Screening models for immunomodulatory agents:- Introduction for immunostimulants and immunosuppressant, Models for immunomodulatory agents, Screening for immunostimulants, screening for immunosuppressant

1. SCREENING MODELS FOR
IMMUNOMODULATARY AGENTS
PRESENTED BY:
Chandragiri Siva Sai

2. IMMUNOMODULATORS
These are the chemical agents which modifies the
Immune responses or the functioning of the
Immune system.
Either by inducing or inhibition of immune
responses.

3. TYPES OF
IMMUNOMODULATERS
• IMMUNOSUPPRESSANTS:- These are the chemical agents which inhibits
(decreases) the immune responses. (prednisone, dexamethasone)
• Ex: Mainly used in organ transplantation.
• IMMUNOSTIMULANTS:-These are the agents enhances body resistance
against infections. (BCG, Interferons,Interleukin-2)
• Ex: In cancer and AIDS cases

4. CLASSIFICATION OF IMMUNOSTIMULANTS
1. Microbial products:
ex: Bacillus Calmette-Guerin(BCG)
2. Immunological products from human or
animals:
ex: Interferons,Interleukin-2

5. 3. Chemical Drugs:
Ex: Levamisole
4. Others:
Ex: polysaccharides,
Active TCM components

6. SCREENING MODELS FOR
IMMUNOMODULATORS
• IN VITRO METHODS:-
• Inhibition of Histamine Release from mast Cells.
• Mitogen induced lymphocyte proliferation.
• IN VIVO METHODS:-
• Delayed Type Hypersensitivity.

7. Anti-anaphylactic activity
(Schultz – Dale reaction).
Acute Systemic anaphylaxis in rats.
Antibody (HA) titre response to SRBC.

8. IN VITRO MODELS

9. INHIBITION OF HISTAMINE RELEASE
FROM MAST CELLS
• PURPOSE
• Hypersensitivity reactions can be elicited by various factors:
• Immunologically induced,
• Non- Immunologically induced,
• Mediation through immune responses.

10. Mediators are responsible for hypersensitivity reactions are released from mast cells.
An important performed mediators of allergic reactions found in these cells of histamine.
Mediators are responsible
for hypersensitivity
reactions are released
from mast cells.
An important performed
mediators of allergic
reactions found in these
cells of histamine.

11. Procedure:-
Preparation of mast cell suspension:-
Wistar rats are decapitated and exsanguinated
50ml of HBSS(Hanks balanced salt solution)
injected into peritoneal cavity
Fluid containing peritoneal cells collect in a
centrifuge tube
and centrifuged at 2000rpm

12. The cells are Resuspended in HBSS.
Finally Brought to concentration of 105mast cells/100ul

13. TEST COMPOUND ADMISTRATION AND
INDUCTION OF HISTAMINE RELEASE
1ml of test drug and mast cells suspension incubated at 37degres
for 15min.
The cells are made up of volume 3ml with HBSS equal to specific
allergen is added.
The suspension incubated at 37degres for 30 min followed by
centrifugation at 2500rpm

14. DETERMINATION OF HISTAMINE RELEASE
• The total sample is transferred to an autosampler
vial.
• Then histamine concentration is determined by a
fluorescence detector

15. EVALUATION
• Percentage histamine release can be Expressed by the
following formulae:-
Sample His.relese. – spontaneous his.rel.
100per His.relese. – spontaneous his.rel.
* 100

16. MITOGEN INDUCED LYMPHOCYTE
PROLIFERATION
• Purpose:-
• Cultured lymphocytes can be stimulated to DNA synthesis by various
mitogens.
• Measurement of DNA synthesis can be accomplished by tritiated
thymidine which is incorporated into the newly synthesized DNA.
• Immunomodulatory properties can be detected either by pre-treatment of
the animals in vivo or by adding the test drug to the lymphocytes.

17. PROCEDURE
• For this procedure Mice or rats are used.
• Animals receive the test compound once a day for 5 days.
• After 5 days sacrificed, spleens are removed and a single cell suspension of
5×106 cells/ml prepared.
• Mitogens are titrated and 0.1ml of the suspensions is added.
• Plates are incubated at 37degrees in 5% Carbon Dioxide in air for 48-60 hrs and
for another 8hrs after addition of 3H-thymidine per well.

18. Cells are harvested on glass fibres filters and drying the
degree of radioactivity.

19. EVALUATION
• Stimulation index = proliferation ratio according to
positive control either with or without mean
spleen weight.

20. IN VIVO

21. DELAYED TYPE
HYPERSENSITIVITY
• PURPOSE:-
• Delayed type hypersensitivity is a reaction of cell mediated
immunity and becomes visible only after 16-24hours.
• The same methods as for testing immediate type
hypersensitivity can be used.

22. PROCEDURE
• Rats are sensitized by Intra muscular administration of 0.5 ml
ovalbumin suspension 7days prior to the start of the
experiment.
• They are challenged by injection of 0.1ml of 0.04% solution of
highly purified ovalbumin in the left hind paw.
• Footpad thickness is measured immediately and 24 hours after
ovalbumin administration.

23. ANTI-ANAPHYLACTIC ACTIVITY
(Schultz-Dale reaction)
• Purpose:-
• Guinea pig 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.

24. PROCEDURE
• Guinea pigs of either sex (300-350gm) are sensitized with alum ppt egg
albumin.
• Alum egg albumin is prepared by dissolving egg albumin (1mg/ml) in six
percent aluminium hydroxide gel, suspended in saline.

25. • The mixture is stirred and kept at room temperature.
• Each animal receives at the same time injections of 0.125 ml of this
mixture in each foot pad and 0.5ml
• After 4weeks the animals are killed and ileum is dissected out.

26. • Take 2-3cm ileum and mounted in organ bath containing Tyrode solution
at 37degrees.
• Leave for resting time for 15min and tested for contractility of ileum by 10-
4gm/ml BaCl2 solution.
• To one organ bath standard and to other vials the test compounds are
added.

27. • One organ bath is serves as control.
• After 3min ovalbumin in a final concentrations of 2×10-6g/ml is added.
• The contractions are recorded with strain gauges by a polygraph.

28. EVALUATION
• The results are expressed as presence or absence of blocking
activity (percentage inhibition).
• If anti-anaphylactic activity is observed, ED50 values using
different doses are calculated.

29. ACUTE SYSTEMIC ANAPHYLAXIS IN
RATS• PURPOSE:-
• Rats are immunized with ovalbumin.
• After 11days the animals are challenged by intravenous injection of
ovalbumin.
• The shock symptoms can by inhibited by corticosteroids and I.V disodium
cromoglycate.

30. PROCEDURE
• Female Sprague-Dawley rats (120gm) are immunized by i.m Injection of
10mg/kg highly purified ovalbumin.
• 1ml of Bordetella pertussis suspension is injected intraperitoneally.
• IgE antibodies are induced and attached surface of mast cells and
basophilic granulocytes.

31. • 11days later by i.v injection of 25 mg/kg highly purified ovalbumin to
animals. This results in formation of antigen-antibody-complexes.
• On the surface of mast cells and basophilic granulocytes in blood with
immediate release of various mediators such as histamine, serotonin,
prostaglandins.

32. EVALUATION
• The shock symptoms are scored and mortality counted.
• Results after treatment are compared with untreated controls.
• Pre-treatment with corticosteroids or disodium cromoglycate can inhibit
death and ameliorate shock symptoms.

33. ANTIBODY (HA) TITRE
RESPONSE TO SRBC

34. PROCEDURE
• On initial day, all groups were sensitized with 0.1 mL of SRBC containing 1×10*8 cells, i.p.
• Group I served as control and was administered 1% Gum acacia suspension in saline.
• Group II received 100 mg/kg bd.wt. of MESI(Methanolic Extract of Sphaeranthus Indicus)
p.o. respectively (1 to 7 days).
• Group III received 200 mg/kg bd.wt. of MESI p.o. respectively (1 to 7 days).
• Group IV received 400 mg/kg bd.wt. of MESI p.o. respectively (1 to 7 days).
• Group V received 50 mg/kg bd.wt. of standard, Levamisole, p.o. respectively (1 to 7 days)

35. • On 7th day before challenge, blood was withdrawn from retro-orbital plexus of
each animal.
• Blood was centrifuged, and serum was separated. Serial two fold dilutions were
made and 50 µL of serum was added to 1st well of 96-well micro titer plate
containing 50 µL normal saline.
• To this 1% SRBC (50 µL) dissolved in normal saline was mixed.

36. From 1st well 50 µl of diluted serum was added to 2nd well containing 50 µl normal
saline and 50µl 1% SRBC. Such dilutions were done till 24th well.
• Plates were incubated at 37ºC for 1h.
• Highest dilution that has shown visible agglutination was considered as
hemagglutination(HA) antibody .

37. REFERENCE
• Hans Gerhard Vogel. Drug discovery and evaluation:
pharmacological assays, vol 2 and 3rd edition,
Page.no: 792-799.
• Harsh Mohan, Text book pathophysiology, 6th
edition, J.P bros., New Delhi, 2010, Page. no:115-120.

38. • IOSR Journal of Pharmacy and Biological Sciences (IOSR-
JPBS) e-ISSN: 2278-3008, p-ISSN:2319-7676.
• Volume 10, Issue 6 Ver. II (Nov - Dec. 2015), PP 91-99
Page Screening of immunomodulatory activity of
Sphaeranthus indicus Linn.