UNIVERSITY OF SANTO TOMAS

FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

IMMUNOPATHOLOGY

HYPERSENSITIVITY REACTIONS
 Hypersensitivity is an exaggerated or uncontrolled response to a typically harmless antigen that
results in injury to the tissue, disease, or even death.
 May be down-regulated or up-regulated
 Considered a malfunction of the immune system
 Types (Originally there are four as proposed by Coombs and Gell in 1963)
 Type I – Immediate or Anaphylactive Hypersensitivity Reaction
 Type II – Antibody-Mediated Cytotoxic Hypersensitivity Reaction
 Type III – Immune Complex-Mediated
 Type IV – Delayed-Type Hypersensitivity
 Type V – Anti-Receptor or Stimulatory Type Hypersensitivity
 Type VI – Miscellaneous
 Type I Hypersensitivity Reaction
 Can range from life-threatening anaphylactic reactions to milder manifestations
associated with allergens (antigens that trigger Type I Hypersensitivity)
 Atopy – (Greek word Atopos = out of control) tendency to develop classic allergic
responses to naturally occurring inhaled or ingested allergens
 Key Immunologic Components: IgE, Basophils, Mast Cells, Eosinophils
 Immunologic Mechanism
‐ Allergen will be processed by Langerhans and Dendritic Cells possessing MHC-
Class II molecules then present the peptide to the CD4 of Th2 Cells
‐ Th2 Cells secrete IL-4 and IL-13 which is necessary for IgE production of B-Cells
‐ B-Cells produce IgE antibodies
‐ Sensitization Phase
‐ IgE binds to cell mebrane of mast cells and basophils and its half life is
increased from 2-3 days to atleast 10 days. Once bound, it becomes the
antigen receptor of mast cells and basophils.
‐ IgE attaches to high-affinity receptors FcεRI of mast cells and basophils.
It binds the Fc region of the epsilon-heavy chain of IgE.
‐ Activation Phase
‐ Degranulation of mast cells and basophils causing release of preformed
mediators namely Histamine, Heparin, ECF-A, NCF-A, and Proteases. It
occurs after the sensitized mast cells and basophils are re-exposed to
the similar antigen causing cross-linking which then initiates multiple
phosphorylation reactions, secretion of cytokines, and an influx of
calcium ions. The influx of calcium causes the rapid degranulation of
mast cells and basophils.
‐ Histamine and Heparin are involved in smooth muscle contraction,
vasodilation and increased vascular permeability
‐ Proteases such as tryptase and chymase are involved in the stimulation
of mucus production, activation of cytokines, and vonversion of C3 to
C3b
‐ Release of these substances is responsible for early-phase symptoms
which occur 30-60 mins after exposure to allergen
‐ Late Phase
‐ After releasing the preformed mediators, the mast cells and basophils are
triggered to synthesize reactants from the breakdown of phospholipids in
the cell membrane. These products are more potent than the preformed
mediators and are referred as secondary mediators
‐ Prostaglandin : involved in vasodilation and increased vascular
permeability
‐ Leukotriene LTB4 : chemotactic factor for neutrophils and eosinophils
‐ Leukotriene LTC4, LTD4, LTE4 : vascular permeability,
bronhioconstriction, mucus secretion
‐ Platelet Activating Factor : platelet aggregation
‐ Cytokines : increase IgE Production and Inflammatory Cells
 Clinical Manifestations
‐ Anaphylaxis
‐ Urticaria / Hives
‐ Rhinitis
‐ Hay Fever
‐ Asthma
‐ Food Allergy
‐ Angioedema
 Testing for Type I Hypersensitivity
‐ Wheal-and-Flare Skin Test : diameter > 3 to 4 mm
‐ Radioallergosorbent Test (RAST)
‐ Allergen-specific IgE testing
‐ Radioimmunosorbent Test (RIST)

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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY
‐ Total IgE measurement
‐ Competitive RIST
o Uses radiolabeled IgE to compete with patient’s serum IgE for
binding sites on a solid phase coated with anti-IgE
 Type II Hypersensitivity Reaction
 IgG and IgM antibodies are directed against antigens found on cell surfaces.
‐ Binding of antibody on cell surface can cause various effects depending on the
situation
‐ Cell Destruction
‐ Cellular Function Inhibition
‐ Cellular Function Amplification
‐ Cell Damage can occur by
‐ Activation of classical complement pathway causing formation of
membrane-attack complex causing cell lysis
‐ Coating of cell surfaces by antibodies can promote opsonization and
subsequent phagocytosis of the cell
‐ Antibody-dependent Cellular Cytotoxicity
 Includes Transfusion Reactions, HDN, AIHA, and Goodpasture’s Syndrome
‐ Goodpasture’s Syndrome – antibodies react with basement membrane protein,
usually the glomeruli in the kidney and the pulmonary alveolar membranes are
the ones affected.
‐ Other examples related to Type II Hypersensitivity reactions to tissue antigens
are Hashimoto’s Disease, Myasthenia gravis, and insulin-dependent diabetes
mellitus
‐ Grave’s Disease and Myasthenia Gravis are classified as Type V
Hypersensitivity Reactions because the autoantibodies bind on cell-
surface receptors mimicking the receptor’s ligand rather than on cellular
surfaces although some would classify it as Type 2 then segragated into
its subcategory
 Testing of Type II Hypersensitivity
‐ Direct Coomb’s Test
 Type III Hypersensitivity Reaction
 Similar to Type II in that IgG and IgM is involved and destruction is complement-
mediated but differs in the nature of antigen to be acted upon – that is the antigen
involved is soluble.
 Normally the soluble antigen complexed with antibody is precipitated out of the serum.
However when the immune system is overwhelemed, the ag-ab complex deposits in the
tissues. When such event takes place, the complex in the tissue would bind
complements causing tissue damage.
 Examples : Arthus Reaction and Serum Sickness
 Viral hepatitis, Group A streptococcus, SLE and RA can trigger Type III hypersensitivity
reactions.
 Type IV Hypersensitivity Reaction
 Delayed-Type Hypersensitivity Reaction / Cell-Mediated
 Involves T-Cells
 Antigen-sensitized Th1 Cells release cytokines that recruit macrophages and induce
inflammatiom or activate cytotoxic T-cells to cause direct cell damage
 Contact Dermatitis, Tuberculin and Anergy Skin Tests, Hypersensitivity Pneumonitis
 Type V Hypersensitivity Reaction
 Associated with autoimmune disorders such as Myasthenia Gravis and Grave’s Disease
 Not complement-fixing
 Occurs during second exposure to antigen

AUTOIMMUNITY
 Horror autotoxicus concept of Paul Ehrlich : fear of self-poisoning
 Phenomena whereby an immune response is directed against self-antigens which result to
tissue or organ damage
 Etiology of Autoimmunity
 Concept of Self-Tolerance
‐ Ability of the immune system to accept self-antigens and not initiate an immune
response against them
‐ Developed by educating lymphocytes which occurs at two levels
‐ Central Tolerance
‐ Occurs in the central or primary lymphoid organs, thymus, and bone
marrow.
‐ Negative Selection : Self-Reactive T-Cells are deleted or converted to T-
regulatory Cells
‐ Receptor Editing : Self-Reactive B-Cells that are not deleted undergo
rearrangement of genes until they achieve the state of anergy
‐ Anergy : state of unresponsiveness of B cells to the antigens
‐ Peripheral Tolerance

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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY
‐ Second level of protection as self-reacting lymphocytes may escape to
the secondary lymphoid organ
 Self-Reactive T-cells can develop anergy, cause binding of
inhibitory receptors such as CTLA-4, inhibition by Tregs or death
by apoptosis.
 Self-Reactive B-Cells can be deleted by apoptosis, be rendered
anergic, or receive inhibitory signals through receptors such as
CD22.
 Autoimmunity can arise if this second level fails to filter self-
reactive lymphocytes.
Genetics
‐ HLA-B27 : higher risk of developing diseases
‐ PTP22, IL2RA, CTLA4, BLK, AIRE genes
 Hormonal influence
‐ Women are 2.7x more likely to acquire autoimmune disorder than men
‐ Estrogen, Prolactin and other female hormones
 Cryptic Antigens
‐ Hidden antigens exposed following tissue trauma
 Molecular Mimicry
‐ Refers to the fact that pathogens contain antigens that closely resemble the
structure or amino acid sequence of self-antigens
 Bystander Effect
‐ immune phenomenon in which T cells are activated in absence of specific T-cell
receptor stimulation, which allows T cells to bypass certain immune regulatory
checkpoints (e.g., central or thymic tolerance and peripheral tolerance
mechanisms) acting after naive T cells have encountered their cognate antigen
(e.g., anergy induction, apoptosis and effects of Treg cells).
 Epitope Spreading
‐ Expansion of immune response to unrelated antigens
 Superantigens
‐ Proteins produced by microbes that have the ability to bind to both Class II MHC
Molecules and TCRs regardless of antigen specificity
‐ Act as potent T-cell mitogens by activating a large number of T-cells with
different antigen specificities
 Epigenetics
‐ Refers to modifications in gene expression that are not caused by changes in the
original DNA sequence triggered by exposure to environmental toxins, ingestion
of harmful food or drugs, or senescence.
‐ Mechanisms include up/downregulation of methylation of cytosine bases,
modifications of histones, and abnormal regulation of microRNAs
SYSTEMIC AUTOIMMUNE DISORDERS
 Systemic Lupus Erythematosus
 A systemic disease with the potential to affect many organ systems
 17 criteria (11 clinical;6 immunological; 4 must be met with atleast 1 clin and 1 immuno)
 Characterized by the production of antinuclear-antibodies (ANAs) that are directed
against nuclear antigens
 Most widely used method of ANA detection is the Fluorescent antinuclear antibody
testing which employs indirect immunofluorescence
 Slide with fixed nucelated (Hep2Cell-line)
 Patterns of Immunofluorescence
1. Homogenous / Diffuse
‐ Uniform staining
‐ Anti-dsDNA ; anti-histone ; anti-DNP
‐ SLE; Drug-induced SLE
2. Periphery / Rim
‐ Greater staining intensity around the outer circle surrounding the
nucleus
‐ Anti-dsDNA; highly specific for SLE
3. Speckled
‐ Discrete fluorescent specks throughout the nuclei
‐ Coarse Specks : anti-RNP and anti-Sm
‐ Fine Specks : anti SS-A/Ro and anto-SS-B/La ; SLE & Sjogren’s
4. Nucleolar
‐ Prominent staining of nucleoli within the nuclei
‐ Antinucleolar
‐ Antibodies to RNA and RNP
‐ Scleroderma
5. Centromere
‐ Discrete speckles in the nuclei
‐ Anti-centromere
‐ CREST syndrome

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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY
ANTINUCLEAR ANTIBODIES
IMMUNOFLUORESCENCE
AUTOANTIBODY ANTIGEN DISEASES ASSOCIATED
PATTERN

Anti-dsDNA dsDNA Peripheral / Homo SLE

Anti-ssDNA ssDNA Not on routine SLE

Drug-induced SLE, RA,

Felty’s, Sjogren, Systemic
Antihistone Histones Homo
Sclerosis, Primary biliary
cirrhosis

Anti-DNP Nucleosome Homo SLE; Drug-induced SLE

Extractable Nuclear Ag
Anti-Sm Coarse Speckled SLE
Uridine Rich RNA

snRNA-complexed
Anti-RNP Coarse-Speckled SLE
CHON

Anti-SS-A/Ro RNA-complexed CHON Fine-Speckled SLE; Sjogren’s

Phosphoprotein
Anti-SS-B/La Fine-Speckled SLE; Sjogren’s
complexed with RNA Pol

Antinucleolar RNA Pol; Finrillarin; PM-1 Prominent Staining SLE; Systemic Sclerosis

Systemic Sclerosis;
Anti-Scl-70 DNA topoisomerase I Atypical Speckled
Scleroderma

Anti-Jo-1 Istidyl-tRNA synthetase Fine Cyto Speckling Polymyositis

Chromosome
Anti-centromere Discrete Speckled CREST syndrome
Centromeres

 dsDNA antibodies are the most specific for SLE

 Histones haver five major classes: H1, H2A, H2B, H3, H4
 Antibodies to H2A and H2B are seen in almost all patients with drug-induced SLE
 Nucleosome antibodies are stimulated by complexes known as nucleosomes or
deoxyribonucleoprotein and are directed against the complexes only and not on the DNA
 Extractable Nuclear Antigens represent a family of small nuclear proteins associated with
uridine-rich RNA. The ENAs include RNP, Sm antigen, SS-A/Ro, anf SS-B/La antigens
 Antibody to the Sm antigen (Smith) is specific for lupus as it is not related to other
autoimmune diseases however it is found only in 20-40% of SLE patients
 RNP forms complexes with Sm antigen in the nucleus, and antisera to these complexes
produce a pattern of partial identity when they are reacted in the Ouchterlony double
immunodiffusion test.
 Anti-RNP is found in 20-30% of px with SLE. It is found at a higher titer in individuals with
mixed CT disease.
 Antifibrillarin is commo in systemic sclerosis/scleroderma and is indicated by clumpy
nucleolar fluorescence.
 Anticentromere antibodies bind proteins in the middle region of chromosomes where the
sister chromatids are joined.
 CREST syndrome is a subset of scleroderma named after its five major features:
Calcinosis, Raynoud’s Phenomenon, Esophageal Dysmotility, Sclerodactyly, and
Teleangiectasia.
 Titer of Significance for ANA : 160 and above
 IIF using Crithidia luciliae (a hemoflagellate) : utilizes the kinetoplast of the parasite
which is composed mainly of dsDNA; higher specificity but of lower sensitivity
 Agglutination Tests : Anti-DNP Slide Test : Classic Passive Agglutination
‐ Uses DNP antigen from Calf’s Thymus
‐ Cross Reacts with RA and Scleroderma
 Mashed Clot Test
‐ LE Cell Detection : Neutrophil Eats another Neutrophil and Lymphocyte
‐ Tart Cells and Rosette Formation of Neutrophils may be mistaken as LE Cell

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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY
Antiphospholipid Antibodies
 Found in up to 60% of SLE px
 May cause false-positive results in nontreponemal serological tests, lupus anticoagulant
assay, and immunoassays for antibodies to cardiolipin and other phospholipids.
 Lupus Anticoagulant : Increased risk of clotting and spontaneous abortion

 RHEUMATOID ARTHRITIS
 Caused by Rheumatoid Factor and/or by an antibody against cyclic citrullinated peptide
(anti-CCP)
 RF is an IgM with Affinity to the Fc Portion of IgG (hence an anti-IgG)
 The antibodies bind to their specific antigen and immune complexes deposit in the joints
resulting in a type III hypersensitivity reaction. Complement cascade occurs.
Chemotactic factors C3a and C5a attract Neutrophils and Macrophages. The continued
presence of these cells and their cytokines leads to chronic inflammation which damages
the synovium.
 20% of those who possess RF does not actually have RA
 Classic S/S : Swan-Neck Deformity; Erosion of Joints
 RF Latex Slide Test : Classic Indirect Agglutination
‐ Carrier : Latex particles with IgG
‐ Limit : 20 IU/L
‐ 1:20 dilution
‐ Positive Result : Coarse Granulation / clumping after 2 mins
 Classic Tests for RF
‐ Rose-Waaler Test : Hemagglutination Test
 Carrier: Antisheep hemolysin-sensitized Sheep RBC
 Quantitative-Significant Titer : 16 Rose Units
 WEGENER’S GRANULOMATOSIS
 Involves inflammation of the small to medium sized blood vessels or vasculitis
 Begins with a localized inflammation of the upper and lower respiratory tract
 Antineutrophil Cytoplasmic Antobody
 Autoantibodies against proteins present in neutrophil granules
ORGAN-SPECIFC AUTOIMMUNITIES
Disease Target Autoantibodies
Addison’s Disease Adrenal Glands Antibody to adrenal cells
AIHA RBC RBC Antibodies
Autoimmune Hepatitis Liver AIH 1 – anti-smooth muscles; ANAs
AIH 2 – anti-LKM-1 and anti-LC-1
Autoimmune Platelet Antiplatelet antibody
Thrombocytic Purpura
Celiac Disease Small Intestine Antitransglutaminase (tTG)
Anti-DPG (deaminated gliadin peptide)
Endomysial Antibodies
Goodpasteur’s Kidneys & lungs Antibody to Ag in Kidneys and Lungs basememnt
Syndrome membrane
Graves Disease Thyroid TSH-Receptor Antibodies
Antithyroglobuline
Anti-TPO
Hashimoto’s Thyroiditis Thyroid Antithyroglobulin and anti-TPO
Multiple Sclerosis Myelin Sheath of Nerves Anti-myelin basic protein
Myasthenia gravis Nerve-Muscle synapse Ab to Acetylcholine Receptors
Anti-muscle-specific kinase
Antibody to Lipoprotein LRP4
Pernicious Anemia Stomach Parietal Cell Ab; Intrinsic Factor Ab
Poststreptococcal Kidneys Streptococcal Ab that cross-react with kidney tissue
glomerulonephritis
Primary biliary Cirrhosis Intrahepatic bile ducts Antimitochondrial antibodies
Rheumatic Fever Heart Strep Ab that cross-react with cardiac tissue
Scleroderma Connective Tissue ANA, anti-Scl-70l anticentromere ab
Sjogren’s Syndrome Eyes and Mouth ANA, RF, antisalivary duct ab, antilacrimal gland ab
T1DM Pancreas Anti-insulin; anti-islet cells; Anti-GAD, Anti-IA-1; Anti-IA-2

TRANSPLANTATION
Transplantation
 May involve transfer of cells, tissues, or organs
 From one part of the body to another part of the body in the same individual
 From one individual to another individual of the same specie
 Also known as grafting
 Graft-transplanted tissue
Types of graft based on mechanism of transfer
 AUTOGRAFT
 Belonging to the same individual
 The donor and the recipient is the same individual
 One part of the body is grafted into another part of the body
 Cells, tissues, or organs normally autografted
 Piece of skin

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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY
 Hair
 Adipose tissue
 Bone
 Example: the skin from the thigh will be grafted into the upper arm

 ISOGRAFT/ SYNGRAFT
 Happens between monozygotic twins, meaning the twins are identical
 Chances of rejection are low since the donor and recipient are genetically similar
 ALLOGRAFT
 Transplant of an organ or tissue from one individual to another of the same species with a
different genotype
 Tissue typing is needed to decrease the risk of rejection
 The source of cell, tissue or organ may come from a relative, or from cadaveric organs
 XENOGRAFT
 Surgical graft of tissue from one species to an unlike species (or genus or family)
 Example: graft from a baboon to a human is a xenograft; bone from the leg of a guinea pig is
grafted to a mouse
Terminologies
 Liver
 Made up of facultative cells- cells that temporarily leave the cell cycle, then they will rest in Go
phase
 When needed, they return to the cell cycle in order to resume replication of cells
 Homovital- there is growth of tissue after transplantation
 Homostatic- no growth occurs in the transplanted tissue
 Orthotopic- organ is transplanted on the same site
 Heterotopic- organ or tissue is transplanted on a different site; bone from leg is placed in the upper
part of the body
Histocompatibility Systems
1. Major Histocompatibility Complex Antigens (MHC)
 HLA proteins are encoded by Chromosome 6
 Class I : HLA-A, HLA-B, HLA-C
 Class II : HLA-DQ, HLA-DR, HLA-DP
 HLA genes are inherited as haplotypes
2. Minor Histocompatibility Antigens (mHAs)
 Non-HLA proteins coded
 Demonstrate the amino acid variation in individuals
 Variation is Recongized by CD4 or CD8 in MHC-restricted manner
 Examples include the protein encoded by Y chromosome, proteins for which the individual
has a homozygous gene deletion, proteins thatare autosomally encoded, and proteins
encoded by mitochondrial DNA
3. MHC-Class-I Related Chain A Antigens (MICA)
 Expressed on keratinocytes, endothelial cells, fibroblasts, epithelial cells, dendritic cells,
and monocytes.
 Serves as target for allograft immune responses
Tests to consider
1. ABO and Rh typing
2. HLA typing- for organ transplantation; most important test

 Privileged tissue or organ- equipped with mechanism which will not allow it to combine with other
substances
 Anterior chamber of the eye
 Cornea (least immunogenic, no need for blood group typing)
 Highly vascularized organs have high risk of rejection: bone marrow, heart, liver, spleen,
kidneys, pancreas
 Privileged sites- part where you can transplant organ or tissue without risk of rejection
 Avascular (no blood supply in the area); increased blood supply increases the chance of
immune response
 Away from the lymphatic drainage
 Uterus- HLA-G, class I
 Brain- has blood brain barrier
 Testes- has blood testes barrier
 MHC class II gene- HLA-D gene product is for major transplantation barrier
 Transplantation has no guarantee that the tissue or organ will be permanently lodged in the body
Rejection
HYPERACUTE REJECTION
 After the tissue is transplanted, as soon as the vascular connection started between the graft
and the circulation, the temperature of the patient immediately starts to decrease
 Chills, fever, change in color of the graft
 Takes only a few hours
 Pre-formed cytotoxic antibodies
 Previous transplantation
 Multiple blood transfusion

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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY
 Multiparous women
ACUTE REJECTION
 Lasts for more or less than a month
 Same symptoms with hyperacute rejection
CHRONIC REJECTION
 Months to years for the graft to be in place without risk of rejection
GRAFT VS HOST DISEASE
 100 days or more
 HLA-mismatch of HSC transplants
 Donor T cells destroy the host cells
Prolonging the survival of the graft
1. Corticosteroids- potent anti-inflammatory and immunosuppressive agents used for
immunosuppresion maintenance
 Treatment of acute rejection episodes
 Steroids- block production and secretion of cytokines, inflammatory mediators,
chemoattractants, and adhesion molecules
 May cause hypertension and diabetes mellitus
2. Antimetabolic agents- interfere with maturation of lymphocytes and kill proliferating cells
 Azathioprine- first agent
 Mycophenolate mofetil
3. Calcineurin inhibitors
 Cyclosporine and FK-506 (tacrolimus)- block signal transduction in T lymphocytes, resulting in
impairment in cytokine synthesis, including IL-2, 3, 4 and interferon-gamma
 Rapamycin (sirolimus)- inhibits T-cell proliferation by binding to specific intracellular proteins
4. Monoclonal antibodies- bind to cell surface molecules on lymphocytes; used as induction agents
and to treat severe rejection episodes
 OKT3- mouse monoclonal antibody that binds to CD3 receptor on human lymphocytes
 Basiliximab and dacluzimab- bind the CD25 (IL-2 receptor), interfering IL-2 mediated T cell
activation
 Alemtuzumab- for induction therapy
5. Polyclonal antibodies- deplete lymphocytes from the circulation
 Thymoglobulin- antithymocyte antibody prepared in rabbits
 ATGAM- polyclonal antiserum prepared from immunization of horses

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