immunobiology and experiment. immunopharmacology: intersection of immunology and...
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IMMUNOBIOLOGY
And EXPERIMENT
Immunopharmacology: intersection of immunology and 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.
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 ElectrophoresisOther techniques
Related
Capillary electrophoresisSDS-PAGETwo-dimensional gel electrophoresis
Temperature gradient gel electrophoresis
P53 ; RT-PCR
Immunocytochemistry vs. immunohistochemistryImmunocytochemistry 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.CounterstainsAfter 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
Microscope
Ab Primer P21 ;C-myc; Bcl2COX-2
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
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 marrowcells
Blood cells derived from bone marrow cells
cells
Innate immAdaptive imm
Blood cells migrate through blood and lymph nodes or home to tissues
cells
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
PCR , IHC, ELISA, FACS-FlowsitometriExperiment Design: Invitro; Invivo
antigenpresenting cell (macrophage,dendritic cell)
cytotoxicT cells
Antigen
CD4T helpercell
primedCD4T helpercell
CD8 T cell
plasmacells
1
2 3
4
4
IL-1 IL-2
IL-2
IL-2
MAJOR STEPS IN IMMUNE RESPONSES
B cell
Antigen
antigenpresenting cell
CD4T helpercell
primedCD4T helpercell
CD8 T cell
cytotoxicT cells
plasmacells
1
2 3
4
4
IL-1 IL-2
IL-2
cytokines
SITES OF ACTION OF IMMUNOSUPPRESSIVE DRUGS
X
X
X X
XA
BD D
EC
X
Immunostimulatory Cytokines
InterleukinsIL-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 UsesInterferon Alpha (prod. by leukocytes)
(antiviral, antiproliferative)malignant melanoma, renal cell carcinoma, hairy cell
leukemia, Kaposi’s sarcomaInterferon 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)
TUMOR CELL
Different Principles in drugs used for cancer vs immunosupressant
TEKNIK DASAR BIOLOGI MOLEKULER
Teknik yang sering digunakan a.l :
Faga, Bakteri, Yeast / Fungi, VIRUS ?Higher eukaryotes
KIT DNA /RNA : Sesuaikan petunjuk penggunaan dari Manufakturing
PROTOKOL ISOLASI DNA/
RNA
Denaturasi 94oC Anealing primer 55oC
Sintesis 72oC
72oC
Produk dominan
Efek Hepatoprotektor, Nefroprotektor, dan
ImunomodulatorUbi jalar (Ipomoea batatas) pada
Tikus Putih Jantan(Rattus novergicus) Galur Sprague
Dawley Sarmalina et al., 2012, Laporan
Hasil Penelitian28 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)
Pengukuran serum (IL-4) Elisa, SGPT, SGOT, Kreatinin dan Ureum dengan Kolori meter. Analisis data anova test dengan signifikansi p<0,05Pengukuran: 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. 2004.
Bakteri, tanaman mamalia
Gel AgarosaGel Poliakrilamid :
Dengan / tanpa SDS
Hubungan antara Laju migrasi dan konformasi DNA plasmid
Sirkuler, linier, superkoil
DNA Kromosom
Gel Poliakrilamid SDS ( vertical )
ss DNA – ss DNA ( Southern )
ss DNA – RNARNA – RNA ( Northern )
Pembentukan duplek antaraPROTEIN - ANTIBODI ( Western )
Pembentukan duplek antara dua untai asam nukleat yang komplemen
DOUBLE HELIX DNA
UNTAI 1 UNTAI 2
TCCGA CCTGA CCCGAAT GGACT GGGCTTA CCCTG
CCTGACCCGAATGGC GGCTTACCGTTAAGTTCC
1
2
CCTGACCCGAATGGCGGACTGGGCTTACCG
3
Denaturasi 94oC Anealing primer 55oC
Sintesis 72oC