bahan kuliah eksperimentasi immunofarmakologi

January 29, 2018 | Author: Anonymous | Category: Science, Health Science, Immunology
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Immunopharmacology: and pharmacology.

intersection of immunology

Focuses on drugs that affect the immune system, whether to suppress it, activate it, or manipulate it in some way. The most well-known immunopharmacology agents include anti-rejection drugs and vaccines.

Fluorescence-activated cell sorting (FACS) Flowcytometry using a BD FACS Calibur.

• Flow-FISH (fluorescent in-situ hybridization) is a cytogenetic technique to quantify the copy number of specific repetitive elements in genomic DNA of whole cell populations via the combination of flow cytometry with cytogenetic fluorescent in situ hybridization staining protocols

Gel electrophoresis apparatus – An agarose gel is placed in this buffer-filled box and electrical field is applied via the power supply to the rear. The negative terminal is at the far end (black wire), so DNA migrates toward the camera. Classification


Other techniques


Capillary electrophoresis SDS-PAGE Two-dimensional gel electrophoresis Temperature gradient gel electrophoresis

P53 ; RT-PCR

Immunocytochemistry vs. immunohistochemistry Immunocytochemistry differs from immunohistochemistry in that the former is performed on samples of intact cells that have had most, if not all, of their surrounding extracellular matrix removed. This includes cells grown within a culture, deposited from suspension, or taken from a smear. In contrast, immunohistochemical samples are sections of biological tissue, where each cell is surrounded by tissue architecture and other cells normally found in the intact tissue. Counterstains After immunohistochemical staining of the target antigen, a second stain is often applied to provide contrast that helps the primary stain stand out. Many of these stains show specificity for discrete cellular compartments or antigens, while others will stain the whole cell. Both chromogenic and fluorescent dyes are available for IHC to provide a vast array of reagents to fit every experimental design, and include: hematoxylin, Hoechst stain and DAPI are commonly used.

Aktivitas Senyawa Semisintetik Kuasinoid dari Buah Makasar (Brucea javanica [L.] Merr) sebagai Antikanker dengan Target Protein P53, Bcl-2, Kaspase-3, COX-2 dan c-Myc


Ab Primer P21 ;C-myc; Bcl2

Components of the immune system • White blood cells • Lymphoid organ – Primary lymphoid organ: • Bone marrow & fetal liver – origin of all immune cells – site for development and education of B cells

• Thymus: – site for development and education of T cells

– Secondary lymphoid organ • Lymph nodes, spleen, lymphoid tissue – induction sites for immune responses

• Body tissues • effector sites for immune responses


All white blood cells originate from the bone marrow

Growth and differentiation factors (cytokines) produced by and present on bone marrow stromal cells determine the type of white blood cell that will emerge, as well as their relative numbers.


Blood cells derived from bone marrow cells

Adaptive imm Innate imm


Blood cells migrate through blood and lymph nodes or home to tissues

Molecules involved for antigen recognition • B cell receptor & product – antibodies (Abs): immunoglobulin (Ig)

• T cell receptor (TCR) – TCR a/b (type II), g/d (type I)

• Major histocompatibility complex (MHC)/HLA – Class I – Class II

Immune responses • Natural/innate/non-specific – Humoral: type I Interferon (IFN-a/b), lysozyme, complement (C) – Cellular: phagocytes, NK cells

• Adaptive/acquired/specific – Humoral: Abs: IgM, IgG, IgA, IgE, IgD – Cellular: T cells: • CD4+ Th, CD8+CTL, CD4+CD25+ T reg.

Lymphocytes: the B and T cells

The cells in adaptive immune responses  Antigen specific lymphocytes  Effector cells  Specialized accessory cells

Lymphocytes  Capable of specifically recognizing and distinguishing different antigenic determinants  Responsible for the defining characteristics of adaptive IR i.e. - specificity - memory

Normal Blood Cell Counts

Experiment Design: Invitro; Invivo




CD8 T cell


cytotoxic T cells

4 2




antigen presenting cell (macrophage, dendritic cell)

CD4 T helper cell

primed CD4 T helper cell

4 IL-2

B cell

plasma cells


1 X A


CD8 T cell 4






BIL-1 antigen presenting cell





CD4 T helper cell


primed CD4 T helper cell


X cytokines

cytotoxic T cells

plasm a cells

Corticosteroid Actions Inhibition of IL-1 and TNF gene expression and synthesis Decreased activation of T lymphocytes by decreasing IL-1 release Decreased neutrophil functions esp chemotaxis Decreased antibody production (high doses) Decreased release of kinins and proinflammatory eicosanoids (prostaglandins and leukotrienes)

Corticosteroid Immunosuppression • Decreased cell-mediated immune reactions that mediate rejection of organ transplants – Mechanisms: • decreased activation of T lymphocytes by inhibition of IL-1 synthesis by macrophages • decreased lymphocyte mobilization out of lymphoid organs (lymphopenia)

TOLEROGEN (reviewed by Mowat, 1987) Oral administration of protein antigens to naive adult animals has been shown repeatedly to induce antigen-specific hyporesponsiveness of systemic immunity (oral tolerance) (reviewed by Mowat, 1987) and may play an important role in the prevention of food hypersensitivity disease. Previous studies have shown

The mucosal response to infection and regulation of mucosal immune responses Commensal bacteria  recognize by epithelial cells EC secretion of cytokines inhibit the expression of proinflammatory cytokines by DC and macrophage  DC & macrophages

secrete signals for the expansion and/or survival of T cells with regulatory capacities, including regulatory T cells, T regulatory type 1 (TR1) cells, T helper 2 (TH2) cells and TH3 cells.

Musketeers Course October 2008


Immunostimulatory Cytokines Interleukins IL-2 (enhance antitumor actions of cytotoxic T cells and NK cells)

Colony Stimulating Factors G-CSF (neutropenia) and GM-CSF (bone marrow transplant patients)

Interferons (uses) alpha (anticancer uses) beta (relapsing type multiple sclerosis) gamma (chronic granulomatous disease)

Interferon Uses Interferon Alpha (prod. by leukocytes) (antiviral, antiproliferative) malignant melanoma, renal cell carcinoma, hairy cell leukemia, Kaposi’s sarcoma

Interferon Beta (prod. by fibroblasts) (antiviral, antiproliferative) relapsing type MS

Interferon Gamma (prod. by lymphocytes) (stimulates NK cells and macrophages) chronic granulomatous disease

Other Hematopoetic Growth Factors • Erythropoietin alpha (Epoetin alpha) (Procrit®) – Produced by recombinant DNA technology – Stimulates division and differention of erythroid progenitor cells – Used for anemia due to renal failure or cancer chemotherapy – Adverse effects include hypertension, headache, hypersensitivity reactions are rare

• Darbopoetin alpha (Aranesp®) – Recombinant long-acting erythropoetin (3X epoetin)

Cytokine Inhibitors • TNF inhibitors (disease modifiers to treat rheumatoid arthritis) – Etanercept (Enbrel) • Recombinant version of TNF receptor – Infliximab (Remicade) • Chimeric human/murine anti-TNF monoclonal antibody

• Anakinra (Kineret) – Human IL-1 receptor antagonist – Disease modifier agent for Rheumatoid arthritis

Other Immunostimulants • Thymic Hormones – Improve primary immune deficiency in children

• Synthetic Stimulants – Levamisole stimulates phagocytosis and T cell production of cytokines

• Adjuvants of bacterial origin – BCG is viable strain of Mycobacterium bovis that enhances macrophage activity – BCG used for bladder cancer and melanomas

Targeted Immunotherapy • Antibody-mediated delivery systems • Radiolabeled antibodies • Types of antibodies in trials – Anti-CD20 for B cell lymphomas – Anti-vascular endothelial cell growth factor – Anti-fibroblast growth factor – Anti-body to F19 on surface of activated fibroblasts

New Approaches for Tolerance • Interference with costimulatory signals required for T cell activation – Two signals required for T cell activation • Signal 1 via T cell receptor • Signal 2 via costimulatory receptor-ligand pair

• Antibodies to costimulator receptors (on T cell) or ligands (on antigen presenting cell) – Anti-CTLA4 (blocks B7 binding to T cell CD28) – Anti-CD40 (inhibits macrophage and endothelial activation by blocking T cell CD40 ligand binding to macrophage CD40)


Different Principles in drugs used for cancer vs immunosupressant


Teknik yang sering digunakan a.l :

Faga, Bakteri, Yeast / Fungi, VIRUS ? Higher eukaryotes

KIT DNA /RNA : Sesuaikan petunjuk penggunaan dari Manufakturing


Denaturasi 94oC Anealing primer 55oC Sintesis 72oC


Produk dominan

Efek Hepatoprotektor, Nefroprotektor, dan Imunomodulator Ubi jalar (Ipomoea batatas) pada Tikus Putih Jantan (Rattus novergicus) Galur Sprague Dawley Sarmalina et al., 2012, Laporan Hasil Penelitian

28 ekor hewan coba; dibagi 7 kelompok; satu kontrol (K). Hari ke 7 diberi parasetamol dosis tinggi (P1), Diberi bakteri salmonella p.o 107 cfu (P2), Ekstrak Ipomea (P3), Ekstrak Ipomea + Salmonella (P4), Ipomea + Parasetamol (P5-6). Hari ke 21-28. Narkose, darah intrakardiak, Sentrifugasi serum-darah.

(Persetujuan Etikal Klirins: FK Unsri Oktober 2012)

Efek Hepatoprotektor, Nefroprotektor, dan Imunomodulator Syrup Ubi jalar (Ipomoea batatas) pada Tikus Putih Jantan (Rattus novergicus) Galur Sprague Dawley Sarmalina et al., 2013, Laporan Hasil Penelitian

28 ekor hewan coba; dibagi 7 kelompok; satu kontrol (K). Hari ke 7 diberi parasetamol dosis tinggi (P1), Diberi bakteri salmonella p.o 107 cfu (P2), Ekstrak Ipomea (P3), Ekstrak Ipomea + Salmonella (P4), Ipomea + Parasetamol (P5-6). Hari ke 21-28. Narkose, darah intrakardiak, Sentrifugasi serum-darah.

(Pemeriksaan Histopatology Ginjal ( RS Kharitas Des 2013)

Pengukuran serum (IL-4) Elisa, SGPT, SGOT, Kreatinin dan Ureum dengan Kolori meter. Analisis data anova test dengan signifikansi p
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