cells in response to viral
infection, it blocks the replication of virus in other cells

3rd Line of Defense

 ● Acquired immunity or adaptive immunity
● Allows the body to react to antigens
● Can result in the elimination of microorganisms and recovery from disease, the host often
acquires a specific immunologic memory
● Happens when microorganism overwhelms the body's natural resistance
○ Recognize a specific antigen
○ Respond to a specific antigen
○ Memory (acquired resistance)
● Allows host to respond more effectively if reinfection occurs
● Adaptive immunity, as with natural immunity is composed of cellular and humoral components
● The major cellular component of acquired immunity is the lymphocyte, the major humoral
component is the antibody
● Lymphocytes selectively respond to nonself materials which leads to immune memory and a
permanently altered pattern of response

Humoral vs. Cell Mediated Immunity

Humoral-mediated Immunity

● Specific antibody response

○ Recognition of foreign substance
○ Subsequent production of specific antibody
● Active immunity: antibody formed by host
● Passive immunity: antibody received from another source

Active vs. Passive

Active Immunity

● Natural Active Immunity: natural exposure to an infection or a natural series of infections

● Artificial Active Immunity: intentional injection of an antigen (vaccination)
○ Antigenic materials for immunization: animal or plant origin
○ Living suspensions weak/attenuated/killed cells
○ Stimulate production of antibodies without clinical signs and symptoms of disease in an
immunocompetent host
○ Result in permanent antigenic memory
○ Booster vaccinations expand the pool of memory cells

Passive Immunity

● Artificial Passive Immunity:

○ Infusion of serum containing high concentrations of antibody or lymphocytes from an
actively immunized individual
○ Provides immediate, temporary antibody protection against microorganisms (Hep A)
○ Recipient will benefit only temporarily for as long as the antibodies persist in the
circulation
○ Immune antibodies are usually of the IgG type with a half life of 23 days
○ Short immunoglobulin (antibody) half-life
● Natural Passive Immunity:
○ Maternal antibodies acquired naturally by the fetus/newborn
○ In utero through placenta
○ After birth through colostrum (thick yellowish milk very rich in antibodies) and breast
milk
○ The amount of and specificity of maternal antibodies depend on the mother’s immune
status to infectious diseases that she has experienced
○ Passively acquired immunity in newborns is only temporary, starts to decrease after the
first several weeks of birth

Cell Mediated Immunity

● Consists of immune activities that differ from antibody mediated immunity.

● Lymphocytes are the unique bearers of immunologic specificity which depends on antigen
receptors
○ However, the full development of immune responses requires that non-lymphoid cells
and molecules act as amplifiers and modifiers
● Cell mediated immunity is moderated by the link between T-lymphocytes and phagocytic cells
(monocytes and macrophages)
 ● B lymphocytes can respond to an antigen of the appropriate fit
● T lymphocytes responds to antigens presented by other cells in the context of major
histocompatibility complex (MHC) proteins
○ T lymphocytes do not directly recognize the antigens of microorganisms or other living
cells (allografts), but recognize when the antigen is presented on the surface of an
antigen-presenting cell (APC)
○ APC's are macrophages
● Lymphocytes are active through various types of cell-to-cell contact and by the production of
soluble factors
○ Nonspecific soluble factors are made by or act on various elements of the immune
system. These molecules are collectively called cytokines
○ Some mediators that act between leukocytes are called interleukins.

Leukocyte Immune Characteristics and Functions

Comparison of Innate and Adaptive Immunity: Summary

Innate Immunity

● Mechanisms of the innate immunity (phagocytes) and the alternative complement pathways are
activated immediately after infection and quickly begin to control multiplication of infecting
microorganisms
○ Innate immune recognition is mediated by germline encoded receptors, which means that
the specificity is genetically predetermined
● The innate immune response may not be able to recognize every possible antigen, but may focus
on a few large groups of microorganisms, called pathogen-associated molecular patterns
(PAMPs)
○ PAMPS are molecules associated with groups of pathogens that are recognized by cells
of the innate immune system
● The receptors of the innate immune system that recognize these PAMPs are called pattern
recognition receptors (PRR's, eg. toll-like receptors)
 ● There are three types of PRRs
○ Secreted PRRs - molecules that circulate in blood and lymph, if bound to PAMP it
triggers the complement cascade
○ Phagocytosis receptors - cell surface receptors that bind the pathogen initiating the
release of cytokines
○ Toll like receptors - set of transmembrane receptors that recognize different types of
PAMPS, they are found on macrophages, dendritic cells and epithelial cells
■ TLR-1 binds to gram positive bacteria
■ TLR-2 binds to gram negative bacteria

Adaptive Immunity

● By comparison, the adaptive immune system is organized around two class of cells, T & B
lymphocytes
● When the lymphocyte encounters an antigen that binds to its unique antigen receptor site,
activation and proliferation of the lymphocyte occurs
○ This is called clonal selection and is responsible for the basic properties of the adaptive
immune system
● Random generation of highly diverse database of antigen receptors allows the adaptive immune
system to recognize virtually any antigen
○ This however can be a problem when the body cannot distinguish self from non-self

Definitions

● Antigen
○ Substance that stimulates antibody formation
○ Ability to bind to an antibody
○ Can bind to an antibody or T cell receptor but may not be able to evoke an immune
response initially

● Immunogens
○ Macromolecules capable of triggering an adaptive immune response
○ Induces formation of antibodies or sensitized T cells
○ Can specifically react with corresponding antibodies or sensitized T cells
○ All immunogens are antigens but not all antigens are immunogens
● Hapten
○ A low molecular weight particle
○ Can bind to antibody but must be attached to a carrier to stimulate an immune response

Antigen: Characteristics

● Antigenic determinant or epitope

○ Part of an antigen
○ Reacts specifically with an antibody or T lymphocyte receptor
 ○ Some substances are much more immunogenic than others

Antigen: Composition and Examples

● Composition: protein, large polysaccharides, or combination of carbohydrate and protein

(glycoprotein)
● Found usually on cellular membrane
● HLA’s are glycoprotein in nature
● Proteins make great antigens because of their high molecular weight and structural complexity
● Lipids are considered inferior because of their relative simplicity and lack of structural stability
● Nucleic acids are poor antigens because of relative simplicity, and rapid degeneration
● Examples:
○ In a blood transfusion: antigen-bearing red blood cells from a donor, are transfused into a
recipient
○ Red cell antigens can be immunogenic
○ Outer surfaces of bacteria (capsule or the cell wall) can be immunogenic.
○ Normal immune system responds by producing antibodies

Physical Nature of Antigens

● Physical nature of antigen influences function

● Characteristics
○ Foreignness – degree of difference from self
○ Degradability – sufficient levels of antigen present (adequate dose of antigen must be
given during vaccination)
○ Molecular weight – high molecular weight = large # of antigenic determinants or bound
to hapten
○ Structural stability – stable molecule is a better antigen
○ Complexity – more complex molecule is a better antigen

Antigen: Major Histocompatibility Complex (MHC)

● Major Histocompatibility Complex (MHC)

○ nucleated cells (e.g. leukocytes) and tissues have cell surface–protein antigens that
readily provoke immune response if transferred into a genetically different individual of
the same species
○ These antigens are the second to ABO antigens in influencing the survival or graft
rejection of transplanted organs
● MHC antigens = potent antigens/readily provoke an immune response.
○ also referred to as Human Leukocyte Antigens (HLAs)

Characteristics of MHC classes I and II: Histocompatibility Antigens

Autoantigens

● Autoantigens = "self" antigens

● Failure to recognize self as non-foreign produces autoantibodies
● E.g. Thyroglobulin

Adjuvant

● Adjuvant: enhances response to immunization

● Adjuvant is a substance, distinct from antigen
● Enhances T cell activation by promoting the accumulation of antigen presenting cells (APC) at
site of antigen exposure
● Enhances the expression of co-stimulators and cytokines by APC

Antibody: General Characteristics

● Antibody: specific proteins, also called immunoglobulins (Ig)

○ Can be isolated gamma globulin fraction of protein by electrophoresis separation
● Found in plasma and other body fluids (tears, saliva, colostrum)
● Primary function: combine with antigen
● May require secondary interaction (e.g. with complement) to remove larger antigens (e.g.
bacteria)
 ● Determining immunoglobulin concentrations: can be of diagnostic significance in infectious and
autoimmune diseases
● Five distinct classes:
○ IgM - Pentamer
○ IgG - Monomer
○ IgA - Monomer or polymer
○ IgD - Monomer
○ IgE - Monomer

Immunoglobulin Classes: Molecule Structure

● Each Ig molecule is bifunctional, one region of the molecule involves binding to an antigen, and a
different region mediates binding of the Ig to host tissues including complement (C1q)
● The core of the antibody consists of the sequence of amino acid residues linked by peptide bond
● 12 domains arranged in two heavy (H) & two light (L) chains
○ 2 types of antigenically different L chains: kappa, lambda
● Chains linked through cysteine residues by disulfide bonds (-s-s-)
● Remainder of chain composed of constant amino acid sequences = constant (c)
● Fab region
○ Antigen-binding portion, hypervariable loops - complementary determining regions
(CDRs)
○ N-terminal
○ Heterogeneity = variable (v)
● Fc region
○ Interacts with other phagocytic cells and complement
● Hinge region
○ flexibility

IgG

● Major immunoglobulin in serum: accounts for 70-75% of the total Ig pool

● Diffuses readily into the extravascular spaces & neutralizes toxins, binds to microorganisms in
extravascular spaces
● Can cross the placenta
● Complement can be activated by IgG complexes
● IgG3 subclass is the largest of IgG molecules
● In infants 3 to 4 months old: IgG levels are much lower than adult levels
 ● Average adult level is achieved before 16 yrs of age
● IgG in cord blood and spinal fluid
● Decreased IgG: primary (genetic) or secondary (acquired) Ig deficiencies
● Increased IgG: Infectious diseases, collagen disorders, hematologic disorders

IgM

● 10% of total Ig, mostly in intravascular pool because of its large size
● Produced early in immune response
● Effective in agglutination and cytolytic reactions
● Has five individual heavy chains
● 4 months of age, 50% of the adult level is present; adult levels are reached by 8 to 15 years
● Some IgM in cord blood; not detectable cerebrospinal fluid
● Decreased : primary (genetically determined) Ig disorders, secondary Ig deficiencies
● Increased: Infectious diseases, collagen disorders, hematologic disorders, monoclonal
gammopathies

IgA

● 15% - 20% of Ig pool

● Predominant Ig in secretions: tears, saliva, colostrum, milk, intestinal fluids
● Synthesized by plasma cells on body surfaces and intestinal wall
● If produced by cells in intestinal wall, IgA may pass directly into the intestinal lumen or diffuse
into the blood circulation
● Binds to secretory component in intestine to protect IgA from proteolytic digestive enzymes
(secretory IgA) critical in protecting the body surfaces against invading microorganisms because
of its presence in seromucous secretions
● Adult levels reached by 16 yrs of age
● Decreased: primary or secondary Ig deficiencies
● Increased: infectious diseases, collagen disorders, hematologic disorders, monoclonal
gammopathy, liver disease

IgD

● Very low concentrations in plasma; less than 1% of Ig pool

● Extremely susceptible to proteolysis
● Cell membrane Ig found on the surface of B lymphocytes in association with IgM

IgE

● Trace plasma protein if no parasites present

● Responsible for immunity to invading parasites
● Mediates hypersensitivity (allergic) reactions, allergies, anaphylaxis
● Binds strongly to antigen and receptor on mast cells and basophils; mediates release of
histamines, and heparin
Antibody Response: Antibody Synthesis

● Production of antibodies is induced when the hosts lymphocytes come into contact with a foreign
antigenic substance that binds to its receptor
● This triggers activation and proliferation (AKA clonal selection)
● Clonal expansion of lymphocytes in response to infection is necessary for an effective immune
response
● Whether a cell mediated or antibody response takes place depends on how the antigen is
presented to the lymphocytes, many immune rxn’s display both types of reponses
● The antigenicity of a foreign substance is also related to route of entry; intravenous and
intraperitoneal routes are stronger stimuli than subcutaneous or intramuscular
● Anamnestic response: Subsequent exposure to the same antigen produces a memory response
● Antibody response:
● antigen encountered
○ cells of the immune system recognize the antigen as nonself
○ elicit an immune response or become tolerant of it
● Immune response:
○ cell-mediated immunity: T cells and macrophages
○ production of antibodies by B lymphocytes and plasma cells

Antibody Response: Primary Response

● IgM antibody response follows four phases:

1. Lag phase: no antibody detectable
2. Log phase: antibody titer increases logarithmically
3. Plateau phase: antibody titer stabilizes
4. Decline phase: antibody catabolized

Antibody Response: Secondary Response/Anamnestic Response

 ● Subsequent exposure produces an antibody response that exhibits the same four phases of
antibody response as primary response.
● clones of memory cells proliferate
● Anamnestic response differs from a primary response:
○ Time: shorter lag phase, longer plateau, more gradual decline
○ Type of antibody: IgG class predominant (some IgM)
○ Antibody titer: antibody levels attain higher titer
● Examples:
● hemolytic disease of the fetus and newborn, booster vaccination

Antibody Response: Functions of Antibodies

● Principal function: to bind antigen

● Secondary effector functions:
1. Complement fixation: mainly IgG1, IgG3
2. Placental transfer: most IgG
● Passive immunity for the newborn
● It is not known if IgG2 is able to cross the placenta

Antibody Specificity

● Antibody ability to combine with a particular antigen, determined by Fab site

● Binding by non covalent forces (e.g. hydrophobic or electrostatic bonds )
● Depends on three-dimensional fit

Heterophile Antibodies

● Cross reactivity: When some of the determinants of an antigen are shared by similar antigenic
determinants on the surface of apparently unrelated molecules, a proportion of the antibodies
directed against one type of antigen will also react with the other type of antigen
● Cross-reactivity occurs between bacteria that possess the same cell wall polysaccharides as
human red blood cells
● (e.g. blood group A-like and B-like antigens)

Antibody Affinity and Avidity

 ● Antibody affinity:
○ Binding strength at a single binding site
● Antibody avidity:
○ Measure of total binding strength
● Immune complex:
○ non covalent combination of antigen with its specific antibody (small/soluble or
large/precipitating)

Antibody Response: Molecular Basis of Antigen-Antibody Reactions

● Antigen + Antibody:
○ Multiple, reversible, intermolecular attractions
○ Bonds weak and across short distances
● 4 Types of bonding:
○ Hydrophobic Bonds: major bonds; exclude water molecules to strengthen bond
○ Hydrogen Bonds: hydrogen bridges
○ Van der Waals Forces: electron clouds and hydrophobic bonds
○ Electrostatic Forces: Goodness of Fit, zeta potential
● Zeta Potential: Electrostatic potential
○ net charge between cell membrane and the surface of shear
○ larger antibodies (e.g. IgM) bridge the zeta potential
○ large cells like red blood cells able to bind and agglutinate

Monoclonal Antibody Production

Monoclonal Antibodies: Application

● Identifying and quantifying hormones

 ● Typing tissue and blood
● Identifying infectious agents
● Identifying clusters of differentiation for the classification of leukemias and lymphomas and
follow-up therapy
● Identifying tumor antigens and autoantibodies
● Delivering immunotherapy

Module 3 & 4

Origin and Development of Blood Cells

● Hematopoiesis: Formation of blood cells

○ 2-8 weeks of gestation: Erythroblasts form in the yolk sac
○ 2-5 months gestation: Liver & spleen - start to see granular leukocytes
○ 4th month of gestation: Bone marrow begins to produce blood cells
○ 5th month of gestation: Bone marrow is the primary site of hematopoiesis
● Blood cells are produced from common multipotential stem cells
● Stem cells differentiates into blast cells
● Blast cells produce:
○ Erythrocytes (RBC's)
○ Megakaryocytes
○ Granulocytes
○ Monocytes-macrophages
○ Lymphocytes
○ Plasma cells
● Further maturation produces thrombocytes (platelets), and specific types of leukocytes (WBC's)
● Leukocytes in normal peripheral blood (from most to least common) are:
○ Neutrophils
○ Lymphocytes
○ Monocytes
○ Eosinophils
○ Basophils

Neutrophils

● Neutrophils: Polymorphonuclear leukocytes (PMN's) meaning they have various shapes and sizes
of the cell's nucleus
● Lifespan of 5.4 days
● Provide host defense against bacterial and fungal infections
○ This is essential in the innate immune response
● Main leukocyte associated with phagocytosis and localized inflammatory response
● We know that they are part of the initial immune response because neutrophils are found in
inflammatory exudate (pus formation)
● Can also prolong inflammation by releasing soluble substances such as cytokines and chemokines
○ Cytokines are small proteins that signal the immune system to “keep going”
● Influence adaptive immune response:
○ Shuttling pathogens
○ Antigen presentation
 ○ Modulation of T helper types responses
● Move to peripheral tissues by diapedesis
○ Diapedesis: movement through blood vessel wall
○ Move from the blood circulation, through blood vessel wall to site of injury where they
perform phagocytosis
○ Phagocytosis in peripheral tissue
● Granzymes:
○ Granules containing antibacterial substances
○ Can also be destructive to host if they release these particles after digestion

Signs and Symptoms of Disorders of Neutrophil Function

● Phagocyte releases oxygen radicals and granule contents into surrounding tissues; cause tissue
damage
○ e.g. Dust inhalation and smoking
● Autoimmune diseases: Inappropriate activation of phagocytosis (body attacks own cells/tissues)
○ Rheumatoid arthritis, multiple sclerosis
● Abnormal Neutrophil function may lead to:
○ Recurrent infections
○ Unusual infections
○ Persistent infections
○ Severe infections
○ Impaired inflammatory response

Eosinophils

● Homeostatic regulators of spread of inflammation

● By suppressing inflammation, it prevents excessive spread of inflammation to other parts of the
body
● Role in host defense against parasites
● Eosinophil membrane interacts with the larval stages of the parasitic worms and damage them
through oxidative mechanisms
○ Eosinophil granules release proteins to damage antibody coated parasites (eosinophils
will be increased in the blood in these conditions)
○ Eosinophil granule protein can also damage endothelial cells in hyper-eosinophilia
conditions

Basophils

● High concentrations of heparin and histamine in granules

● Basophils play a role in acute systemic hypersensitivity reactions
● During allergic reactions, degranulation occurs when an antigen (E.g. pollen) binds to two
adjacent IgE antibody molecules that are located on the surface of the mast cells
● The result of degranulation causes:
○ Increased vascular permeability
○ Smooth muscle spasm
○ Vasodilation (faster blood flow potentially leading to anaphylactic shock)
○ Possible anaphylactic shock
Process of Phagocytosis

● The physical occurrence of damage to the tissue, whether from trauma or microbial invasion, is
what triggers and initiates the process of phagocytosis

1. Chemotaxis

● When phagocytic cells migrate to the site of injury or invasion

● The release of substances that attract phagocytic cells as the result of traumatic or microbial
damage
● Neutrophils and macrophages arrive to the site of injury
○ Neutrophils are the first to arrive to the site of injury (within less than one hour)
● Macrophages are located in tissues of the body
● Additional macrophages from the bone marrow and other tissues can be released in response to
severe infections
● Neutrophils can activate the complement pathway when it is stimulated by cytokines and
coagulation derived factors
● Neutrophils are essential to innate immune defense
○ can active alternative complement pathway (C5)
○ C5 will increase further neutrophil response
● Opsonization: Coating of an organism by molecules (AKA opsonins) that speed up
phagocytosis
○ Binding of antibody or C3b to pathogen
○ Fc portion of antibody or C3b binds to an Fc or C3b receptor on the phagocyte,
facilitating phagocytosis
○ The core receptor and antibody complex create byproducts, C3b & C4b that participate in
the complement system

2. Adherence

● Cell surface receptors - mediate cell to cell binding or adhesion of white blood cells
1. Immunoglobulin family - Antigen specific receptors (e.g. T cell receptor and surface
immunoglobulins)
 2. Selectin family - Adhesion molecules help leukocyte adhere (e.g. Leukocyte adhesion
molecule-1 (LAM-1))
3. Integrin family - Interacts with the cell surface and extracellular matrix to enhance
leukocyte adhesion. Integrin molecules also play a role in the spread of malignant
tumor cells. If there are integrin receptors on tumor cells, it helps the tumor cells
attach to other cells in other parts of the body

Disease States Involving Leukocyte Integrins

● Integrins: Aid adhesion

● Leukocyte adhesion deficiency (LAD) genetic mutation defect that is characterized by impaired
leukocyte adhesion (genetic)
● Leads to recurrent and often fatal bacterial and fungal infections
● Adhesion defects can also be caused by two common drugs:
○ Epinephrine
○ Corticosteroids

3. Engulfment

● At the site of infection, phagocytes engulf and destroy pathogens

● After the phagocytic cells arrive at the site of injury, bacteria is engulfed through active
membrane invagination
● Pseudopodia (protrusions that extend around the pathogen) around the pathogen (pulled by Fc
receptors and Fc antibody portions on the opsonized bacterium)
● Pseudopodia meet and fuse, internalizing/enclosing bacterium in phagosome
● For effective phagocytosis: bacteria must be more hydrophobic than the phagocyte
○ Some bacteria like streptococcus pneumoniae which is the causative agent that causes
pneumonia, possesses a hydrophilic capsule
○ This will prevent the bacteria from being engulfed by phagocytic cells
○ Strep pneumo can cause pneumonia, ear & sinus infections as well as meningitis

4. Phagosome Formation, 5. Fusion & 6. Digestions and Destruction

● Granules in the phagocyte fuse with phagosome to form phagolysosome

● Digestion follows ingestion of the foreign particle
● Digestions requires energy primarily provided by anaerobic glycolysis
● Types of degrading enzymes in granules
○ Primary/azurophilic containing enzymes (lysozyme, myeloperoxidase)
○ Secondary/specific containing substances such as lactoferrin
○ Tertiary containing substances such as caspases
● Neutrophil degranulation releases antibacterial substances (lactoferrin, lysozyme, defensin) and
enzymes (elastase) to increase membrane permeability which causes bacterial cell wall to burst,
elastase can also cause damage to host tissues
● Monocytes are effective as phagocytic cells because:
○ They have large amounts of lipase in cytoplasm (e.g. lipase attacks bacteria with a lipid
capsule, such as mycobacterium tuberculosis)
○ They have the ability to bind and destroy cells that are coated with either complement or
antibody, this is because the surface of the monocytes have receptors that are specific for
these components
Phagocytic Engulfment Test

● Screening test for phagocytic engulfment:

○ Mixture of bacteria and phagocytes incubated and examined for the presence of engulfed
bacteria
○ Take a patients blood and spin it down - add 8 drops of the patients buffy coat to 3 drops
of bacterial broth culture
○ Incubate it, then prepare blood smear, stain and read it
● Negative: (Normal) phagocytic engulfment test, many bacteria in various stages of destruction are
evident within the cell
● In an abnormal test there is no engulfment of bacteria, no bacteria will be seen in phagocytic cells
● Diagnosis if abnormal: Impaired neutrophilic function

Neutrophil Extracellular Traps (NETS)

● Additional defense mechanism

● Neutrophils form neutrophil extracellular traps (NETs) - release of nuclear contents into
extracellular space
● NETs composed of chromatin components, including histones, and neutrophil antimicrobial
proteins
● When bacteria are trapped in NETs:
○ They are exposed to high concentrations of antimicrobial proteins
○ Bacteria killed or injured
○ Functions as part of the innate immune response

Monocytes and Macrophages: Location

● Monocytes migrate freely from blood to tissues

● Macrophages fixed or wandering cells
○ Wandering cells are found in the circulation
○ Fixed cells may be in the lining of the lungs, linings of endothelium, capillaries, sinuses
of organs such as bone marrow, spleen and lymph node

Monocytes and Macrophages: Function

● The most important step in the maturation of macrophages is the conversion from a normal
resting macrophage to the activated macrophage
● Cytokine driven conversion of the normal resting macrophage to the activated macrophage
● Macrophages can be activated:
1. during infection by release of macrophage activating cytokines: e.g. interferon-gamma
(IFN-y) and granulocyte colony stimulating factor (G-CSF)
2. From T-lymphocytes specifically sensitized to antigens from the infecting
microorganisms = basis of cell mediated immunity monocyte

Monocytes and Macrophages: Host Defense Functions

 1. Phagocytosis (primary role)
● activated macrophage ingest & kill microorganisms (can result in the release of parasite
lethal mediators, important in host immunity against malaria)
● dispose if damaged/dying cells (macrophages line the sinusoids of the spleen and ingest
aging RBC’s)
● phagocytic activity increases when there is tissue damage and inflammation
2. Antigen presentation
● process antigens and physically present this biochemically modified and more reactive
form of antigen to lymphocytes (particularly helper T cells) as an initial step in the
immune response
● Recognition of the antigen on the macrophage surface by T lymphocytes requires the
additional matching of the surface MHC class 2 gene product
3. Induction of the immune response
● macrophage secretes a lymphocyte-activating factor (interleukin-1 or IL-1), lymphocyte
proliferation occurs, and the immune response (T cell–B cell response) is facilitated
4. Secretion of biologically active molecules
● synthesize a number of biologically important compounds, including transferrin,
complement, interferon, pyrogens, and certain growth factors
● IL-1 – supports B cells proliferation, antibody production, T lymphocyte production of
lymphokines
● Lymphokines can in turn further trigger B lymphocyte proliferation when exposed to
toxins that are produced by bacteria
● Macrophages also release tissue necrosis factor alpha, which will then stimulate the
production of IL-1
○ Both of these can induce fever and could also promote the synthesis of acute
phase reactants, which are both characteristics of the inflammatory response

Signs and Symptoms of Monocyte or Macrophage Disorders

● Depressed migration of monocytes: prone to more infections

● Symptoms are dependent on the defect
● Patients with chronic granulomatous disease have a defect of phagocytic killing, which will result
in the formation of an abscess
● Qualitative monocyte-macrophage disorders (the amount of monocytes and macrophages that are
present) manifest as lipid storage disease with impaired resistance to infection

How do white blood cells actually get to the site of infection?

● 5 steps for effective leukocyte recruitment:

1. Capture
2. Rolling
3. Slow rolling
4. Firm adhesion
5. Transmigration

Acute Inflammation & Sepsis

● Signs of inflammation:
○ Redness
○ Heat
 ○ Pain
○ Swelling
● Stages of inflammatory response:
1. dilation of capillaries to increase blood flow
2. microvascular structural changes and escape of plasma proteins from bloodstream
3. leukocyte transmigration through endothelium and accumulation at the site of injury
● Primary goal of this inflammatory response is to localize and remove the pathogen and to repair
the surrounding tissue, however, it could lead to sepsis.

Sepsis

● Inflammation when infectious agent overwhelms adaptive immune system

● fever or hypothermia
● tachycardia and tachypnea
● increased respiratory rate
● leukocytosis or leukopenia

What happens in your body?

● Toll-like receptors (TLR) cause antigen presenting cell (APC) to produce pro-inflammatory
cytokines: TNF( tumor necrosis factor), IL-1, IL-6
● Liver is stimulated to produce C-reactive protein (C-RP)
● cytokines activate circulating polymorphonuclear leukocytes (PMNs)
● APCs (adaptive immune) present bacterial antigen to T-cell receptor: using a Class II major
histocompatibility complex (MHC) protein and co-stimulation of CD28

Lymphocytes and Plasma Cells

● Lymphocytes and plasma cells are the primary cells that act in the adaptive immune response in
both the humoral and cellular systems
● Two classes of specialized cells:
1. T lymphocytes
2. B lymphocytes

Lymphocytes

● Recognize foreign antigens

● Directly destroy some cells
● Produce antibodies as plasma cells
● Developed in the thymus and bone marrow (in adults)

Primary Lymphoid Tissues

1. Thymus
2. Bone marrow, fetal liver

Thymus: Function

● Function: development of T cells

 ● Note: Lymphocytes are produced in the bone marrow, but travel to the thymus where they will
mature to becomes specialized T cells
● Small gland in the lymphatic system that is located in between the lungs
● Thymus produces most of your T cells before birth and continues production throughout
childhood, you have all the T cells you need by the time you hit puberty
○ Because of this, the thymus loses 95% of its mass in the first 50 years of life
● Characteristics of thymus gland change with aging:
○ Reduction in thymus production of naïve T cells
○ Intrinsic defects in mature T cell function
○ Alterations in the life span of naïve T cells & memory T cells
○ Decline of T cell response in older persons
○ Increased susceptibility to infections, autoimmune disease, neoplasms

Thymus: Role in T Lymphocyte Maturation

● Thymus gland is part of the endocrine system

○ Regulates immune function by secretion of soluble hormones
○ Thymosin – helps make specialized types of T cells, thymus progenitor cells
proliferate/differentiate to thymocytes
○ 97% of thymus cells die in the thymus before they become mature T cells, they do this
through phagocytosis
■ This is a safety mechanism to eliminate lymphocyclones directed against self
antigens
○ Thymocytes – lymphocyte precursors with acquired surface membrane antigens
○ Reticular structure of thymus allows lymphocyte to pass through it & into bloodstream
○ Viable cells migrate to secondary tissues
● Absence thymus development:
○ Results in T lymphocyte deficiency
○ Presents as changes in immune function
● Abnormal thymus development:
○ dysfunction of T and B lymphocytes
○ elevated levels of circulating immune complexes
○ increases in autoantibodies, and monoclonal gammopathies

Bone Marrow - Function

● Bone marrow is source of progenitor cells:

○ can differentiate into lymphocytes and other hematopoietic cells
● Bone marrow supports differentiation of mature T and B lymphocytes, probably from the
common lymphoid cell progenitor

Secondary Lymphoid Tissues

● Lymph nodes
○ Contain lymph fluid which contains proteins, minerals and lymphocytes which will attack
and destroy the foreign antigens
○ Filters, recycles and replenishes lymph fluid according to our bodies needs
● Spleen
○ filters blood by removing cellular waste and getting rid of old/damaged blood cells
● Gut-associated lymphoid tissue (GALT)
○ Peyer’s patches in intestines and liver
 ○ Important for the development of tolerance against ingested antigens
○ Patches of lymphocytes in the GI system, specifically pre B lymphocytes, these meet
antigens from the gut and enter general circulation and re-enters back into the gut
○ lymphocyte recirculation
● Thoracic duct
○ thoracic duct lymph – rich source of mature T cells
● Bronchus-associated lymphoid tissue (BALT)
○ lymphoid tissue in lower respiratory tract
○ Mainly associated with IgA production in response to inhaled antigen
● Skin-associated lymphoid tissue
○ Antigens introduced through the skin are presented by epidermal cells
○ epidermal cells then interact with lymphocytes in skin and in draining lymph nodes
● Blood
○ Mature lymphocytes in circulation
○ An increase or decrease in lymphocytes in the blood means there is something going on
with the thymus, bone marrow, or any of the other secondary tissues

Secondary Lymphoid Tissues - Distribution

T lymphocytes (T cells) populate:

1. perifollicular and paracortical regions of the lymph nodes

2. medullary cords of the lymph nodes
3. periarteriolar regions of the spleen
4. thoracic duct of the circulatory system

B lymphocytes (B cells) multiply in and populate:

1. follicular and medullary (germinal centers) of the lymph nodes

2. primary follicles and red pulp of the spleen
3. follicular regions of GALT
4. medullary cords of the lymph nodes

Secondary Lymphoid Tissues: Function

● Secondary lymphoid organs allow migration and interaction between antigen-presenting cells
(APCs), T and B lymphocytes, follicular dendritic cells (FDCs) and other stromal cells
○ Results in effective generation of humoral immune responses
○ Proliferation of T and B lymphocytes in secondary/peripheral lymphoid tissues is
primarily dependent on antigenic stimulation
● Tumor necrosis factor (TNF), lymphotoxins and cytokines can signal the production of T&B
lymphocytes

T Cell Maturation

● Most of the lymphocytes found in circulating blood are T cells

● T cells derived from the bone marrow progenitor stem cell that mature in the thymus gland are
responsible for the cellular mediated immune response
○ Also involved in humoral because they regulate antibodies
 ● Differentiation of T lymphocyte begins in thymus as thymocyte (immature T cell)
○ Early surface markers on T cell thymocytes: CD44 and CD25, this tells the thymocyte
that they’re going to become mature T cells
○ As thymocytes develop, there is a rearrangement of genes coding for an antigen receptor
● T cell maturation: 3 weeks; cells filter through thymus cortex to medulla
● Mature T lymphocytes survive for several months or years but the average life of B lymphocytes
is only a few days
● Naïve lymphocytes: have not encountered their specific antigen
● Memory cells: long-lived T or B cells that have been stimulated by antigen; quick response to a
previously encountered antigen

Cluster of Differentiation

● Surface marker that identifies cell line with a defined structure

● Molecules on surface of lymphocytes that identify the cell to other immune system cells
● E.g. CD4 vs CD8
● T lymphocytes that show CD4 markers are MHC Class 2 restricted, which means they are
programmed to be T helper cells
● T lymphocytes that show CD8 markers are MHC Class 1 restricted, which means they are pre
programmed for cytotoxic functions (cytotoxic/killer T cells)
● CD44 marker will be upregulated after increase of activation of naive T cells
● CD25 is important in T cell proliferation

T Cell Maturation: Surface Markers

● Double-negative thymocytes:
○ Early thymocytes that lack T cell receptors: CD4 and CD8
○ These cells proliferate in the outer cortex of the thymus under the influence of
Interleukin-7 (IL7) - critical for growth and differentiation
○ They develop beta (β), gamma (γ) and delta (δ) chains depending on their genetic makeup
○ chains classify thymocytes as positive or negative; which ultimately affects the role of
what the T cells take on
○ increased numbers of double negative lymphocytes (CD4-CD8-): individuals with
autoimmune and lymphoproliferative disorders and graft versus host disease
● Double-positive thymocytes:
○ cells with both CD4+ and CD8+
○ increased numbers of double-positive thymocytes: immune inflammatory diseases, viral
infections and cancer

T Lymphocyte: Subsets

Helper T (Th) lymphocytes

● Assist other lymphocytes to mature and activate

● Th1: cell-mediated effector mechanisms
● Th2: regulation of antibody production
● Treg (regulatory T cells): immunoregulatory type of Th cell

Cytotoxic T (Tc) lymphocytes:

 ● capable of directly destroying virally infected target cells
● suppressor T (Ts) lymphocytes: down-regulate actions of other T and B cells
● Normal function of the immune system requires balance between helper cells and suppressor cells

Natural killer (NK) cells:

● lysis of virus-infected cells through antibody-dependent cellular toxicity

● granzymes: serine proteases; released by cytoplasmic granules within cytotoxic T cells and
natural killer (NK) cells
● A patient with decreased NK cells will have uncontrolled viral replication
● Main difference between cytotoxic and natural killer cells is how they recognize their targets
○ Natural killer cells can attack their target in the absence of any surface antigen markers
○ Cytotoxic T cells recognize their target and bind to it

Alterations in Lymphocyte Subsets

● Ratio of T helper to T suppressor lymphocytes (Th/Ts ratio) 2:1

● 2:1 Th/Ts ratio changes with aging:
○ decrease in the number of suppressor cells, increase in helper cell
● With age, serum IgM decreases, serum IgA and IgG increases

T Lymphocytes: Function

● T cells are aided by antigen presenting cells (APCs)

● Two main pathways of antigen processing for APCs:
● Endogenous pathway
○ Endogenous antigens are generated within the APC and presented on the membrane with
MHC class I molecules
○ This will allow immune system to recognize and kill the infectious cell through the
cytotoxic T cells
● Exogenous pathway
○ Exogenous antigens are taken up by the APCs through endocytosis and presented on the
surface along with MHC class II molecules
○ This will activate helper T cells which will create cytokines to stimulate B cells to
become plasma cells to secrete antibodies
● T cells clonally restricted determined by TCR (T cell receptor): T cell interact with specific
peptide
● T cells recognize protein antigens in the form of peptide fragments presented at the cell surface
by MHC I or II
● T cell activation leads to:
○ proliferation
○ differentiation
○ production of cytokines
○ development of effector function

B Lymphocytes: Overview

● Represent ~10-15% of circulating peripheral blood lymphocytes

 ● Mature in bone marrow and GALT

Function

● Produce antibodies: beneficial immunity/harmful immune diseases

● Antibody-independent pathogenic role e.g. presenting antigen
● They have the potential to expand clonally which makes them the dominant APCs
● Produce cytokines and chemokines: signal immune effector cells

Subsets

● B1 cells: CD5 marker, appear to form self-renewing set, respond to common microbial antigens,
occasionally generate autoantibodies
● B2 cells: most of adult B lymphocytes, generate a greater diversity of antigen receptors, responds
effectively to T-dependent antigen

B Lymphocytes: Plasma Cells

● humoral immune response

● Reacts to antigenic stimuli through division and differentiation into plasma cells
● Plasma cells or antibody-forming cells are terminally differentiated B cells
● Plasma cells produce antibody = primary defense against microorganisms
● B cells mediate hyper-acute rejection of transplanted organ
● Continuous antigenic stimulation prompts B cells to multiply

Plasma Cells

● When B cells are stimulated by interleukins, they mature into plasma cells
● Function is synthesis and excretion of the 5 classes immunoglobulins (IgM, IgG, IgD, IgA, IgE)
● Increased: nonmalignant disorders, viral disease (e.g. rubella, infectious mononucleosis), allergic
conditions, chronic infections, and collagen diseases
● Plasma cell dyscrasias plasma cells greatly increased or infiltrate bone marrow completely (e.g.
multiple myeloma, Waldenström's macroglobulinemia)

Categories of Immunodeficiency Disorders

1. Primary disorders:

● Could be deficiency of lymphocytes, phagocytic cells and/or complement proteins

● Involve dysfunction of immune organs caused by genetic disorders of the innate and adaptive
systems
● rare genetic disorders: DiGeorge’s Syndrome, Severe Combined Immunodeficiency (SCID),
Bruton’s X-Linked Agammaglobulinemia
● Of the primary disorders:
○ 40% are T cell disorders
○ 50% B cell disorders
○ 6% phagocytic abnormalities
○ 4% complement alterations
○ The most common T cell abnormality occurs concurrently with B cell abnormality
2. Secondary Disorders:

● Immune deficiency as a result of other diseases or conditions: malnutrition, chemotherapy drugs,

autoimmune diseases, overwhelming infections

3. Disorders mediated through immune mechanisms

● Include disorders in which the immune mechanisms are out of control

● Hypersensitivity, transfusion reactions autoimmune reactions

Evaluation of Immunodeficiency Syndromes

● Symptoms: recurrent upper and lower respiratory tract infections and/or diarrhea, abscesses,
sepsis, or meningitis
● Laboratory testing: complete blood cell count (CBC), platelet count, erythrocyte sedimentation
rate (ESR)
● Screen for common immunodeficiencies: immunoglobulin testing, complement testing, cell-
mediated immunity testing, and the neutrophil function test
● Additional laboratory testing: should include a general metabolic panel to assess overall
general health, HIV types 1 and 2, protein electrophoresis, sweat chloride, and pneumococcal
antibody IgG titers pre- and post-vaccine in patients with only recurrent sinopulmonary
infections.
● If all initial test results normal: IL-1 receptor-associated kinase-4 (IRAK-4) deficiency screening
or a Toll-like receptor function assay:
● abnormal results = innate immune deficiency

Complement Pathways
Complement Pathways

● Complement in complement pathway is made up of plasma proteins

● These circulate through the blood in their inactive form
● Process is initiated through 3 pathways, classical, alternative and mannose lectin pathway
● The ultimate goal of all three complement pathways are the same:
○ Activate inflammation
○ Tag pathogen for destruction
○ Kill the pathogen

Stage 1: Recognition (Classical pathway is initiated by an antigen antibody complex)

1. C1 complex is made up of C1q, C1r, & C1s, it recognizes one bound IgM or two IgG
○ C1q first binds to Fc portion of IgG or IgM
○ C1r and C1s attach to form activated C1 Complex (first enzyme of pathway)
 2. Activated C1 enzymatically cleaves C4 into C4a and C4b
3. Activated C1 also cleaves C2 into C2a and C2b
4. C4b binds to adjacent proteins and carbohydrates on antigen surface and also binds to C2a
forming C4b2a (C3 convertase)
○ Classical complement pathway is now activated

Stage 2. Amplification or proteolytic complement cascade

● C3 convertase can cleave hundreds of molecules of C3 into C3a and C3b

Stage 3. Membrane Attack Complex or MAC

1. C3b molecule bind to C4b2a on the antigen surface to form C4b2a3b (C5 convertase)
2. C5 convertase cleaves C5 to C5a and C5b
3. C5b binds to surface of target cells and subsequently binds C6, C7, C8 and a number of
monomers of C9 to form C5b6789 (MAC)
4. C5bC6C7C8 complex polymerizes C9 to form a tube structure,
a. the tube inserts itself into the membrane of the antigen
b. It spans the membrane of the cell thats being attacked which allows ions to flow freely
between the inside and outside of the cell
c. This enhances osmotic cytolytic reaction
d. Influx of sodium and water leads to disruption of osmotic balance
e. MAC complex - think of it like a “pin” that causes a balloon to burst
5. Cell lysis occurs
● Example of classical pathway: Immediate severe transfusion reaction (transfusion of wrong ABO
type)

Complement Pathways: Alternative Pathway

● Non-antibody initiated pathway

● Activated by microbial/mammalian cell surfaces by factors such as:
○ Inulin - polysaccharides found in plants
○ Symosin - polysaccharides found on the surface of E cells
○ Bacterial polysaccharides
○ Endotoxins
● C1, C4 and C2 do not participate in the cascade (as they do in the classical pathway)
1. Conversion of another set of serum proteins, which leads to activation of C3
2. C3b and factor B combine to form C3b,B - C3 convertase
3. For the rest of the pathway, refer to MAC stage in classical pathway, all of the pathways
are the same from the MAC stage forward

Regulation

● Once these antigens are destroyed we do not want the pathways to continue as it will cause injury
to our own cells and tissues
● Factor H which prevents the association between C3b and factor B
○ Competes for site on C3b
○ Eventually leading to C3 inactivation

Complement Pathways: Mannose-Binding Lectin Pathway

 ● Mannose-binding lectin:
○ Member of a family of calcium-dependent lectins - collectins (collagenous lectins)
○ Homologous or similar to the structure of C1q
○ Mannose-binding lectin associated serine protease (MASP) enzymes activate
complement by interacting with two serine proteases called MASP1 and MASP2
○ Rest of the pathway is the same as the classical pathway

Complement Pathways - Function

● Biological functions of the complement system fall into two categories

1. Cell lysis by membrane attack complex (MAC)
○ MAC leads to osmotic lysis of a cell
2. Biological effects of proteolytic fragments of complement
○ Encompasses other effects of complement in immunity and inflammation that are
mediated by proteolytic fragments generated during complement activation

Alterations in Complement Levels

● Abnormal complement levels can indicate certain disease states

● Complement activity can cause tissue damage to healthy tissues
● Complement activation associated with intravascular thrombosis
○ E.g. rheumatoid arthritis, SLE, some genetic disorders

Elevated Complement Levels

● Inflammatory conditions
○ E.g. trauma, acute illness (myocardial infarction)
● Testing of limited clinical significance - there are other tests that are more specific to
inflammatory diseases

Decreased Complement Levels

● Complement has been excessively activated recently

● Complement is currently being consumed
● A single complement component absent (genetic defect)
● More susceptible to infection
 ○ Deficiency of the opsonic activities of complement
○ Deficiency of the lytic activities of complement
○ Deficient function of the mannose-binding lectin pathway

Other Soluble Immune Response Mediators

● Biological response modifiers (BRM’s)

○ Modulate individuals own immune response
● Four main sources of major BRMs secreted by mononuclear leukocytes
○ B lymphocytes: antibodies
○ T lymphocytes: soluble mediators (e.g. IL-2, GM-CSF, IFN-𝛾 , TNF-β)
○ Natural killer (NK) lymphocytes: IFN-ɑ
○ Monocytes/macrophages: IFN-ɑ, IL-1, TNF-ɑ, GM-CSF, M-CSF
● BRMs used therapeutically
○ Active: use microbial or chemical to modulate (e.g. vaccine)
○ Adoptive: use of soluble mediators
○ Passive: transfer pre-formed antibodies

Mediators of the Immune Response: Cytokines

● Synthesized/secreted by cells of innate and adaptive immunity in response to antigen exposure

● Role of cytokines is to transmit signals between cells
● Act on numerous cell types
● Cytokines in innate immunity:
○ Mediate early inflammatory reactions to microbial organisms
○ Stimulate adaptive immune responses
● Cytokines in adaptive immunity:
○ Stimulate proliferation/differentiation of antigen-stimulated lymphocytes
○ Activate specialized effector cells (e.g. macrophages)

Mediators of the Immune Response: Interleukins

● Type of cytokines
● Plays an important role in normal proliferation, differentiation and activation of several
hematopoietic and lymphoid cell lines
● Made by and act on lymphocytes
● Function:
○ Mediate local interactions between leukocytes by secreting peptides and proteins but do
not bind antigen
○ Modulate inflammation and immunity by regulating growth, mobility and differentiation
of lymphoid cells

Mediators of the Immune Response: Interferons

● Proteins that are part of our natural defense

● They tell our immune system that germs or other cancer cells are present in our body and they
trigger killer immune cells to fight off those invaders
 ● They can alert our immune system so it can go after a virus or cancer cells, they tell our immune
system to attack
● Interferon = INF (interfere with viral replication)
● Natural defensive responses to microbes, tumors, antigens
○ Physiologic regulators
○ Enhance expression of specific genes
○ Inhibit cell proliferation
○ Augment immune effector cells
● Clinical use:
○ Antiviral agents
○ Immunomodulators
○ Antineoplastic agents

Type I IFNs

● Mediate early innate immune response to viral infections

● Two distinct groups of proteins:
○ IFN-ɑ and IFN-β
○ The release of these two interferons act as a warning signal to our immune system

Interferon gamma (IFN-𝛾)

● Principal macrophage-activating cytokine

● Stimulates expression of MHC class I & II and APCs
● Promotes differentiation of naive CD4+ T cells to the helper T cell type 1 (Th1)
● Acts on B cells to promote switching to certain IgG subclasses
● Activates neutrophils
● Stimulates cytolytic activity of natural killer (NK) cells, antagonistic to IL-4
● Diverse effects on the immune response: used to augment cytokines in clinical disease trials

Mediators of the Immune Response: Tumor Necrosis Factor (TNF)

● Principal mediator of acute inflammatory response to gram-negative bacteria

● Responsible for many systemic complications of severe infection
● Stimulates gene transcription or induces apoptosis in a variety of cells
● Functions of TNFs are
○ Stimulate the recruitment of neutrophils and monocytes to sites of infection
○ Activate these cells to get rid of bacteria
● Low concentration of TNF:
○ Acts on leukocytes and endothelium to induce acute inflammation
● Moderate concentration TNF:
○ Mediates systemic effects on inflammation
● High concentration TNF:
○ Causes severe disease (e.g. septic shock from gram negative bacteria)

Mediators of the Immune Response: Hematopoietic Stimulators and Chemokines

● Hematopoietic Stimulator
○ Stem cell factor that acts on immature stem cells in the bone marrow and thymus to
promote proliferation
● Chemokines
 ○ Large family of cytokines
○ Stimulate leukocyte movement from the blood to the tissue site of infection
○ Regulate migration of PMNs and mononuclear leukocytes within tissue

Acute Phase Proteins

● Produced by innate immune system

● Nonspecific indicator of an inflammatory process
● Acute-phase proteins rise at different rates and in varying levels in response to tissue injury (e.g.
inflammation, infection, malignant neoplasia, trauma, surgical procedures, and drug response)

Acute Phase Proteins: C-Reactive Protein

● Clinical significant of measurement of CRP:

● Differentiate between bacterial and viral infection (bacterial = extremely elevated CRP levels)
● Useful when serial CRP measurements performed
● CRP measurement is useful for monitoring the effect of treatment and for early detection of
postoperative complications on concurrent infections
● CRP level reflects short-term and long-term disease activity and response to drugs in
inflammatory diseases (e.g. Rheumatoid arthritis (RA), Crohn’s disease)
● CRP values may be normal when other acute-phase proteins are altered (e.g. In SLE, ulcerative
colitis)
● Both CRP and low-density lipoprotein (LDL) cholesterol levels are known to be elevated in
persons at risk for cardiovascular disease; CRP level may be a stronger predictor of
cardiovascular events than LDL cholesterol

HLA & MHC

● HLA - human leukocyte antigen

● MHC - Major histocompatibility complex
● Genes encoding HLA antigens are part of MHC gene system located on chromosome 6
● MHC genes are divided into 3 classes
○ Class I: A, B, C loci
○ Class II: DR, DP, and DQ loci
○ Class III: code for complement proteins and cytokines

HLA Nomenclature

● HLA antigens:
○ Letter designating the locus
 ○ Number indicating the antigen

HLA/MHC: Testing and Identification

● HLA class I antigens are found on surface of platelets, leukocytes and most nucleated cells
● HLA class II antigens are found on antigen-presenting cells e.g. macrophages, dendritic cells, and
B cells
● In the circulation, the number of cells expressing Class I antigens is greater than the number of
cells with Class II antigens
● Because Class II antigens are not found on platelets, it is not necessary to match class II antigens
when HLA-compatible platelets are requested HLA-compatible platelets are requested

HLA/MHC: Testing and Identification

● Serologic identification requires lymphocytotoxicity test method

● Complement and dye are used to determine whether there is antigen-antibody recognition

● Serologic typing of the HLA antigens is being replaced by molecular methods because:
○ Antibodies can react to more than one epitope (or cross-reactive antibodies)
○ Specific antibodies have not been developed to recognize many different HLA antigens

HLA/MHC: Antibody Detection and Identification

● For successful transplant:

 ○ ABO compatibility
○ Matching of HLA antigens in patient with existing antibodies
● Organ transplant candidates periodically screened for newly developing HLA antibodies so
suitable match can be determined when an organ becomes available
● Patients may become sensitized to HLAs by the following exposures:
○ Pregnancy
○ Blood transfusions
■ CBS provides leukocyte-reduced blood: decreases chances for patient
sensitization to donor HLA antigen
○ Previous transplant
■ About 90% of patient develop HLA antibodies within 2 weeks of failed graft
● Testing for HLA antibodies prior to organ transplant:
1. Serologic tests on microtiter trays of panel of common antigens
2. Panel-reactive antibody (PRA): Number of HLA antibodies detected
a. High PRA: Recipient has many reaction (HLA antibodies) = less likely
compatible with available donor organs
3. Calculated panel-reactive antibody (CPRA): newer solid phase method/more sensitive
and specific/determines probability of compatibility to donor organ
a. CPRA number based on antigens that the organ candidate is reactive against,
called unacceptable antigens
b. CPRA number provides a statistical guide for determining reactivity within the
general donor population
c. Helps to rank the recipient on the waitlist for available organs
● Crossmatch - recipient serum with the T&B cells from the potential donor to avoid rejection
caused by antibodies to he donor tissue

Hematopoietic Progenitor Cell (HPC)

● HPCs can be obtained from bone marrow, peripheral blood, and cord blood
● Used to treat diseases: e.g. aplastic anemia, leukemia, lymphoma, and Hodgkin’s disease
● HLA matching at the allelic level is important to avoid rejection and graft versus-host disease
(GVHD)
○ Preformed HLA antibodies and ABO compatibility less important in HPC transplants
compared with solid organ transplants

Platelet Antigens

● platelets display HLA class I antigens: A, B, and C

● Platelet antigens that can elicit an immune response
○ Platelet antibodies less frequent because there is less platelet antigen variability in
population

Platelet Antibodies

● Antibodies to platelets may cause:

1. Neonatal alloimmune thrombocytopenia (NAIT)
a. Destruction of newborn platelets by maternal antibody
b. Therapy: washed maternal platelets or antigen-negative donor platelets
2. Post Transfusion Purpura (PTP)
a. Destruction of platelets by platelet antibodies after transfusion
● Platelet recipients can become refractory
 ○ Unresponsive to platelet transfusion
○ Can receive HLA-matched platelets or serum crossmatched to detect platelet antigen
incompatibility before transfusion

Graft Versus Host Disease (GVHD)

● Donor lymphocytes (graft) multiple and attack the patient cells (host)
● Recipient are often on immunosuppressants
○ Donor cells able to multiple
○ Organ rejection is more likely to occur
● Allele-level matching important for optimal graft survival
● GVHD can occur in immunocompromised patients receiving blood components
○ Irradiation prevents viable donor leukocytes within the blood components from
replicating by destroying mitotic activity and blast formation
○ Leukocyte reduction in blood components not enough to avoid GVHD because small
amounts of “live” leukocytes can proliferate
● Laboratory results:
● Initially present as decreased total lymphocyte concentrations: indicating immunosuppression or
immunodeficiency
● as GVHD progresses: evidence of inflammation:
○ increased C-reactive protein (CRP) level
○ elevated leukocyte count with granulocytosis
○ increased erythrocyte sedimentation rate (ESR)
○ complications of anemia and liver disease: o increased levels of bilirubin and blood
enzymes (e.g. transaminases, alkaline phosphatase)
○ presence of opportunistic pathogens (e.g. CMV)
● Pathologic features:
○ lymphocytic and monocytic infiltration into perivascular spaces of dermis and dermo-
epidermal junction of the skin and into the epithelium of oropharynx, tongue, and
esophagus
○ infiltration into base of intestinal crypts of small and large bowels and into periportal area
of liver, with secondary necrosis of cells in infiltrated tissues