B. Tech Biotechnology Dissertation - SRM University - May 2007

Dissertation titled "in vivo assessment of the Immunomodulatory Activity of plant-derived drug, Silymarin, and its effects on stress". Work performed at Indian Institute of Integrative Medicine in Jammu Tawi, India (Jan - March 2007)

B. Tech Biotechnology Dissertation work by Vivek

Transcript

1. Vivek Krishnakumar (10903253)
   School of Biotechnology
   SRM University
   Cell Biology Lab,
   Pharmacology Division,
   Indian Institute of Integrative Medicine (CSIR)
   Jammu Tawi
   15/05/2007
   Immunomodulatory Activity of
   Silymarin and its Effect on Stress
   Advisors
   Dr. Sarang Bani (Scientist, IIIM, Jammu Tawi)
   Dr. R. S. David Paul Raj (Asst. Professor, SRM University)
   

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2. 15/05/2007
   OVERVIEW
   1. Introduction
   2. Motivation
   3. Objectives of work
   4. Immunomodulatory study
   a. Protocol
   b. Results
   5. Stress related study
   a. Protocol
   b. Results
   6. in vitro TNFα estimation
   a. Protocol
   b. Results
   7. Inferences & Conclusion
   8. References
   

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3. Immunomodulation
   15/05/2007
   Introduction
   A global term that refers to a wide range of drug or
   immunologic interventions that alter normal or abnormal
   immune responses by
   • Deletion of specific T or B cells
   • Immune deviation
   • Modification of various inflammatory pathways
   (e.g., chemotaxis, adhesins, or intracytoplasmic
   signaling).
   Immunomodulators (agents that exhibit immunomodulatory
   properties) can be categorized into
   1. Immunosuppressants
   2. Immunostimulants
   

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4. 15/05/2007
   Introduction
   Silybum marianum
   Silybum marianum (Blessed milk thistle) is a milk thistle
   which belongs to the Asteraceae family which is a taxon of
   dicotyledonous plants.
   A fairly typical thistle with red and purple flowers and shiny
   green leaves with white veins.
   Originally, a native of Southern Europe through to Asia, it is
   now found throughout the world.
   The medicinal parts of the plant are the seeds and the
   fruits.
   

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5. 15/05/2007
   Introduction
   Silymarin (C25
   H22
   O10
   )
   A plant-derived flavonoid isolated from the seeds and fruits
   of the Milk thistle, Silybum marianum.
   Active principle was first isolated and characterized in
   1968-1974 (Wagner et al, 1968)
   Consists of 4 flavonolignan isomers (Wagner at al, 1974):
   • Silybinin (70-80%)
   • Isosilybinin (5%)
   • Silydianine (10%)
   • Silychristine (20%)
   

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6. 15/05/2007
   Silymarin
   Properties
   1. Hepatoprotective (liver protection)
   2. Antidepressant (alleviate mental depression)
   3. Antioxidant (maintains ROS levels)
   4. Anti-inflammatory (inhibits PG, LTB synthesis; inhibits
   Neutrophil migration)
   5. Chemopreventive (anti-cancer properties)
   6. Immunomodulator
   7. Demulcent (soothes inflamed mucus membranes)
   8. Laxative (stimulates bowel evacuation)
   Introduction
   

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7. 15/05/2007
   Motivation
   The work already done with Silymarin reveals to us its
   multifarious utilities.
   We know that the liver consists of Kuppffer cells
   (specialized macrophages that form part of the RET
   system). Since Silymarin is known to possess liver
   restorative capacities, it can directly be linked to be
   capable of exhibiting immune-stimulating activity.
   This study focuses on determining the effect of
   Silymarin on the Immune system and confirm
   whether it does possess immune-stimulatory
   activity or not.
   Introduction
   Motivation
   

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8. 15/05/2007
   Objectives of Work
   • Study the immunomodulatory activity of
   Silymarin (in vivo)
   • Study the effect of Silymarin on stress-
   induced mice (in vivo)
   • Study the effect of Silymarin on intracellular
   TNF-α secretion by isolated murine
   neutrophils (in vitro)
   Motivation
   Objectives
   of work
   Introduction
   

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9. 15/05/2007
   Animals
   Before Immunophenotyping
   or Intracellular Cytokine
   estimation can be performed,
   the animals need to be
   prepped.
   Grouped into cages with 5/6
   animals per cage and
   marked with picric acid to
   differentiate between the
   mice.
   Grouping
   Head, Back, Tail
   Head-Back, Back-Tail, No Color
   Proper handling of mice
   Immunomodulatory
   Study
   Motivation
   Objectives
   of work
   Introduction
   

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10. 15/05/2007
    Protocol for drug administration
    Ø Challenge with antigen:
    Challenging activates the immune
    system.
    Ø Day 0: Animals were challenged by
    i.p. injection of SRBC (5 × 109
    cells)
    Ø Drug preparation: Using DW and
    Gum Acacia
    Ø Drug Admin: 2hrs after challenge.
    For a period of 7 days.
    Ø Dosage is measured as mg/kg
    body wt. of the mice.
    Immunomodulatory
    Study
    Motivation
    Objectives
    of work
    Introduction
    

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11. 15/05/2007
    Protocol (contd.)
    Ø Challenge with SRBC. Performed to assess primary
    response.
    Ø Cyclosporine (a known immunosuppressant) and
    Levamisole (a known immunostimulant) used as
    negative and positive controls respectively.
    Ø 48hrs after challenge
    (Day 8), blood drawn
    from the ROP.
    Ø Blood processed as
    per the standardized
    protocol and the sample
    analyzed by Flow
    Cytometry.
    Immunomodulatory
    Study
    Motivation
    Objectives
    of work
    Introduction
    

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12. 15/05/2007
    Study of Immunomodulatory activity
    Immunomodulatory activity of Silymarin was studied by
    using the following technique:
    IMMUNOPHENOTYPING AND INTRACELLULAR
    CYTOKINE ESTIMATION BY FLOW CYTOMETRY
    Both IMMUNOPHENOTYPING and INTRACELLULAR
    CYTOKINE ESTIMATION focus on the assessment of
    lymphocyte populations in Acquired immunity.
    Immunomodulatory
    Study
    Motivation
    Objectives
    of work
    Introduction
    

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13. 15/05/2007
    Flow Cytometry
    PRINCIPLE
    • Uses principles of light scattering, light excitation and
    emission of flourochrome molecules to generate
    specific multi-parametric data from particles and cells
    in the size range of 0.5µm to 40µm diameter.
    • Lasers are most often used as a light source
    WORKING
    • Each suspended particle passing through the beam
    scatters the light in some way. A combination of
    scattered and fluorescent light is picked up by the
    detectors and by analyzing fluctuations in brightness
    at each detector (one for each fluorescent emission
    peak) it is then possible to extrapolate various types of
    information about the particles.
    Immunomodulatory
    Study
    Motivation
    Objectives
    of work
    Introduction
    

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14. 15/05/2007
    Immunophenotyping
    A method that involves determining the cells of the immune
    system by phenotyping them based on the cell surface
    markers.
    The surface markers analyzed in this study were:
    1. CD8+ (T-cytotoxic cells)
    2. CD4+ (T-helper cells)
    To determine the presence of a particular type of cell, the
    surface markers are tagged with flourochromes. When run
    in a flow cytometer, the cells are separated and counted
    based on the attached flourochrome.
    Immunomodulatory
    Study
    Motivation
    Objectives
    of work
    Introduction
    

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15. 15/05/2007
    Intracellular Cytokine Estimation
    Cytokines : Signaling compounds secreted by Immune
    cells, which help in regulating the activity of the immune
    system by autocrine, paracrine and endocrine routes.
    Estimation of the secreted cytokines (extracellular) is not
    practically possible because they are not localized.
    On the other hand, intracellular cytokines are localized
    within the Golgi and hence can be targeted with
    Flourochrome + MAbs directed toward the cytokines.
    The cytokine analyzed in this study was IFNγ.
    Immunomodulatory
    Study
    Motivation
    Objectives
    of work
    Introduction
    

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16. 15/05/2007
    The flourochromes used for the experiments were:
    1. Flourescein Iso Thio Cyanate (FITC)
    2. Phycoerythrin (PE)
    The flourochromes were attached to monoclonal
    antibodies directed against the cell surface markers,
    namely, CD4+, CD8+ and CD244 and the cytokines,
    namely, IL12, IFNγ and TNFα
    The combinations of flourochromes and MAbs used were:
    • FITC – anti-CD4+
    • PE – anti-CD8+
    • FITC – anti-IFNγ
    Flourochromes
    Immunomodulatory
    Study
    Motivation
    Objectives
    of work
    Introduction
    

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17. 15/05/2007
    Results
    Immunophenotyping
    Immunomodulatory
    Study
    Motivation
    Objectives
    of work
    Introduction
    

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18. 15/05/2007
    Results
    Intracellular Cytokine Estimation (IFNγ)
    Immunomodulatory
    Study
    Motivation
    Objectives
    of work
    Introduction
    

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19. 15/05/2007
    Stress
    Stress
    The constellation of events that begins with a stimulus
    (stressor), which precipitates a reaction in the brain (stress
    perception) that subsequently results in the activation of
    certain physiologic systems in the body (stress response).
    It refers to the condition where coping with the various
    actual or perceived stimuli alters the homeostatic state of
    the organism including behavioral, endocrine and
    immunological changes.
    2 types of stress are:
    a. Acute Stress
    b. Chronic Stress
    Immunomodulatory
    Study
    Motivation
    Objectives
    of work
    Introduction
    

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20. 15/05/2007
    Stress & the Immune System
    All lymphocytes have adrenergic receptors. Differential
    density and sensitivity of these receptors on lymphocytes
    may affect responsiveness to stress
    Sympathetic fibers release a wide variety of substances
    that influence immune responses by binding to receptors
    on white blood cells.
    Acute stress generally has positive effect while chronic
    stress typically provokes immunosuppression.
    Psychological stress raises catecholamine levels, which
    suppresses the immune system.
    Stress
    Immunomodulatory
    Study
    Motivation
    Objectives
    of work
    Introduction
    

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21. 15/05/2007
    Protocol
    Ø Mice subjected to restraint stress in well-ventilated 50 ml
    polypropylene tubes for 6 hours during the light cycle
    during the experimental period of 12 days.
    Ø After 6 hours of restraint, mice removed from tubes. 2
    hours after de-stressing, drug is administered.
    Ø After 12 days, blood drawn from ROP and processed as
    per the standardized procedure and analyzed by flow
    cytometry.
    Stress
    Immunomodulatory
    Study
    Motivation
    Objectives
    of work
    Introduction
    Ø Groups:
    Naïve control (not
    subjected to any
    stress);
    Restraint stress
    (RST) control;
    RST + Drug
    

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22. 15/05/2007
    Results
    Immunophenotyping (CD8+/CD4+)
    Stress
    Immunomodulatory
    Study
    Motivation
    Objectives
    of work
    Introduction
    

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23. 15/05/2007
    Immunophenotyping (CD244)
    Results
    Stress
    Immunomodulatory
    Study
    Motivation
    Objectives
    of work
    Introduction
    

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24. 15/05/2007
    Intracellular Cytokine Estimation (IFNγ, IL12)
    Results
    Stress
    Immunomodulatory
    Study
    Motivation
    Objectives
    of work
    Introduction
    

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25. 15/05/2007
    Intracellular Cytokine Estimation (TNFα)
    Results
    Stress
    Immunomodulatory
    Study
    Motivation
    Objectives
    of work
    Introduction
    

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26. 15/05/2007
    Motivation
    Objectives
    of work
    Introduction
    in vitro TNF-α secreted by
    Neutrophils
    Neutrophils are the most abundant type of WBCs and
    are the first immune system cells to rush to the site of
    an infection. Neutrophils are known as phagocytes
    because they engulf foreign organisms and use
    prepackaged chemicals to destroy them.
    TNFα (Tumor Necrosis Factor) is a cytokine involved
    in systemic inflammation and is a member of a group
    of cytokines that stimulate the acute phase reaction.
    On the in vitro activation of neutrophils by an agent,
    TNFα is secreted.
    The measure of the amount of TNFα secreted is a
    good indicator of the level of activation of the immune
    system.
    Stress
    Immunomodulatory
    Study
    in vitro TNFα
    

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27. 15/05/2007
    Motivation
    Objectives
    of work
    Introduction
    Protocol
    Blood is drawn from naïve mice for this purpose. It is
    processed and the neutrophils are isolated.
    The isolated supernatant is incubated along with LPS
    (Lipopolysaccharide - derived from E. coli) and the
    drug at varying doses in a CO2
    incubator.
    A separate group called LPS control was taken, to
    which no drug was added.
    Following the 3 hr incubation, the flourochrome
    tagged anti – TNFα MAb was added and the sample
    was run in the flow cytometer to determine TNFα
    levels.
    Stress
    Immunomodulatory
    Study
    in vitro TNFα
    

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28. 15/05/2007
    Motivation
    Objectives
    of work
    Introduction
    Results
    Stress
    Immunomodulatory
    Study
    in vitro TNFα
    

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29. 15/05/2007
    Motivation
    Objectives
    of work
    Introduction
    Results
    Stress
    Immunomodulatory
    Study
    in vitro TNFα
    

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30. 15/05/2007
    Immunomodulatory
    Study
    Motivation
    Objectives
    of work
    Introduction
    Inferences & Conclusion
    From the Immunophenotyping and Intracellular
    Cytokine estimation data, Silymarin displayed
    commendable dose dependent
    immunopotentiating activity.
    Most effective dose was 100 mg/kg body wt.
    Its immune-potentiating activity was confirmed by
    studying TNF-α secretion by activated Neutrophils in
    vitro.
    These studies actuated the test of its activity on mice
    subjected to stress. The deleterious effect of chronic
    stress was countered by Silymarin, which was able to
    hype up the immune parameters to respectable
    levels, thus demonstrating good stress-busting
    activity.
    Stress
    in vitro TNFα
    Conclusion
    

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31. 15/05/2007
    Stress
    Immunomodulatory
    Study
    Motivation
    Objectives
    of work
    Introduction
    in vitro TNFα
    References
    References
    Conclusion
    Wagner H., Horhammer L., Munster R., 1968, The chemistry
    of silymarin (silybin), the active principle of the fruits of
    Silybum marianum (L.) Gaertn. (Carduus marianus) (L.).
    Arzneimittelforschung, 18, 688-696
    Wagner V.H., Diesel P., Seitz M., 1974, Chemistry and
    analysis of silymarin from Silybum marianum (L.) Gaertn,
    Arzneimittelforschung, 24, 466-471
    

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32. 15/05/2007
    Acknowledgements
    Dr. K. Ramasamy
    Dr. R. S. David Paul Raj
    Dr. Sarang Bani
    Ms. Beenish Khan
    Ms. Prabhavathy Devan
    Mr. Deepak Bhagwat
    Ms. Anjali Pandey
    Ms. Kiranjeet
    Ms. Romaisa Latef
    Thank you !!
    

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