The document summarizes various screening methods used to evaluate immunomodulators. It discusses in vivo methods like acute systemic anaphylaxis in rats and delayed type hypersensitivity reaction in rats. It also discusses in vitro methods like inhibition of histamine release from mast cells and neutrophil locomotion assays. The document provides details of various protocols used for screening immunomodulators.

1. SCREENING METHODS OF
IMMUNOMODULATORS
PRESENTED BY:-
AANCHAL ARYA
M. PHARM
PHARMACOLOGY

2. IMMUNE RESPONSE
 The immune system evolved to discriminate self from nonself.
 Multicellular organism shows immune response by destroying
infectious invaders (microbes) while leaving normal cells intact.
 Immunity system shows two types of responses that are-
1. Innate immunity
2. Adaptive immunity

3. 1.Innate, or natural immunity - It is primitive, does not require
priming, and have relatively low affinity, but is broadly reactive. The
major effectors of innate immunity are granulocytes,
monocytes/macrophages, natural killer cells and mast cells.
2. Adaptive, or learned, immunity - It is antigen specific, depends
upon antigen exposure and have very high affinity. The major
effectors of adaptive immunity are B and T lymphocytes. B
lymphocytes make antibodies; T lymphocytes function as helper,
cytolytic cells.

4. IMMUNOMODULATORS
 Immunomodulators are the agents that modulates the
immune system by suppress or stimulate the immune
response. So, these are also divided into two parts that are-
1.Immunosuppressants &
2.Immunostimulants

5. 1.IMMUNOSUPPRESSANTS-
Immunosuppressive drugs are used to dampen the immune response
in organ transplantation and autoimmune disease.
(a)Specific T-cell inhibitors-Cyclosporine, Tacrolimus
(b)Cytotoxic drugs-Azathioprine, Cyclophosphamide,
Methotrexate, Chlorambucil.
(c)Glucocorticoids-Prednisolone
(d)Antibodies- Muromonab CD3, Antithymocyte globulin(ATG),
Rho(D) immunoglobulin

6. 2.IMMUNOSTIMULANT-
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- gamma1b & beta1a
(e) Interleukin-2

7. screening method
(IN VIVO)
ACUTE SYSTEMIC ANAPHYLAXIS IN RATS
ANTI-ANAPHYLACTIC ACTIVITY
PASSIVE CUTANEOUS ANAPHYLAXIS
ARTHUS TYPE IMMEDIATE
HYPERSENSITIVITY
DELAYED TYPE HYPERSENSITIVITY
REVERSED PASSIVE ARTHUS REACTION
ADJUVANT ARTHRITIS IN RATS

8. Acute systemic anaphylaxis in rats
STRAIN: 10-20 FEMALE SPRAGUE-DAWLEY RAT(120G)
FIRSTLY IMMUNIZED BY I.M. INJECTION OF 10MG/KG HIGHLY PURIFIED
OVALBUMIN.
SIMULTANEOUSLY 1ML OF BORDETELLA PERTUSSIS SUSPENSION INJECTED
INTRAPERITONEALLY.
AFTER 11 DAYS ANIMALS INJECTED WITH I.V. INJECTION OF 25MG/KG HIGHLY
PURIFIED OVALBUMIN
18HR PRIOR TO CHALLENGE;
TEST – DEXAMETHASONE 1-10MG/KG S.C. CONTROL-VEHICLE

9. Evaluation-
 after treatment compared treated and control group
for their shock symptoms and mortality counted.
Modification-
 By Davis and Evans(1973)-This experiment have
also been performed in guinea pigs and in mice.
Anaphylactic bronchospasm can be measured in
isolated guinea pig lungs.

10. Passive cutaneous anaphylaxis
Animal-male rats
Body wt.-100g
 Principle-
Formation of antigen-antibody complex induces the release
of mediator from mast cells. This results increase in
permeability of the vessel walls and leakage of plasma

11. Procedure-
Antiserum are injected intradermally in to the 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 sacrificed.
Amount of Evans blue dye that leaked at the site of reaction is extracted
and determined colorimetrically at 620milimicron wavelength.

12. Evaluation-
 Amount of Evans blue that extracted from passive cutaneous
anaphylactic reaction of control group is compared with the treated
group.
Modification-
 Goose and Blair(1969)- used Bordetella pertusis as antigen in passive
cutaneous anaphylaxis experiments in the rat.

13. Arthus type immediate
hypersensitivity
Animal-rats of both sex
Strain-Wistar or Sprague-Dawley
Body wt.-220-280g
Principle-
Antigen-antibody induced reaction leading to an inflammatory factors
that characterised by edeme, hamorrhage and vasculitis.

14. procedure
Seven days prior to experiment rats are sensitized by i.m. administration
of 0.5ml of the ovalalbumin suspension
1stgroup(treated group)- 1hr prior test compound are administered and
challenged with 0.1ml of ovalalbumin in left hind paw
2ndgroup(positive control)-sensitized animals treated with solvent
alone.
3rdgroup(negative control)-nonsensitized animals treated with solvent.

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

16. Delayed type hypersensitivity
Principle-
Delayed type hypersensitivity is a reaction of cell mediated immunity
and becomes visible after 16-24hr.
Procedure-
7days prior, rats are sensitised by i.m. administration of 0.5ml
ovalbumin.
After 7 days again 0.1ml of 0.04% of ovalbumin injected in the left hind
paw.

17.  Footpad thickness is measured immediately and24hr after of
administration.
Modification-
 Mizukoshi et al.(1994)-They use sheep red blood cells for
immunization.

18. In-vitro methods
In-vivo methods
1. Inhibition of histamine release from mast cell
2. Neutrophil locomotion and chemotaxis assay
3. Cell lines
1. Acute systemic anaphylaxis in rats
2. Murine models
3. Collagen type II induced arthritis in rats
4. Fish model

19. Inhibition of histamine release from mast cell
An important preformed mediator of allergic reactions found in cells is histamine which release
from mast cells.
Procedure
1. Preparation of Mast Cell Suspension
• Wistar rats are decapitated and exsanguinated.
• Hank’s balanced salt solution is injected into the peritoneal cavity and following massage of
the body, the abdominal wall is opened.
• The fluid containing peritoneal cells is collected in a centrifuge tube and centrifuged at 2000
rpm.
• The cells are re-suspended in HBSS.

20. Test Compound Administration and Induction of Histamin Release
• The test drug is added to the mast cell suspension and the mixture is incubated at 37°C for 15min.
• The cells are made up to a volume of 3ml with HBSS, an equal volume of allergen is added.
• The suspension is incubated at 37°C for 30 min. followed by centrifugation at 2500 rpm. Test Compound
Administration and Induction of Histamine Release
Extraction of histamine
• 1 ml of the top layer is transferred to a tube containing 300 mg NaCl and 1.25 ml butanol followed by
alkalization to extract adding 1 ml 3 N NaOH.
• The sample is centrifuged for 5 min, One ml of the top layer is pipetted into a tube containing 2 ml of n-
heptane and 0.4 ml of 0.12 N HCl.
• 0.5 ml of the aqueous phase is transferred to another tube induction of o- phthalaldehyde.

21. Determination of Histamine Release
Histamine concentration is determined by a fluorescence detector (using excitation and emission wave
lengths of 350 and 450nm respectively.
Percent histamine release
sample hist. release- spontaneous hist. release
100%hist. release- spontaneous hist. release
LIMITATIONS OF THE PROCEDURE
Specimen collection has a significant effect on the test results.

22. Neutrophil cell suspension
were prepared in phosphate
buffer saline (PBS).
• One chamber is added with PBS and used as
control.
• Chamber 2 is added with casein and is used as
standard.
• The remaining chambers were filled with
different concentration of the test drug.
The bottoms of the upper compartment
were placed with wet filter paper of 3 mm
pore size and filled with predetermined
concentration of neutrophil cell suspension
Neutrophil locomotion and chemotaxis assay
• In general, cells are placed in the upper compartment and are allowed to migrate through the
pores of the membrane into the lower compartment, in which chemotactic agents are present.
• After an incubation time, the membrane between the two compartments is fixed and stained, and
the number of cells that have migrated to the lower side of the membrane is determined.

23. These upper compartments were then placed on the lower compartments and incubated for 180 min at 37
0 C
After incubation the neutrophil suspension was emptied by inverting the upper compartments and the filter
papers were removed.
The lower surfaces of the filter papers of all the chambers were fixed with ethanol and stained with
haematoxylin dye for 5 min.
Determination of neutrophil locomotion and chemotactic ability
• The neutrophils locomotion and chemotactic abilities of the test were determined by observing the lower
surface of stained filter papers under 100x .
• The number of neutrophil cells reached the lower surface of filters were counted.

24. Cell lines for immunomodulatory testing
1. THP-1 (Human leukaemia monocytic cell line
This cell line has become a common model to estimate modulation of monocyte and
macrophage activities.
The THP-1 cell line is isolated from the peripheral blood of a 1-year old male patient suffering
from acute monocytic leukaemia.
THP-1 monocytes maximally expressed IL-1ß,IL-6, IL-8, IL-10 and TNF-a genes after 3 h of LPS
stimulation, while gene expression appears to be maximal after 6hr.
In addition NF-KB expression also occurs resulting in further inflammation.
Advantages
1. The average doubling time in THP-1 monocytes is around 35 to 50 h .Under growing conditions
with the use of RPMI 1640 supplemented with 10% FBS, THP-1 cells can quadruple within three and
a half days.

25. 2. There is no reported evidence for the presence of infectious viruses or toxic products in THP-1 cells,
making this cell line relatively easy and safe to use.
3. THP-1 is an immortalized cell line that can be cultured in vitro up to passage 25 (approx. 3 months)
without changes of cell sensitivity1 and activity.
Some of other cell lines for immunomodulatory studies
• K562cell line:- Stimulation of NK cells activity against K562 cells
• J 779 macrophage cell line :- Inhibition of chromate-induced cytotoxicity
• K562 cell line :- Decreased production
• Cutaneous squamous cell carcinoma cell line :- Increased cytotoxic T lymphocytes

26. Humoral antibody response is studied by injecting prepared erythrocytes of sheep blood. After exposure to
antigens, the antigen-specific immune response is observed.
Sheep blood is collected in
Alsever’s solution, and cells
are isolated by
centrifugation at 1000 rpm
for 15 min
Plasma and the buffy
coat is removed
followed by washing of
cells with 0.9% NaCl
thrice
The so formed pellet is suspended in
0.05 M Tris-HCl with 0.1 mM EDTA
(pH 7.6) and mixed thoroughly and
again centrifuged at 25,000 for 30
min.
Finally,the membrane antigens are
dialyzed against 0.1% SDS in and
stored at- 200 C
The process may be repeated till the
supernatant becomes clear. The pellet is
suspended in 0.1%sodium dodecyl sulphate
(SDS) with 0.02% sodium aside.
Murine model(in-vivo)
Wistar rats and Sprague-Dawley rats , BALB/c mice, Swiss albino mice are used
immunomodulatory studies.

27. Macrophage phagocytosis by carbon clearance method
• Carbon clearance test evaluates the effect of drugs of the reticuloendothelial system
• RES comprises of a ‘diffuse system’ that make up of phagocytic cells. Once the colloidal carbon
particles are directly injected into the blood, they are cleared.
• Rapid removal of carbon particles has been associated with an increase in phagocytic activity.
1. Animals are divided into several suitable groups. The control
group is treated with cyclophosphamide (30 mg/kg, i. p.) while
other groups receive test compounds.
2. On day six, all the groups are administered with 0.1 ml of carbon
ink suspension through the tail vein.
3. Blood is collected 0 and 15 min immediately after injection of
carbon suspension.
4. Blood (25 ml) is lysed with 2 ml of 0.1% sodium carbonate
followed by measurement of absorbance spectrophotometric at
675 nm to determine optical densities.
The rate of carbon clearance, termed as
phagocytic index (K), is calculated by
using equation:- K = (lnOD1- lnOD2) / t2 -
t1

28. Rapid in onset; characterized by life-threatening airway, breathing, and/or circulatory problems; and
usually associated with skin and mucosal changes.
On the surface of mast cells and basophilic granulocytes in blood various mediators of anaphylaxis,
such as histamine, serotonin, SRS-A, prostaglandins are released in shock symptoms and 80% lethality
is found
Acute systemic anaphylaxis in rats
Post-mortem diagnosis of induced fatal anaphylaxis in rats assesses the serum
levels of mast cell , IgE and histamine, and histopathological changes in organs
using light and electronmicroscope examinations
Male adult albino rats (200-300 g)

29. The first (control) group receives
0.5 ml of distilled water
Every mouse in the second and third group was actively sensitized
twice by a single s.c. injection of ovalbumin (1 mg) and PenicillinG (0.2
ml), respectively with a one week time interval.
Assessment of shock
1. Not responding to painful
stimuli
2 . Hair ruffled
3. Moderate drop in body
temperature
4. Severe drop in body
temperature
5. Dead in 4 hr.
6. Dead in 24 hr.
• One week after the skin test, the second and the third
group received a single i.v. dose of 1 mg
ovalbumin dissolved in 0.5 ml of distilled water , and 0.2
ml of penicillin G (10.000 IU)
• One week after the rat sensitization, a skin test Is
performed.

30. Histamine in the blood was measured by a
radioimmunoassay using the succinylglycinamide
derivatization of histamine in the samples to
mimic the immunogen used to generate the
monoclonal Antibody.
At the end of the experiment, after the
sacrifice of the first-group rats the tissues
samples from the larynx, trachea, lung, hearts
and spleen were taken and fixed in
10%formalin. •
Serum immunoglobulin E (IgE) was measured by an ImmunoCAP-specific IgE blood test that
was a fluorescent enzyme immunoassaywhich measured allergen-specificIgE in the serum;
• At the end of the experiment, the blood samples were obtained from the right femoral
artery were left at room temperature for 15-30 minutes to clot.
• The samples were centrifuged at 2000rpm for 10minutes at 4°C to remove the clot and
separate the serum samples that were stored at -20°C until the assay.

31. The joint inflammation which develops in CIA resembles inflammation in human patients with RA.
CIA is mediated by both T-cells and antibodies (B-cells). Macrophages are believed to play an important role in
mediating tissue damage during disease development.
1. Immobilize the mouse using a restrainer.
2. Clean the tail with 70% ethanol, wipe the area dry with sterile gauze.
3. Position the syringe containing collagen/CFA emulsion parallel with the tail.
4. Puncture the skin approximately 25 mm (1 inch) distal of the hair line. Insert needle
subcutaneously 7 to 10 mm toward the body of the mouse.
Collagen type II induced arthritis
Disease induction Either male or female DBA/1 mice
may be used. All mice should be 8 to 12 weeks old at
immunization

32. Booster dose with collagen/IFA emulsion - Day 18
to 21.
Blood vessels in the tail will be dilated as a result
of the initial immunization.
EVALUATION
Paw score Clinical observations
0 Normal paw.
1 One toe inflamed and swollen.
2 More than one toe, but not
entire paw, inflamed and
swollen,
3 Entire paw inflamed and
swollen.
4 Very inflamed and swollen paw
or ankylosed paw

33. Fish model
Evaluation
Phagocytic ratio =
Number of phagocytic cells with engulfed
bacteria/Number of phagocytes X 100
Alcohol fixed for next 5 min and then stained with
Giemsa, and observed for bacteria ingested by
phagocytes.
Phagocytosis by fish blood lymphocytes
(Catla catla)
Test compound supplemented
with fish feed for 30 days
Infected with pathogenic Pseudomonas aeruginosa
After 5 days
Blood Serum sample is mixed with an equal amount of
suspension of bacteria and incubated at room
temperature for next 20 min

34. Thank you