This document summarizes various screening methods used to evaluate potential immunomodulators. It describes 5 methods: 1) acute systemic anaphylaxis in rats, 2) anti-anaphylactic activity, 3) passive cutaneous anaphylaxis, 4) Arthus type immediate hypersensitivity, and 5) delayed type hypersensitivity. Each method is outlined, including the principles, procedures, evaluations, and potential modifications. The overall purpose is to evaluate candidate immunomodulators for their ability to suppress or stimulate immune responses.

1. P r e s e n t e d b y : S h i v a m
M. Pharm I year Pharmacology
Under the Guideline of: Dr. Phoolchandra
D e p a r t m e n t o f P h a r m a c o l o g y
SCREENING OF
IMMUNOMODULATORS
School of Pharmaceutical Science
I F T M U n i v e r s i t y
M o r a d a b a d

2. IMMUNOMODULATORS
• Immunomodulators are the agents that
modulates the immune system by suppress
or depress the immune system. So, these are
also divide into two parts
• 1- Immunosuppressant
• 2- Immunostimulants

3. IMMUNESUPRESSENTS
• Immunosuppressive drugs are used to
dampen the immune response in the organ
transplantation and autoimmune disease.
a) Specific T-cell inhibitors: Cyclosporine, Tacrolimus
b) Cyclotoxic drugs: Azathioprine, Cyclophospamide,
Methottraxate, Chlorambusil, Mycophenolate
c) Glucocorticoids: Prednisolone
d) Antibodies: Muromonab CD3, Antithymocyte
globulin(ATG), Rho(D), Immunoglobulin

4. IMMUNOSTIMULANTS
• These drugs are used to stimulate the immune
response in case of immunodeficiency.
• a) Levamisole (ergamisole)
• b)Thalidomide (Thalomid)
• c) Bacillus Calmette- Guerin (BCG)
• d) Interferons- gamma 1b & 1a
• e) Interleukin-2

5. Screening Methods
• Acute systemic anaphylaxix in rats
• Anti-anaphylactic activity (schuitz-dale reaction)
• Passive cutaneus anaphylaxis
• Arthus type immediate hypersensitivity
• Delayed type hyper sensitivity
• Reversed passive arthus reaction
• Adjuvant arthritis in rats

6. 1. Acute systemic anaphylaxis in rats
• Strain 10-20 female Sprague-Dawley rat (120g)
• Firstly immunized by i.m. injection of 10 mg/kg
highly purified ovalbumin.
• Simultaneously 1 ml of Bordetella pertusis
suspension injected intraperitoneally.
• After 11 days animals injected with i.v. injection
of 25 mg/kg highly purified obalbumin
• 18hr prior to challenge, test dexamethasone 1-
10mg/kg s.c. control vehicle.

7. • Evalution:
after treatment compared treated and
control group for their shock symptoms and
mortality counted.
Statistical calculation is performed using
the chi-square test.

8. Modification
• By Devis and Evans(1973)- this experiment have also
been performed in guniea pigs and in mice. Anaphylactic
bronchospasm can be measured in isolated guniea pig
lungs.
• By Ufkes and Ottenhoff (1984)- study on brown norway
rats.
Given a suspension of trinitrophenyl heptenized ovalbulin
together with aipo4.
Bronchial and cardiiovascular function were studied after
treatment with antiallergic agents and antigen challenge.

9. 2. Anti anaphylactic Activity
• Animal guinea pig of both sex
• Body weight 300-350g
• Sensitized with alum precipitated egg
albumin
• Give injection of 0.125 ml of egg albumin
in each foot pad and 0.5 ml
subcutaneously.

10. • After four weeks animal are killed and the
ileum is dissected out.
• About 2-3 cm long are mounted in an organ
bath containing Tyrode solution at
37cetigrate.
• Contractility of the ileum strips is tested by
adding standard in one organ bath and test
compound in another organ bath.

11. Biochemical estimation
• Carbon clearance test- for phagocytic
response
phagocytic Index = K(sample)/K(control)
• Hemagglutinating antibody titer.

12. Evalution-
• The concentration are recorded by a
polygraph
Modification-
• This method modified by testing histamine
release lung after challenging with egg
albumin.
• Koppel et al.(1981) developed a method to
induce concentration of mouse trachea by
antigen.
• Omote et al. (1994) modified method by using
sensitized guinea pigs.

13. 3. Passive coetaneous anaphylaxis
• Animal- male rats
• Body weight- 100 g
principle:
Formation of antigen antibody complex induce
the release of mediator from mast cells. This
results increase in permeability of the vessel
walls and leakage of plasma.

14. Procedure
• Antiserum are injected intradermally in to shaved dorsal
skin of rats.
• After 24hr each animal is challenged with the
intravenous administration of 0.1ml of 2.5% Evans blue
dye containing 25mg/ml of egg albumin.
• Test compound is also administer along with the antigen.
• After 30 min animals are scarified.
• Amount of Evans blue dye that leaked at the site of
reaction is extracted and determined calorimetrically at
620 milimicron wavelength.

15. Evaluation
• Amount of Evans blue that extracted from
passive coetaneous anaphylactic reaction of
control group is compared group.
Modification
• Gose and Blair (1969)- used Bordetella pert sis
as antigen in passive coetaneous anaphylaxis
experi

16. 4. Arthus type immediate hypersensitivity
• Animal-rats of both sex
• Strain-Wistar or Spargue- Dawley
• Body weight 220-280g
Principle-
• Antigen antibody induced reaction to an
inflammatory factors that characterized by
edeme, hemorrhage and vasculitis.

17. Procedure
• Seven days prior to experiment rats are sensitized
by im administration of 0.5 ml of the ovalabulin
suspension.
• 1st group (treated group) 1hr prior test compound
are administered and challenged with 0.5ml of
ovalabulin in left hind paw
• 2nd group(positive group)-sensitized animal
treated with solvent alone.
• 3rd group (negative group) non sensitized animals
treated with solvent.

18. Evaluation
• The change in footpad thickness is expressed
as percent from the vehicle control group.
Thickness can be measured by calipers.
Modification
• Omote et al.(1994)-sheep red blood cell
suspension used for immunization.

19. 5. Delayed type hypersensitivity
Principle
• Delayed type hypersensitivity is a reaction of
cell mediated immunity and becomes visible
after 16-18hr.
Procedure
• 7 days prior, rats are sensitized by im
administration of 0.5 ml ovalbulin.
• After seven days again 0.1 of 0.04% of
ovalbulin injected in the left hind paw.

20. • Footpad thickness is measured immediately
and 24hr after of administration.
Modification
• Mizukoshi et al.(1994)- they use sheep red
blood cells for immunization.