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IMMUNOBIOLOGY And EXPERIMENT. Immunopharmacology: intersection of immunology and pharmacology.pharmacology The most well-known immunopharmacology agents.

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Presentation on theme: "IMMUNOBIOLOGY And EXPERIMENT. Immunopharmacology: intersection of immunology and pharmacology.pharmacology The most well-known immunopharmacology agents."— Presentation transcript:

1 IMMUNOBIOLOGY And EXPERIMENT

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

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

5 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 protocolscytogeneticDNAflow cytometryfluorescent in situ hybridization

6 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. ClassificationElectrophoresis Other techniques Related Capillary electrophoresis SDS-PAGE Two-dimensional gel electrophoresis Temperature gradient gel electrophoresis

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8 P53 ; RT-PCR

9 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.immunohistochemistrysamples extracellular matrixculture suspensionsmearbiological 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. hematoxylinHoechst stainDAPI

10 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; Bcl2COX-2

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13 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

14 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. All white blood cells originate from the bone marrow cells

15 Blood cells derived from bone marrow cells cells Innate imm Adaptive imm

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

17 Molecules involved for antigen recognition B cell receptor & product – antibodies (Abs): immunoglobulin (Ig) T cell receptor (TCR) – TCR  (type II)   /  (type I) Major histocompatibility complex (MHC)/HLA – Class I – Class II

18 Immune responses Natural/innate/non-specific – Humoral: type I Interferon (IFN-  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.

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25 Lymphocytes: the B and T cells

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

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

28 Normal Blood Cell Counts

29 Experiment Design: Invitro; Invivo

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34 antigen presenting cell (macrophage, dendritic cell) cytotoxic T cells Antigen CD4 T helper cell primed CD4 T helper cell CD8 T cell plasma cells IL-1 IL-2 MAJOR STEPS IN IMMUNE RESPONSES B cell

35 Antigen antigen presenting cell CD4 T helper cell primed CD4 T helper cell CD8 T cell cytotoxic T cells plasm a cells IL-1 IL-2 cytokines SITES OF ACTION OF IMMUNOSUPPRESSIVE DRUGS X X X X X A B DD E C X

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42 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)

43 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

44 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)

45 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

46 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

47 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

48 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)

49 TUMOR CELL

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54 Different Principles in drugs used for cancer vs immunosupressant

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60 TEKNIK DASAR BIOLOGI MOLEKULER

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62 Faga, Bakteri, Yeast / Fungi, VIRUS ? Higher eukaryotes KIT DNA /RNA : Sesuaikan petunjuk penggunaan dari Manufakturing

63 PROTOKOL ISOLASI DNA/ RNA

64 Denaturasi 94 o C Anealing primer 55 o C Sintesis 72 o C

65 72 o C

66 Produk dominan

67 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 10 7 cfu (P2), Ekstrak Ipomea (P3), Ekstrak Ipomea + Salmonella (P4), Ipomea + Parasetamol (P5-6). Hari ke Narkose, darah intrakardiak, Sentrifugasi serum-darah. (Persetujuan Etikal Klirins: FK Unsri Oktober 2012)

68 Pengukuran serum (IL-4) Elisa, SGPT, SGOT, Kreatinin dan Ureum dengan Kolori meter. Analisis data anova test dengan signifikansi p<0,05 Pengukuran: Kadar SGPT, SGOT, kreatinin dan ureum. sekresi interleukin 4 (IL-4). Laboratorium Kes Daerah Palembang (Akreditasi Lab Pengujian) Refference; Roller, M., Rechkemmer, G., and Watzl, G

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72 Bakteri, tanaman mamalia mamalia

73 Dengan / tanpa SDS

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75 Hubungan antara Laju migrasi dan konformasi DNA plasmid Sirkuler, linier, superkoil DNA Kromosom

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78 Gel Poliakrilamid SDS ( vertical )

79 ss DNA – ss DNA ( Southern ) ss DNA – RNA RNA – RNA ( Northern ) Pembentukan duplek antara PROTEIN - ANTIBODI ( Western ) Pembentukan duplek antara dua untai asam nukleat yang komplemen

80 DOUBLE HELIX DNA UNTAI 1 UNTAI 2

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82 TCCGA CCTGA CCCGAAT GGACT GGGCTTA CCCTG CCTGACCCGAATGGC GGCTTACCGTTAAGTTCC 1 2 CCTGACCCGAATGGC GGACTGGGCTTACCG 3

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88 Denaturasi 94 o C Anealing primer 55 o C Sintesis 72 o C

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