IMMUNOLOGY

RESISTANCE
 Non-specific Resistance (Innate defenses)
– Present at birth
– Immediate but general protection

 Specific Resistance (Immunity)

– Develops in response to antigen exposure
– Slow but Specific (self/non-self)
– Memory
 INNATE DEFENSES

FIRST LINE OF DEFENSE

(Physical and Chemical Barriers)
 Skin-epidermis, sweat, sebum
 Mucus, cilia and hairs
 Tears, saliva, urine, vaginal secretions
 Gastric juice
 Defecation and vomiting
SECOND LINE OF DEFENSE
 Antimicrobial Proteins:
 Interferons
 Complement System
 Transferrins
 Natural Killer Cells: IFN, perforins, apoptosis
 Fever
 Inflammation
 Phagocytes: neutrophils & macrophages
 INFLAMMATORY RESPONSE
Pathogen Pin
Macrophage
Chemical signals
Phagocytic cells
Capillary
Red blood cell
1 Chemical signals released
by activated macrophages
and mast cells at the injury
site cause nearby capillaries
to widen and become more
permeable.
Blood clot
Blood
clotting
elements
Phagocytosis
2 Fluid, antimicrobial proteins, 3 Chemokines released by various 4 Neutrophils and macrophages
and clotting elements move kinds of cells attract more phagocytose pathogens and
from the blood to the site. phagocytic cells from the blood cell debris at the site, and the
Clotting begins. to the injury site. tissue heals.
 PHAGOCYTOSIS
1 Pseudopodia
surround
Microbes microbes.

2 Microbes
are engulfed
into cell.
MACROPHAGE

3 Vacuole
containing
microbes
forms.

Vacuole Lysosome
containing 4 Vacuole
enzymes and lysosome
fuse.

Toxic
5
compounds
and lysosomal
enzymes
destroy microbes.

6 Microbial
debris is
released by
exocytosis.
 SPECIFIC RESISTANCE:
IMMUNITY
Antigens
 Properties- Immunogenicity & Reactivity
 Hapten – drug allergy
 Epitopes- antigenic determinants
 Chemical nature – proteins (T-cells)
 Diversity of antigen receptors- genetic recombination
Maturation of cells
TYPES OF IMMUNE RESPONSES

Cell-Mediated Immune Responses

 Cytotoxic T cells
 Intracellular pathogens, cancer cells, foreign tissue
transplants

Antibody-mediated Immune Responses

 Antibodies
 Extracellular pathogens, antigens in body fluids
PATHWAYS OF ANTIGEN PROCESSING

 Major Histocompatibility Complex (MHC) Antigens

 MHC I : all nucleated body cells (except RBCs)
 MHC II : APCs (macrophages, B cells and dendritic
cells), thymic cells and activated T cells (antigen)

 Histocompatibility testing (Tissue Typing)

APCs- macrophages, B lymphocytes, dendritic cells.
 Class II MHC molecules
– Display antigens to helper T cells

Microbe Antigen-
presenting
1 A fragment of cell
foreign protein
(antigen) inside the Antigen
cell associates with fragment
an MHC molecule
and is transported
to the cell surface.
Class II MHC
molecule
1
2 The combination of T cell
MHC molecule and receptor
antigen is recognized 2
by a T cell, alerting it
to the infection.

Helper T cell
(b)

Figure 43.9b
 Class I MHC molecules
– Display peptide antigens to cytotoxic T cells

Infected cell
1 A fragment of
foreign protein
Antigen (antigen) inside the
fragment cell associates with
an MHC molecule
and is transported
to the cell surface.
Class I MHC
1
molecule
2 The combination of
T cell MHC molecule and
receptor antigen is recognized
2
by a T cell, alerting it
to the infection.

Figure 43.9a (a) Cytotoxic T cell

CELL-MEDIATED IMMUNITY
 HELPER T (TH) CELLS – CD4+ T Cells
(cluster of differentiation)
 CYTOTOXIC T (TC) CELLS – CD8+ T Cells
 Perforin (cytolysis)
 Lymphotoxin
 Gamma-interferon (phagocytosis)
 MEMORY T CELLS
 SUPPRESSOR T CELLS
 Role of helper T cells in acquired immunity
1 After a dendritic cell engulfs and degrades a bacterium, it displays
bacterial antigen fragments (peptides) complexed with a class II
MHC molecule on the cell surface. A specific helper T cell binds
to the displayed complex via its TCR with the aid of CD4. This
interaction promotes secretion of cytokines by the dendritic cell.
Cytotoxic T cell
Dendritic Peptide antigen
cell Helper T cell Cell-mediated
Class II MHC
Bacterium molecule immunity
(attack on
TCR infected cells)

2 3
Humoral
1 CD4 immunity
(secretion of
Dendritic Cytokines B cell antibodies by
cell plasma cells)
2 Proliferation of the T cell, stimulated 3 The cells in this clone
by cytokines from both the dendritic secrete other cytokines
cell and the T cell itself, gives rise to that help activate B cells
a clone of activated helper T cells and cytotoxic T cells.
(not shown), all with receptors for the
same MHC–antigen complex.

Figure 43.15
 Activated cytotoxic T cell
1 A specific cytotoxic T cell binds to a 2 The activated T cell releases perforin 3 The granzymes initiate apoptosis within the
class I MHC–antigen complex on a molecules, which form pores in the target cells, leading to fragmentation of the
target cell via its TCR with the aid of target cell membrane, and proteolytic nucleus, release of small apoptotic bodies,
CD8. This interaction, along with enzymes (granzymes), which enter the and eventual cell death. The released
cytokines from helper T cells, leads to target cell by endocytosis. cytotoxic T cell can attack other target cells.
the activation of the cytotoxic cell.

Cytotoxic T cell Released

cytotoxic
Perforin T cell
Cancer
cell
Granzymes
Apoptotic
1 TCR CD8 3
target cell
Class I MHC 2
Pore
molecule

Target
cell Peptide
antigen Cytotoxic
T cell
Figure 43.16
 ANTIBODY-MEDIATED IMMUNITY
1 After a macrophage engulfs and degrades
a bacterium, it displays a peptide antigen
complexed with a class II MHC molecule.
A helper T cell that recognizes the displayed
complex is activated with the aid of cytokines
secreted from the macrophage, forming a
clone of activated helper T cells (not shown).
2 A B cell that has taken up and degraded the
same bacterium displays class II MHC–peptide
antigen complexes. An activated helper T cell
bearing receptors specific for the displayed
antigen binds to the B cell. This interaction,
with the aid of cytokines from the T cell,
activates the B cell.
3 The activated B cell proliferates
and differentiates into memory
B cells and antibody-secreting
plasma cells. The secreted
antibodies are specific for the
same bacterial antigen that
initiated the response.

Bacterium
Macrophage

Peptide
antigen

Class II B cell
MHC
molecule
2 Secreted antibody
3 Clone of plasma cells
1 molecules
TCR CD4 Endoplasmic
reticulum of
plasma cell
Cytokines

Helper T cell Activated

helper T cell Clone of memory
B cells
Figure 43.17
 ANTIBODY (Igs)

 Neutralize antigen
 Immobilize bacteria
 Agglutinize and precipitate
antigen
 Activate complement
 Enhance phagocytosis
TYPES OF ANTIBODIES
COMPLEMENT SYSTEM
IMMUNOLOGICAL MEMORY

 Basis for immunization by vaccination

 SELF-RECOGNITION
 MHC proteins
 Positive selection
 SELF-TOLERANCE
 Lack reactivity
 Negative selection
 Deletion (apoptosis)

 Anergy (unresponsive)

 Loss- autoimmune diseases

Blood Groups & Transfusions

 Certain antigens on red blood cells

 Determine whether a person has type A, B, AB or
O blood

 Antibodies to non-self blood types

 Already exist in the body

 Transfusion with incompatible blood

 Leads to destruction of the transfused cells
 Recipient-donor combinations
 Can be fatal or safe

Table 43.1

 Another red blood cell antigen, the Rh factor

 Creates difficulties when an Rh-negative mother
carries successive Rh-positive fetuses
Tissue and Organ Transplants
 Injured/diseased organ
 Grafts- Autografts, Isografts, Allografts, Xenografts
 MHC molecules (IFN and TNF) - Graft Rejection

 Lymphocytes in bone marrow transplants

 May cause a graft-versus-host reaction in recipients

 The chances of successful transplantation are increased

 If the donor and recipient MHC tissue types are well
matched
 If the recipient is given immunosuppressive drugs
 Allergic response: exaggerated
(hypersensitive) responses
• Type I (anaphylactic) reaction - anaphylactic shock
IgE
Allergen Histamine
1
3
2

Granule
Mast cell

• Type II (cytotoxic) reaction - incompatible blood transfusion

• Type III (immune complex) reaction - glomerulonephritis,
rheumatoid arthritis
• Type IV (cell-mediated) reaction (Delayed hyper. rea.) –
T.B. and certain haptens (poison ivy toxin)
 Autoimmune Diseases
 Immune system loses self-tolerance
 Mechanisms:
1. autoantibodies (bind, block/activate self-antigens)
Graves disease, Myasthenia gravis
2. activation of cytotoxic T cells (destroy body cells)
type I diabetes mellitus, Multiple sclerosis
Rheumatoid arthritis
Hemolytic and pernicious anemias
Systemic lupus erythematosus

 Treatment: Thymectomy, immunosuppressants, plasmapheresis

PRIMARY (INBORN)
IMMUNO-
DEFICIENCY
DISEASES
 B-Cell Deficiencies
Recurrent pyogenic infections – iv gammaglobulin
 X-linked agammaglobulinaemia (XLA)
 (B cell maturation fails) – B cell cytoplasmic tyrosine kinase
 IgA Deficiency – type III hypersensitivity
 Failure in differentiation of B cells
 Immunodeficiency with increased IgM (HIgM)
 Isotype switching does not occur
 Deficient in IgA and IgG
 Common Variable Immunodeficiency (CVID)
 B cells fail to receive proper signals from T cells
 Transient hypogammaglobulinaemia of infancy
 B cells lack help from helper T cells in synthesizing antibodies
 T-Cell Deficiencies
 Susceptible to opportunistic infections
 Humoral (B cell) deficiency also results
 Severe Combined Immunodeficiency (SCID)
 Most profound hereditary deficiency
 Defective gene- encodes IL-2, IL-4, 7, 11 and 15 receptor (few/no lymphocytes)
 Genetic deficiency of purine degradation enzymes- metabolites toxic to
lymphoid tissue
 Prolonged diarrhoea- rotavirus or bacterial infection of GIT
 Develop pneumonia – protozoan P. carinii
 Luxuriant growth of Candida albicans (mouth and skin)
 Incompatible with life (death within two years)
 Rescue-bone marrow transplantation
 MHC-II Deficiency
 Deficiency of MHC-II, CD4+ T cells (depend on positive selection)
 DiGeorge Anamoly
 Arises from a defect in thymus embryogenesis (3rd and 4 th pharyngeal pouches)
 X-linked proliferative syndrome (XLP)
 Failure to control the normal proliferation of Tc cells following EBV infection
 Fatal infectious mononucleosis/ complete B cell destruction/ fatal lymphoid malignancy/
aplastic anaemia
 Hereditary Ataxia-Telangiectasia (AT)
 Chromosomal breaks occur in TCR and immunoglobulin genes
 Infants develop ataxia (wobbly gait) and telangiectasia (dilated capillaries)
 Develop severe sinus and lung infections
 Wiskott-Aldrich syndrome (WAS)
 X-linked disease
 T-cell cytoskeletal defects and abnormal Ig levels
 Small and profoundly abnormal platelets, thrombocytopenia
 Develop severe eczema, pyogenic and opportunistic infections
 Increased IgA and IgE, normal IgG, decreased IgM
Defects in Complement Proteins
 Deficiency of C1, C2, C4 – immune complex diseases
(systemic lupus erythematosus)
 Deficiency of C3 (opsonin), factor H/I – pyogenic infections
 Deficiency of C5,6,7,8 and Factors D, P – infection with
Neisseria genus (N.gonorrhoeae, N.meningitidis)
 Deficiency of C1 inhibitor deficiency – Hereditary
angioneurotic edema
 Uninhibited activation of complement and contact systems – C2 kinin
and bradykinin – retract endothelial cells - forms gaps
 Edema – intestine (abdominal pain and cramps), upper airway (resp.
obstruction – death)
Defects in Phagocytes
 Chronic Granulomatous Disease (CGD)
 Defective NADPH oxidase

 NADPH + 2O2 → NADP+ + 2•O2- + H+

 Cell-mediated response to persistent intracellular microbial

antigens – granulomas (pneumonia, lymphadenitis and abscesses)

 Leukocyte Adhesion Deficiency

 Integrin CR3 deficiency – receptor for opsonized microorganisms
 Defective cell adhesion molecules (no emigration of phagocytes)
 LAD type 2 - Failure of syn of selectin ligands-
Defective conversion of mannose to fucose
SECONDARY
(ACQUIRED)
IMMUNO-
DEFICIENCY
DISEASES
Acquired Immunodeficiency Syndrome
(AIDS)
 Infection by the human immunodeficiency virus (HIV)
 Are highly susceptible to opportunistic infections and
cancers
 Arises from the loss of helper T cells
 Both humoral and cell-mediated immune responses are
impaired
 AIDS
 Causes:
 anal, vaginal or oral sex,
 blood transfusion,
 contaminated hypodermic needles,
 exchange between mother and baby during
pregnancy, childbirth, or breastfeeding

 The spread of HIV - a worldwide problem

 Best approach- slowing the spread of HIV
Pathogenesis of AIDS
SIGNS AND SYMPTOMS
WHO (1990) - a staging system for patients infected with HIV-1

 Stage I: HIV infection is asymptomatic and not categorized as AIDS

 Stage II: includes minor mucocutaneous manifestations and recurrent

upper respiratory tract infections

 Stage III: includes unexplained chronic diarrhea for longer than a

month, severe bacterial infections and pulmonary tuberculosis

 Stage IV: includes toxoplasmosis of the brain, candidiasis of the

esophagus, trachea, bronchi or lungs and Kaposi's sarcoma; these
diseases are indicators of AIDS.
 TREATMENT OF AIDS
 Reverse transcriptase inhibitors
 Nucleoside: eg. Zidovudine, Stavudine, Abacavir

 Nucleotide: eg. Tenofovir, Adefovir

 Non-nucleoside: eg. Nevirapine, Etravirine

 Protease inhibitors: eg Saquinavir, Ritonavir, Indinavir

 HAART
 Integrase inhibitors: Raltegravir (1st to receive FDA approval,
Oct 2007).
 Entry inhibitors (or fusion inhibitors): Maraviroc, enfuvirtide
 Maturation inhibitors: Bevirimat, Vivecon (under investigation)
 Broad spectrum inhibitors: natural antivirals (mushroom)
 KP-1467: Viral mutagen (Phase Ia and Clinical trial)
IMMUNOMODULATORS
 Immunosuppressants
 Immunostimulants

 IMMUNOSTIMULANTS
 Specific immunostimulants: vaccines
 Non-specific immunostimulants: adjuvants (inorganic,
organic and oil-based) and non-specific immunostimulators
(female sex hormones, prolactin, growth hormone and vit. D)
 Synthetic immunostimulants
 Macrokine, a stimulator of macrophages
 Levamisole, Thalidomide, BCG, Interferons, IL-2
 Herbal immunostimulants
 Aloe vera
 Beta-glucan (activates phagocytes and NK cell activity)
IMMUNOSUPPRESSIVE DRUGS

 GLUCOCORTICOIDS
 DRUGS ACTING ON IMMUNOPHILINS
 CYTOSTATICS
 ANTIBODIES
GLUCOCORTICOIDS
 Induces lipocortin-1 synthesis- inhibits PLA2 binding
to substrate arachidonic acid-inhibits eicosanoid
synthesis
 Releases lipocortin-1 into extracellular space- inhibits
COX-1 and COX-2 pathway of PG and leukotriene
synthesis.
DRUGS ACTING ON IMMUNOPHILINS
 CYCLOSPORINE:
 cyclic polypeptide, fungus species Beauveria nivea
 Mechanism: inhibits calcineurin
 Increases expression of TGF-β (inhibitor of IL-2 stimulated T-cell
proliferation
 Nephrotoxicity, tremor, hirsutism, hypertension,
hyperlipidemia
 CYP3A- inh- Ca channel blockers, antibiotics,
glucocorticoids, HIV-protease inhibitors and grape fruit
 Interaction between cyclosporine (nephrotoxicity) and
sirolimus (hyperlipemia and myelosuppression) and
NSAIDs
 TACROLIMUS:
 Macrolide antibiotic – Streptomyces tsukubaensis
 Mechanism: inhibits calcineurin
 Nephrotoxicity, neurotoxicity, hypertension,
hyperkalemia, hyperglycemia, diabetes
 Metabolized by CYP3A

 SIROLIMUS:
 Macrocyclic lactone – Streptomyces hygroscopicus
 Dose-dependent increase in serum chol. and TGL,
anemia, leukopenia, thrombocytopenia
 Metabolized by CYP3A
CYCLOSPORINE, TACROLIMUS & SIROLIMUS
CYTOSTATICS
 Alkylating agents
 nitrogen mustards (cyclophosphamide)
 Nitrosoureas
 platinum compounds, and others

 Antimetabolites: interfere with the synthesis of

nucleic acids
 folic acid analogues: methotrexate
 purine analogues: azathioprine and mercaptopurine
 pyrimidine analogues, Leflunomide
 protein synthesis inhibitors
 AZATHIOPURINE (purine anti-metabolite)
 bone marrow suppression, hepatotoxicity, alopecia,
pancreatitis, GI toxicity
 metabolized by Xanthine oxidase (Allopurinol)

 MYCOPHENOLATE MOFETIL
 2-morpholinoethyl ester of MPA (inh. IMPDH)
 ↓ antibody formation, cellular adhesion, migration
 diarrhea, vomiting, leukopenia
 Interact with Al and Mg contg. antacids
ANTIBODIES
 POLYCLONAL ANTIBODIES
(bind to CD and HLA I and II)
 1. Antithymocyte Globulin (ATG)
 2. Antilymphocyte Globulin (ALG)

 Serum sickness, hypotension, GN, anaphylaxis,

leukopenia, thrombocytopenia
 No drug interactions
 Anti-antibodies develop
 Variation in efficacy & toxicity
 MONOCLONAL ANTIBODIES
 Hybridoma technology
 Defined and limited target specificity

 Muromonab-CD3 (OKT3)
 Internalization of TCR
 ↓ cytokines (IL-2)
 Acute organ transplant rejectionCytokine-release syndrome
(Cytokine storm) –FcR-mediated crosslinking---- adm. of
glucocorticoids
 Anaphylaxis, Anti-antibodies develop
 New Generation Anti-CD3 Antibodies
GENERATION OF MONOCLONAL ANTIBODIES.
Anti-IL-2 Receptor
(Anti-CD25) Antibodies
 Daclizumab: humanized CDR/human IgG1 chimeric
monoclonal antibody
 Basiliximab: human chimeric monoclonal antibody

 bind to IL-2 receptor on activated T-cells

 Prophylaxis – acute organ rejection
 No cytokine release syndrome
 No drug interactions
 Anaphylactic reactions
Anti TNF-α antibody
 Infliximab (monoclonal antibody)
 Prevents binding of TNF-α
 Rheumatoid arthritis, Crohn’s disease
 Fever, hypotension, dyspnea, resp. infections
 antinuclear antibodies, lupus-like syndrome

 Etanercept (fusion protein)

 Ligand binding portion-Fc of IgG1
 Serious infections, injection-site reactions
 VACCINES
 Safe, affordable, induction of right immunity (antibody for toxins and
extracellular org; cell-mediated immunity for intracellular org)
 Types
 Natural living org. – vaccinia (small pox)
 attenuated org. – BCG, polio, measles, mumps, rubella
 killed (intact) – rabies, influenza, hepatitis A, typhoid
 Subunit vaccines (subcellular fragments) – pneumococcus (capsular
polysaccharides), hepatitis B (surface antigen)
 Toxoids – tetanus, diphtheria
 Recombinant DNA-based – gene cloned & expressed (hepatitis B-yeast
derived), genes expressed in vectors (expt.), naked DNA (expt.)
 Transgenic plants expressing vaccine antigens
 Anti-cancer vaccines (purified cytokines-IFNα, APCs expressing tumor
antigen, dendritic cells pulsed with MHC-I peptides of tumor sp. antigens)
 PASSIVE IMMUNIZATION
SERA
 Derived from pooled plasma of adults (IgG)
 Immunodeficient, high degree of risk (inadequate time
for vaccination), specific need, high titre of sp. Igs
req., already circulating toxins (tetanus, snake bite)
 Non-specific or highly specific (hepatitis B, tetanus,
CMV)
 Rho(D) immune globulin
 Antibodies against Rh(D) antigen
 Prophylaxis against hemolytic disease of the newborn
SCREENING METHODS
FOR
IMMUNOMODULATORS
In vitro methods
1. Inhibition of histamine release from mast cells
 Wistar rats are decapitated

 Hank’s balanced salt solution (HBSS) are injected into the

peritoneal cavity
 fluid containing peritoneal cells is collected

 centrifuged at 2000 rpm

 cells are resuspended in HBSS (10 5 mast cells/100 μl)

 Test drug + mast cell suspension

 mixture is incubated at 37 °C for 15 min.

 calcium ionophore 48/80 (induces release of histamine from

mast cells) is added

 Extraction and determination of histamine
2. Mitogen induced lymphocyte proliferation
 Animals are sacrificed and their spleens removed
 single cell suspension of 107 cells/ml placed in each microtiter
well (4 replicates/group)
 test compound is added
 mitogen is added (Sheep red blood cell (SRBC) specific
antigen and/or the following mitogens: Lipopolysaccharide,
Dextran sulfate, etc).
 Plates are incubated for 48–60 h
 3
H-thymidine per well is added and incubated for 8 hrs
 Radioactivity is determined using a β-counter.
3. Inhibition of T cell proliferation

 Peripheral blood leukocytes + equal numbers of

gamma-irradiated allogenic peripheral blood
leukocytes + various conc. of test compounds.
 After 6 days of coculture, the cells are pulsed for 6 h
with [3H]thymidine per well.
 [3H]Thymidine incorporation is measured
4. Chemiluminescence in macrophages
 stimulation of macrophages by antigen, complement,
phorbol-esters leads to elaboration of O2– and other
oxygen metabolites
 oxygen metabolites can produce light-emitting
reactions (chemiluminescence), which is measurable
if amplified with suitable agents, such as the cyclic
hydrazide luminol.
5. PFC (plaque forming colony) test in vitro
6. Inhibition of dihydro-orotate dehydrogenase

 catalyzes the fourth committed step in the de novo

biosynthesis of pyrimidines (leflunomide)
 rapidly proliferating human T cells have an exceptional
requirement
 oxidation of the substrate dihydro-orotate, reduction of
the co-substrate quinone - coupled to the reduction of
the chromogen 2,6- dichlorophenolindophenol (DCIP)
 loss of absorbance of the blue DCIP is monitored
 In vivo methods
1. Acute systemic anaphylaxis in rats
 Immunized with highly purified ovalbumin (i.m., 10

mg/kg)
 Simultaneous adm. of an adjuvant [Bordetella

pertussis suspension (2 × 1010 organisms), i.p.]

 11 days later- challenge with highly purified

ovalbumin (25 mg/kg, i.v.).

 Drug (immunosuppressant- 18 hrs prior to challenge)

 Shock symptoms are scored and mortality counted

2. Anti-anaphylactic activity
(Schultz-Dale reaction)
 Guinea pigs are sensitized against egg albumin.
 Challenge after 3 weeks causes in isolated organs
release of mediators, e.g. histamine, which induce
contraction in isolated ileum (lungs, trachea).
 Results are expressed as presence or absence of
blocking activity (percentage inhibition)
3.Passive cutaneous anaphylaxis
 animals are sensitized with egg albumin using aluminium
hydroxide gel (200 mg) as adjuvant.
 After 8 days, the animals are bled and antiserum is collected
 antiserum are injected intradermally into the shaved dorsal skin
of normal male rats
 After 24 h of latent period each animal is challenged with the
intravenous administration of Evans blue dye containing egg
albumin.+ drug (1 hr prior to challenge)
 animals are sacrificed 30 min after the challenge
 amount of Evans blue dye leaked at the site of passive
cutaneous anaphylactic reaction is extracted and determined
colorimetrically at 620 mμ wavelength.
4. Arthus type immediate hypersensitivity
 Arthus reaction- acute inflammatory responses in
joints of rheumatic patients
 inflammatory focus characterized by edema,
hemorrhage and vasculitis
 7 days prior - rats are sensitized by i.m. adm. of
ovalbumin suspension
 24 hrs and 1 hr prior to induction of the Arthus
reaction, test compounds are adm.
 Challenge with highly purified ovalbumin in the left
hind paw
 After 2-8 hrs, footpad thickness is measured
5. Delayed type hypersensitivity
 Same as above; but takes 16-24 hrs
6. Reversed passive Arthus reaction
 Antigen is injected i.v. followed by a local injection –
either intradermally or into the pleural space – of the
respective antibody
 Arthus reaction in the rat pleural cavity results in a
classic acute inflammatory response.
 Animals receive BSA (i.v.) – drug – anti-BSA
(pleural cavity)
 Animals are sacrificed at various intervals after anti-
BSA injections
 Eicosanoids in the pleural exudate are quantitated by
commercial RIA kits
7. Adjuvant arthritis in rats
 exhibits many similarities to human rheumatoid
arthritis
 complete Freund’s adjuvant [mycobacterium
butyricum suspended in heavy paraffin oil (6mg/ml)]
 primary lesion (3-5 days)
 Secondary lesions (11-12 days) - characterized by
inflammation of non-injected sites (hindleg, forepaws,
ears, nose and tail), a decrease of weight and immune
responses.
 21st day-secondary lesions are graded [absence-0,
presence-1 of inflammation.
8.Collagen type II induced arthritis in rats
 intradermal injection of homologous or heterologous
type II collagen in incomplete Freund’s adjuvant
results in an inflammatory polyarthritis in rats
 similarities of the symptoms in patients with
rheumatic polyarthritis (antibodies to collagen)
 7 days post-immunization, the animals receive
identical booster injections
 From days 20–40, the animals receive the test
compounds p.o. once a day
 On days 20 and 41, the paw volumes are recorded.
9. Experimental autoimmune thyroiditis
 Immunization of rats or mice with porcine
thyroglobulin (followed by challenge) results in
thyroiditis
 Sera are tested for the levels of anti-PTg antibodies
 Histopathology of thyroid glands

10. Prevention of experimentally induced

myasthenia gravis in rats
 Heterologous AChR, or with recombinant α subunits
(two) of the AChR
11. Acute graft versus host disease (GVHD)
in rats
 The i.v. inj. of a mixture of parental splenocytes into
healthy inbred F1-rats results in graft versus- host
(GVH) induced immune abnormalities.
 host F1 T-cells are genetically unable to recognize
antigens of the parental donor as foreign
 response involves only donor recognition of host MHC
 lead to clinical symptoms of an acute, lethal GVHD
(profound immunodeficiency, anemia,
hypogammaglobulinemia)
11. Inhibition of allogenic transplant
rejection
 skin pieces, kidney, rat heart, rat small intestine and
corneal buttons
 strain combination with a major histocompatibility
variant
 Rat tail skin (donor) is cut into square pieces of 0.5 to
1.0 cm and transplanted to the tails of recipient rats.
 Rejection is defined as the day when the skin graft is
of red-brown color and hard consistency
 The immunosuppressive agents, e.g., cyclosporine or
leflunomide are given orally up to 20 days
PARAMETERS ESTIMATED
 B-lymphocyte proliferation
 Cytokine production by T-helper lymphocytes
 T-lymphocyte cytolytic function
 Natural killer (NK) cell function and macrophage
function
 NO and TNF- release
 Fibroblast-lysis assay (tumour necrosis factor (TNF)
activity) and Fibroblast-virus protection assay (IFN
activity)
 Expression of iNOS , expression of CD14, TLR4 and
CR3
 HA titer,
 Number of PFCs
IMMUNOLOGICAL TECHNIQUES

 ANTIGEN-ANTIBODY INTERACTIONS
 Precipitation reactions (immunoelectrophoresis, single
radial immunodiffusion)
 Haemagglutination and complement fixation
 Direct and Indirect Immunofluorescence
 Immunoassay (RIA and ELISA)
 Immunoblotting