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 cytogenetic fluorescent in situ hybridization flow cytometry staining protocols with

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 performed on samples in that the former is 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

All white blood cells originate from the bone marrow

cells 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

cells Adaptive imm Innate 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

Experiment Design: Invitro; Invivo

MAJOR STEPS IN IMMUNE RESPONSES Antigen 1 antigen presenting cell (macrophage, dendritic cell) 2 IL-1 IL-2 CD8 T cell 4 3 IL-2 CD4 T helper cell primed CD4 T helper cell 4 IL-2 B cell cytotoxic T cells plasma cells

SITES OF ACTION OF IMMUNOSUPPRESSIVE DRUGS Antigen A X 1 B 2 X IL-1 antigen presenting cell IL-2 X E X C D 3 X IL-2 CD4 T helper cell D cytokines primed CD4 T helper cell X 4 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 pro inflammatory 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 94 o C Anealing primer 55 o C Sintesis 72 o C

72 o C

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 10 7 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 10 7 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

SGOT,

serum

Kreatinin

dan

(IL-4) Elisa, Ureum SGPT,

dengan Kolori meter. Analisis data anova test dengan signifikansi

p

<0,05 Pengukuran:

kreatinin dan ureum. sekresi interleukin 4 (IL-4).

Kadar SGPT, SGOT, 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

asam nukleat dua untai

yang komplemen

ss DNA – ss DNA ( Southern ) ss DNA – RNA RNA – RNA ( Northern ) Pembentukan duplek antara PROTEIN - ANTIBODI ( Western )

UNTAI 1 UNTAI 2 DOUBLE HELIX DNA

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

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