IN- vitro and In- vivo model

1. Screening models for
testing of immunological
factors
Priyanka Ranta
Pharmacology & Toxicology
PC/2017-X/175

2. Introduction
In vitro methods
In vivo methods
References
Flow of presentation 14/05/2018
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3. Introduction
Immunological factor
Biologically active substances whose activities
affect or play a role in the functioning of the
immune system.
Immunity is the balanced state of having adequate biological defences to fight
infection, disease, or other unwanted biological invasion.
The immune system can be seen as a more or less concerted system of armies
carrying different varieties of weapons to act against different enemies.
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http://www.reference.md/files/D007/mD007155.html

4. Components of innate immunity
 Mechanical and chemical barrier
 Phagocytosis
 Fever
 Inflammation
 Complement activation
 Acute phase proteins
Innate immunity 14/05/2018
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Kuby’s immunology , 7th edition

5. Cont.
Entry of
pathogen
Recognition
of pathogen
Induction of
inflammation
Attachment of
cells to infection
site
Phagocytosis
and killing
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Kuby’s immunology , 7th edition

6. Adaptive immunity
T-H
T-C
CD4+
Plasma
cells
CD8+
Antigen
Antigen presenting cell + MHC
T-cell differentiation
Cytotoxic T-cells
Expressed by cytotoxic cells
Cytotoxic T-
lymphocytes mediated
clearing of antigens
Expressed by helper cells
B-cells Cytokines mediated B-cell activation
Plasma cell proliferate to
produce antibodies
Memory cells responds to
second encounter of antigen
Helper T-cells
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Kuby’s immunology , 7th edition

7. 6
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https://en.wikipedia.org/wiki/Immunity_(medical)

8. Immunological factors
or immunomodulators
Immunostimulant
Immunosuppressan
t
Immunological
adjuvants
Immunostimulant are the
substances that stimulate
the immune system by inducing
activation or increasing activity of
any of its components.
 Bacterial/Viral Vaccines
 Colony stimulating factors
 Interferon's
 Interleukins
immunosuppression is a
reduction of the activation or
efficacy of the immune
system.
 Glucocorticoids
 Antibodies
An immunologic adjuvant is any
substance that acts to accelerate
or enhance antigen-specific
immune responses when used in
combination with specific vaccine
antigens.
 Alum
 Cytokines
 Freud’s adjuvant
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Nagarathna P.K.M1, R. K. (Oct 2013). Review on Immunomodulation and Immunomodulatory Activity of Some Herbal Plants. Int. J. Pharm. Sci. Rev.

9. In-vitro methods
1. Inhibition of histamine release from mast cell
2. Neutrophil locomotion and chemotaxis assay
3. Cell lines
In-vivo methods
1. Acute systemic anaphylaxis in rats
2. Murine models
3. Collagen type II induced arthritis in rats
4. Fish model
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10. Inhibition of histamine release from
mast cell
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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.
Hans gerhard Vogel. Drug discovery and evaluation: pharmacological assays, vol 2, 3rd edition,

11. 10 14/05/2018
• 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-phthaladehyde complexing reaction:
• Sample + 100 μl 1 N NaOH + 100 μl 0.2% phthalaldehyde solution, after 2 min, add 50 μl 3 N HCl.
A.L. Rönnberg Reaction of histamine with o-phthalaldehyde: Isolation and analysis of the fluorophore, Analytical

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

13. 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.
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
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14. • 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.
.
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
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15. Cell lines for immunomodulatory testing14 14/05/2018
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.
Wasaporn Chanput a, ⁎. J. (Aug.2014). THP-1 cell line: An in vitro cell model for immune modulation approach. Elsevier.

16. • 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 of NO
• Cutaneous squamous cell carcinoma cell line :- Increased cytotoxic T lymphocytes
2. There is no reported evidence for the presence of infectious viruses ortoxic 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
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17. Murine model(in-vivo)
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 25,000g for 30 min.
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.
Finally,the membrane antigens
are dialyzed against 0.1% SDS in
and stored at- 200 C
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 Wistar rats and Sprague-Dawley rats , BALB/c mice, Swiss albino mice are used immunomodulatory
studies.
Aditya Ganeshpurkar a, b. A. (Sep.2017). Experimental animal models used for evaluation of potential immunomodulatory agents. Science direct.

18. • The delayed type hypersensitivity reaction
plays a significant role against intracellular
living and proliferating pathogens.
• Augmentation in the delayed type
hypersensitivity indicates that the test
compound has stimulatory effect on
lymphocytes and activation of B cell.
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.
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19. The rate of carbon clearance, termed as
phagocytic index (K), is calculated by using
equation:-
K = (lnOD1- lnOD2) / t2 - t1
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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.
Cont..

20. Acute systemic anaphylaxis in rats
 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
 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
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Said Elshama, A. E.-M.-K. (FEB.2018). Postmortem Diagnosis of Induced Fatal Anaphylactic Shock in Rats.

21. Male adult albino rats (200-300 g)
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.
Cont.…
Assessment of shock
1. Not responding to painful stimuli, score of 1 mark;
2. Hair ruffled, 1 mark;
3. Moderate drop in body temperature, 2 marks
4. Severe drop in body temperature, 3 marks;
5. Dead in 4 hr., 5 marks;
6. Dead in 24 hr., 3 marks
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• One week after the rat sensitization, a skin test Is performed.
• 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 [10], and 0.2 ml of penicillin G (10.000 IU)

22. 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.
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, 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.
• 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.
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Cont.…
Immunoanalysis of histamine through a novel chemical derivatization Pub med

23. Collagen type II induced
arthritis
 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.
Disease induction
 Either male or female DBA/1 mice may be used.
 All mice should be 8 to 12 weeks old at immunization.
 Mice must be acclimated for at least 7 days before immunization.
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.
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Pandey*, S. (June.2010). VARIOUS TECHNIQUES FOR THE EVALUATION OF ANTI ARTHRITIC ACTIVITY . J. Adv. Pharm. Tech. Res.

24.  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.
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Cont.…

25. Fish model
Phagocytosis by fish blood lymphocytes
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.
Alcohol fixed for next 5 min and then stained
with Giemsa, and observed for bacteria
ingested by phagocytes.
Catla catla)
 Evaluation
Phagocytic ratio =
Number of phagocytic cells with engulfed
bacteria/Number of phagocytes X 100
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Aditya Ganeshpurkar a, b. A. (Sep.2017). Experimental animal models used for evaluation of potential immunomodulatory agents. Science direct.

26. 25 14/05/2018References
1. Aditya Ganeshpurkar a, b. A. (Sep.2017). Experimental animal models used for evaluation of
potential immunomodulatory agents. Science direct.
2. Nagarathna P.K.M1, R. K. (Oct 2013). Review on Immunomodulation and Immunomodulatory
Activity of Some Herbal Plants. Int. J. Pharm. Sci. Rev.
3. Wasaporn Chanput a, ⁎. J. (Aug.2014). THP-1 cell line: An in vitro cell model for immune
modulation approach. Elsevier.
4. Kuby’s immunology , 7th edition .
5. Hans gerhard Vogel. Drug discovery and evaluation: pharmacological assays, vol 2, 3rd edition.
6. Pandey*, S. (June.2010). VARIOUS TECHNIQUES FOR THE EVALUATION OF ANTI ARTHRITIC ACTIVITY . J. Adv.
Pharm. Tech. Res.
7. Said Elshama, A. E.-M.-K. (FEB.2018). Postmortem Diagnosis of Induced Fatal Anaphylactic Shock
in Rats. Reasrech gate
8. A.L. Rönnberg Reaction of histamine with o-phthalaldehyde: Isolation and analysis of the
fluorophore, Analytical Biochemistry