Manifestation of Novel Social Challenges of the European Union in the Teaching Material of Medical Biotechnology Master’s Programmes at the University of Pécs and at the University of Debrecen

(1)

Manifestation of Novel Social Challenges of the European Union in the Teaching Material of

Medical Biotechnology Master’s Programmes

at the University of Pécs and at the University of Debrecen

Identification number: TÁMOP-4.1.2-08/1/A-2009-0011

(2)

ANTI-CYTOKINE THERAPY

Zoltan Balajthy

Molecular Therapies- Lecture 8

in the Teaching Material of

Medical Biotechnology Master’s Programmes

at the University of Pécs and at the University of Debrecen

Identification number: TÁMOP-4.1.2-08/1/A-2009-0011

(3)

Learning objectives of chapter 8. As the reason of many common diseases is the appearance of inflammatory process in our body (rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis,

insulin-dependent diabetes melitus, psoriasis, sepsis), the significance of anti-cytokine therapy is interpreted through the understanding both the processes and mechanisms of inflammation.

Topics in chapter 8

8.1. Development of septic shock and subsequent failure of organ Migration of neutrophil from vascular space to tissues Effects of IL-1 , -β and TNF

Stream Of cytolkines in sepsis 8.2. Development of inflammatory response

Synthesis of lipid mediators

Iniciation of cytokine synthesis of inflammation Synthesis of NO controlled by cytokine

8.3. Role of the Liver in Maintenance of Homeostasis

Acute phase response, acute phase proteins (APP) 8.4. Time-Course of the Inflammatory Response During Sepsis

Endothelial Activation, Coagulation and Fibrin Clot Formation

8.5. Collapse of Homeostasis 8.6. Anti-Cytokine Therapy

8.7. Cancer therapy and monoclonal antibodies 8.8. How Can Immunity Be Applied for Treatment

(4)

Cytokines share the following characteristics: they have very high affinity for their specific receptor; they act in a paracrine or autocrine fashion and are potent at picomolar to nano- molar concentrations.

Today, cytokines have been classified into several classes based on structural or functional similarities: monokines, lymphokines, interleukins, colony stimulating factors, interferons, tumor necrosis factors and chemokines.

Characteristic feature of cytokines: they take part of natural immunity, hematopoiesis, activation of lymphocyte, differentiation and initiation of inflammation.

In many common diseases cytokine overproduction would happen giving the key targets for anti-cytokine treatments.

Cytokine receptor family: hematopoietin, interferon, TNF, IL-1, TGF-β and chemokine receptors.

Characterization of cytokines

Cytokines are proteins of size ranging from 50 to 500 amino-acids in average, and are major molecular messengers for communication between cells. More than 200 cytokines

have been identified and cloned.

(5)

Infection, Trauma, Burns Relase of Endotoxines or Ecosanoids, Complement

Activation

Inflammatory Cytokine Storm

Small Mediator Molecules, vascular resistance ↓

Hypotension, Acidosis, Decreased Tissue Oxygenation

Myocardial Suppression, Refractory Shock, Organ Ischemia

Multiple Organ Failure, Disseminated Intravascular Coagulation, Death 8.1. Development of Septic Shock and Subsequent Failure of Organ

(6)

endothelium smooth

muscle

systemic circulation TNF

IL-1

extravascular space capillary leak

PAF PG NO ELAM IL-8

■■■ ■■■

●

● ●

● ● ● ● ● ● ● ● ● ●

● ● ●

●

● ● ● ● ● ● ● ● ● ●

●

● ● ● ●

●

● ● ●

neutrophil emigration into tissue

neutrophil degranulation and tissue damage circulating

neutrophils

Migration of Neutrophil From Vascular Space to Tissues

(7)

HYPOTENSION

TISSUE INJURY

ORGAN FAILURE

DEATH TNF

IL-1

IL-6 Stream Of Cytolkines in Sepsis

(8)

Results of IL-1  and β

Fever

Myalgias and Joint Pain Headach

Loss of Appetite and Confusion

Mild Hypertension at Low Concentration of IL-1 Hypotension at Increased Concentration of IL-1 Hemodynamic shock at High Concentration of IL-1

IL-1α, IL-1β, and the (IL-1RA) (interleukin-1 receptor antagonist) play important role in regulation of immune functions and inflammation process.

Both IL-1α and IL-1β are pro-inflammatory cytokines produced by macrophages, monocytes, fibroblasts and dendritic cells

(9)

Effect of Adrimistration of TNF

Core Body Temperature Increases

Neutrophil Count Decreasing at the Early Phase

White Blood Cells Count Increases in the Late Phase Mean Arterial Blood Pressure Decrease (Hypotensison) Coagulation Parameters Increase

Mental Status Declines Myalgias / arthralgias

TNF (Tumor necrosis factor-alpha) is a cytokine involved in systemic inflammation and is a member of a group of cytokines that stimulate the acute phase reaction.

The primary role of TNF is in the regulation of immune cells. TNF is able to induce apoptotic cell death, to induce inflammation, and to inhibit tumorigenesis . A TNF can

bind to TNF-R in homotrimer form that can activate MAPK, NFκB or cell death signaling.

(10)

IL-1 and TNF Synergism

Hypotension ans Schock Prostaglandin Production Cytotoxicity / Tumor necrosis Muscle proteolysis

Lactic acidosis

Anti-cytokines to Reduce Mortality in Sepsis Antibodies to TNF

Soluble Receptors of TNF

IL-1 receptor antagonist

(11)

Inflammatory Cytokines and Anti-Cytokines

(12)

phospholipids of plasma membrane

arachidonate

PLA2, Phospholipases A2 cleaves

leukotrienes (LTA₄ - LTE₄)

lipoxygenases

prostglandin H2 (PGH2)

other prostaglandins (PGD2, PGE2) prostacyclin

(PGI2)

thromboxanes (TXA2)

cyclooxigenase (COX -1) prostaglandin synthetase

prostacyclin synthase

thromboxane synthase

Arachidonate is the major precursor of several classes of signal molecules: prostaglandins, prostacyclins, thromboxanes, and

leukotrienes

8.2. Development of Inflammatory Response Synthesis of Lipid Mediators

(13)

lysed bacterial cells

LPS

LPS binding protein (plasma)

LPS - LPS binding protein complex (LBP)

TLR-4 CD14

LPS-LPB LPS-LPB

NFκB pathway

CD14, CD11/18, TLR-2/TLR-4 LPS Receptors

Inflammatory cytokines

Development of Inflammatory Response I.

r r r

r r r rr r

r

r r r

r r

r r r r

r

lipoteichoic

acid peptidoglycan - lipoteichoic acid

cytoplasm membrane

(TLR) lipopolysaccharide

(LPS)

neutrophil

monocyte

(14)

adaptor protein

β   p65 p50

IκB

IκB proteasomal degradation

p65 p50

p65 p50 transcription nukleus

IκK complex

phosphorilation p p

nuclear translocation receptor

activation

transcription of

inflammatory genes (IL8, IL6, TNF)

Toll-like receptor

NFκB

Iniciation of Cytokine Synthesis of Inflammation

(15)

cytokine

L-arginine

nitric oxide

NO activates

soluble guanylate cyclase

cGMP GTP

degreased cytoplasmic Ca²⁺

receptor

Vasodilatation

Smooth muscle cell

Endothelial cells

iNO synthase

L-arginine

nitric oxide

cGMP

receptor

iNO synthase

cGMP GTP

degreased cytoplasmic Ca²⁺

NO activates

soluble guanylate cyclase

Blood stream

Synthesis of NO controlled by cytokine

(16)

Cells associated with initiating the acut phase response are neutrophils monocytes and macrophages

Released IL-1, TNF and IL-6 reach the central nervous system and the liver.

Acut phase proteins produced by liver cells:

Complement proteins Coagulation proteins Proteinase inhibtors Opsonins

Serum amyloid A (SAA) C-reactive protein (CRP)

8.3. Role of the Liver in Maintenance of Homeostasis, (Inflammation)

(17)

Blood vessel Blood vessel

Bone marrow

Prostaglandins

Cytokines

Fever

Sickness behaviour

IL1β,IL6, TNF

Infection

Cytokines, chemokines prostaglandines

Acute phase response

Monocytes, neutrophils Complement proteins

Coagulation proteins Proteinase inhibtors

Opsonins

Lungs

Liver

Acute Phase Response

(18)

LIVER TNF-

IL-1 IL-6

ACUTE PHASE PROTEINS Complement proteins:

C3, C5 and C1 inhibitor (6-8 X) Coagulation proteins:

Fibrinogen (2-4 x) Von Willebrand factor Protein inhibitors:

Plasminogen activator inhibitor I

2-antiplasmin

Metal-binding proteins:

Haptoglobulin, ceruloplasmin (1.5-2 x) Negative APRs:

albumin, transferin (0.4-0.5 X) Major APRs:

Serum amyolid A (100-1000 X) C-reactive protein

Serum amyloid P component (SAP) Inflammatory mediators that modulate hepatic APR synthesis

(19)

8.4. Time-Course of the Inflammatory Response During Sepsis

(20)

Induction of proinflammatory cytokines

tissue factor

TLR-4 CD

14

TL

R-4

CD 14 LPS-LPB

LPS-LPB

LPS-LBP

Activation of NFκB Pathway 

tissue factor VII a

+

TNF

IL-1

Platelet-activating factor (PAF), arachidonic acid, leukotriens,

Plasminogen activator inhibitor

Direct tissue injury

Microvascular coagulopathy

Gram-negative bacteria

Gram-pozitive

bacteria _receptor^{CD 14}

Toll-like receptor

lipopolysaccharide, endotoxin (LPS)

lipoteichoic acid

inflammatory response

Endothelial Activation, Coagulation and Fibrin Clot Formation I.

(21)

Factor IXa Factor VIIIa + INTRINSIC TENÁZ Thrombin (IIa)

Protrombin (II)

Fibrinogen Fibrin

fibrin clot formation

VII a Tissue Factor + EXTRINSIC TENÁZ (X-ÁZ) Factor Xa

Factor Va + PROTHROMBINASE

Factor VIII +

Factor V

Factor IX

Factor X +

Profactors (V és VIII) activation

Zimogenes (IX és XIII) activation

Coagulation Response

(+)

VII Tissue Factor +

(+) TFPI

Tissue Factor

Pathway Inhibitor (-)

Fibrinolysis

Aktivated pFrofactors Inactivation of (Va és VIIIa)

PAI-1

(Plasminogen activator inhibitor 1) Plasmin

Plasminogen

Plasminogen activators

activated protein C

(-)

Pathways of Procoagulation Anticoagulation Pathway

(22)

Endothelial Activation, Coagulation and Fibrin Clot Formation II.

b

Tissue Factor

c

Factor IXa Factor VIIIa + Thrombin

Factor IX Prothrombin

Fibrinogen Fibrin Fibrin Clot

endothelium

TLR-4 CD

14

TL

R-4

CD 14

TLR-4

14 CD

TLR-4

CD

14 LPS-LPB

LPS-LPB LPS-LPB LPS-LPB

LPS-LBP

Inflammatory Response to Infection

Thrombotic Response to Infection

Fibrinolytic Response to Infection organism

Tissue Factor VII a

+

Factor X Factor Xa

Factor Va +

Factor VIII Factor V Activation of

NFκB Pathway 

(23)

b

Tissue Factor

c

Factor IXa Factor VIIIa +

Thrombin Protrombin

Fibrinogen Fibrin

endothelium

TLR

-4 CD

14

-4RTL

CD 14

TLR-4

CD

TLR-4 14

CD14 LPS-LPB

LPS-LPB LPS-LPB LPS-LPB

LPS-LBP

organism

Tissue Factor VII a

+ Factor Xa Factor Va +

Factor VIII Factor V

TAF1

(thrombin activated fibrinolysis inhibitor)

PAI-1

(plasminogen activator inhibitor 1) TNF

IL6, IL1 TNF

IL6, IL1 inflammatory

response

Coagulation, Fibrin Clot Formation and Inhibition of Fibrinolysis

Fibrinolytic Response to Infection

(24)

b

Tissue Factor

c

Factor IXa Factor VIIIa +

Thrombin Prothrombin

Fibrinogen Fibrin

endothelium

TLR-4 CD

14

TL

R-4

CD 14

TLR-4

CD

TLR-4 14

CD14 LPS-LPB

LPS-LPB LPS-LPB

LPS-LPB

LPS-LBP

organism

Tissue Factor VII a

+ Factor Xa

Factor Va +

Factor VIII Factor V

TAF1

(thrombin activated fibrinolysis inhibitor)

PAI-1

(plasminogen activator inhibitor 1) TNF

IL6, IL1 TNF

IL6, IL1

thrombospondin protein S protein C

Protein C Catalyzed Inactivation

Degreased

rolling

activated protein C

Endogenous Activated Protein C Has Multiple Mechanisms of Action

Fibrinolytic Response to Infection

(25)

TLR -4 CD

14

T

-4LR

CD 14 LPS-LPB

LPS-LPB

HMGB1

RAGE VCAM ICAM NF-κB

PAI-1 tPA TNF-

MCP-1 IL-8

TNF-

MCP-1 IL-8

ICAM

Neutrophil  adherence Regulation of fibrinolysis

TNF-

IL-1a IL-1b

IL-6 Pro-inflammatory cytokines and chemokines monocytes

macrophages LPS

Contribution of high-mobility group box 1(HMGB1) to sepsis

(26)

Proinflammatory mediators Tissue factor expression Thrombin production Endothelial injury

Increased PAI-1 Increased TAF

Reduced Protein C

PAI : plasminogen activator inhibitor

TAFI: thrombin activated fibrinolysis inhibitor

Homeostasis

8.5 Collapse of Homeostasis

(27)

Infection SIRS Sepsis

Sever Sepsis

Septic Shock

Development of Septic Shock and Organ Failure

Mitochondrial Disfunction Organ

Disfunction

Multiple Organ Failer

Death

Microvascular Coagulation/

Thrombosis

SIRS: systemic inflammatory response syndrome

(28)

antibodies (anti-IL-6R)

Anticomplement Monoclonal Antibody Soluble Receptor (Enbrel)

Receptor Antagonists (IL-1RA) IL-6 Antagonist

Cytokines Antagonists (IL-10, IL-11, IL-13) TACE, ICE inhibittors

Drotrecogin alfa 8.6. Anti-Cytokine Therapy

See: Anti-cytokine therapeutics: history and update.

Ratsimandresy RA, Rappaport J, Zagury JF.

Curr. Pharm. Des. 2009;15(17):1998-2025. Review.

(29)

8.7 Cancer therapy and monoclonal antibodies

Monoclonal antibodies have been seen as a breakthrough in cancer therapy.

However, there are problems with their use. These include the targeting of the antibody to the tumor. There are few if any molecules that are cancer-cell-

specific and many markers of cancer are merely up-regulated or mutated forms of natural cellular products. Additionally, cancers are heterogeneous with respect to antigen production and therefore an antibody may not recognize every cancer cell. Sadly, removal of all cancer cells is a prerequisite for a cure. One of the first tasks is therefore the recognition of a target molecule on the cancer.

Herceptin in breast cancer therapy

see: Cell Cycle and Cancer Therapy, p53

(30)

8.8. How Can Immunity Be Applied for Treatment?

Immunity is a specific system dealing with defensive mechanism. It is useful for prevention, via destroying of foreign body that enters or invades into the human body before it can generate further problems.

However, the present concept transform to the usage of immunity for medical treatment.

The immunotherapy is a new highlight in immunology. There are many forms of immune-related treatments.

A. Immunomodulation Therapy:

Immunomodulation therapy is a new way of treatment making use of modification of immunity systern to help curative process of some diseases. This is widely used for several treatments.

Immunosuppressants: inhibits immune response in organ transplantation and autoimmune diseases.

The drugs are:

- Calcineurin inhibitors (Specific T-cell inhibitors). Cyclosporine (Ciclosporin),

- Antiproliferative drugs (Cytotoxic drugs). Azathioprine, Cyclophosphamide, Methotrexate, - Glucocorticoids. Prednisolone and others

- Antibodies. Muromonab CD3, Antithymocyte globin (ATG), Rho (D) immuneglobin, Efalizumab.

(31)

Immunostimulants

Increase the immune response, useful in infections, immunodeficiency (for example, AIDS) and cancers.

Immunostimulant drugs:

They stimulate the immune system to fight against immunodeficiencies (like AIDS), infections and cancers.

- Levamisole. An antihelmintic drug that also restores functions of B lymphocytes,

T lymphocytes, monocytes and macrophages. Hence it has been used in colon cancer along with 5-FU.

- Thalidomide. Different effects of this old drug have been utilized in conditions such as:

Erythema nodosum leprosum: Anti-inflammatory effect Multiple myleoma: Anti-angiogenesis

Rheumatoid arthritis: Anti TNF effect.

- BCG. Used in carcinoma bladder.

- Recombinant cytokines.

Interferons: In tumors and chronic hepatitis B and C

interleukin 2 (aldeslukin): has been used in renal cell carcinoma and melanoma

(32)

Antibody treatment is any treatment making use of administration of immunoglobulin or antibody. This is famous for a long time. Direct passive immunization is the good example. The most well-know situations are tetanus antitoxin injection and rabies immunoglobulin for preventive and therapeutic purposes. See chapter 6.

C. Therapeutic Vaccination

Indeed vaccine is used for prevention and described as an active immunization.

However, the present role of vaccine changes to more usefulness in curative treatment process. Vaccination against human papilloma virus infection to prevent the

development of tumors.

D. Cytokine Treatment

Cytokine treatment or cytokine therapy is another mode of immune - related treatment.

Indeed, cytokine is a product in cellular process of cell-mediated immunity process.

Cytokine treatment is used for medical treatment of many diseases especially for many viral infections. This is comparable to antibody treatment, which makes usefulness of humoral immunity for treatment.

E. Gene Therapy

See chapter 4. and 10.