bahan kuliah eksperimentasi immunofarmakologi

Report
IMMUNOBIOLOGY
And EXPERIMENT
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
Electrophoresis
Other techniques
Related
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
COX-2
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
cells
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.
cells
Blood cells derived from bone marrow cells
Adaptive imm
Innate imm
cells
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
MAJOR STEPS IN IMMUNE RESPONSES
Antigen
IL-2
CD8 T cell
1
cytotoxic
T cells
4
2
3
IL-1
IL-2
antigen
presenting
cell
(macrophage,
dendritic cell)
CD4
T helper
cell
primed
CD4
T helper
cell
4
IL-2
B cell
plasma
cells
SITES OF ACTION OF
IMMUNOSUPPRESSIVE DRUGS
Antigen
1
X
A
IL-2
CD8 T cell
4
X
E
C
X
2
BIL-1
antigen
presenting
cell
X
D
X
3
IL-2
CD4
T helper
cell
D
primed
CD4
T helper
cell
4
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
45
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)
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 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<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. 2004.
Bakteri, tanaman
mamalia
Gel Agarosa
Gel Poliakrilamid :
Dengan / tanpa SDS
Hubungan antara Laju migrasi
dan konformasi DNA plasmid
Sirkuler, linier, superkoil
DNA
Kromosom
Gel Poliakrilamid SDS ( vertical )
Pembentukan duplek antara dua untai
asam nukleat yang komplemen
ss DNA – ss DNA ( Southern )
ss DNA – RNA
RNA – RNA ( Northern )
Pembentukan duplek antara
PROTEIN - ANTIBODI ( Western )
UNTAI 2
UNTAI 1
DOUBLE HELIX DNA
1
2
TCCGA
CCTGA
CCCGAAT
GGACT CCCTG GGGCTTA
CCTGACCCGAATGGC
GGCTTACCGTTAAGTTCC
CCTGACCCGAATGGC
GGACTGGGCTTACCG
3
Denaturasi 94oC
Anealing primer 55oC
Sintesis 72oC

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