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THE COMPLEMENT SYSTEM ▪ Because of its potential for far-reaching effects,

complement activation needs to be carefully


regulated.
LEARNING OUTCOMES: ▪ Any deficiencies to the complement system can
result in increased susceptibility to infection or the
After completing this topic, you should be able to: accumulation of immune complexes resulting in
1. Describe the roles of the complement system. possible autoimmune disorders.
2. Differentiate between the classical, alternative,
and lectin pathways and indicate proteins and
activators involved in each.
PROTEINS OF THE COMPLEMENT SYSTEM:
3. Discuss the formation of the three principal units
Complement proteins were named as they were
of the classical pathway: recognition, activation,
isolated before the sequence of activation was known—
and membrane attack units.
hence the irregularity in the numbering system.
4. Describe how the initiation of the lectin pathway
occurs.
Proteins of the CLASSICAL PATHWAY (arranged
5. Explain how C3 plays a vital role in all pathways.
in their order of activation in the cascade):
6. Describe regulators of the complement system and
their role in the complement system.
1. The Recognition Unit: C1
7. Discuss the complement-related kidney disorders
▪ Molecular weight: 740,000 Daltons
and applicable complement testing.
▪ Consists of three subunits—C1q, C1r, and C1s
8. Relate biological manifestations of complement
▪ Calcium-dependent (to maintain its structure)
activation to the generation of specific complement
▪ It is composed of one (1) C1q subunit and two
products.
(2) each of C1r and C1s subunits.
9. Describe the deficiencies of complement
components and the diseases they cause.
10. Differentiate tests for functional activity of
complement from the measurement of individual
complement components.
11. Analyze laboratory findings and indicate disease
implications in relation to complement
abnormalities.

INTRODUCTION / HISTORY:
▪ Jules Bordet was awarded the Nobel Prize in
1919 for his role in elucidating the nature of the
complement. a. C1q
▪ The complement system is a complex series of more ▪ Molecular weight: 410,000 Daltons
than 30 proteins that play a significant part in ▪ Concentration in serum: 150 µg/mL
amplifying the inflammatory response to destroy ▪ Structure: composed of six strands that
and clear foreign antigens—hence, their name form six globular heads with a collagen-like
‘complement.’ tail portion. This structure has been
o Complement was originally recognized in the likened to a bouquet of tulips with six
1890s; Paul Ehrlich coined the term blossoms extending outward.
complement because the substance ▪ Function: C1q “recognizes” the fragment
‘complements’ the action of antibodies in crystallizable (Fc) region of two adjacent
destroying microorganisms. antibody molecules, but at least two of the
▪ Complement proteins are part of the innate globular heads of C1q must be bound to
immune system and are composed of soluble and initiate the classical pathway.
cell-bound proteins.
▪ Complement is considered an acute-phase b. C1r
reactant because levels rise during an infection. ▪ Molecular weight: 85,000 Daltons
▪ It also has the unique property of being easily ▪ Concentration in serum: 50 µg/mL
inactivated (i.e., heat-labile) by heating serum to ▪ A serine protease enzyme, initially an
56°C for 30 minutes. inactive proenzyme—becomes activated
▪ They have the following essential roles: once C1q is bound to the antibody
o Membrane attack—lyse foreign cells (MAC). molecules.
o Phagocytosis—opsonize and tag the invaders ▪ It activates the C1s subunit.
for clearance. C3b has the most important ▪ Activated C1r is extremely specific—its
opsonizing activity. only known substrate is the C1s subunit.
o Inflammation—complement proteins serve
as an important link between the innate and c. C1s
adaptive immune responses (C3a, C4a, C5a): ▪ Molecular weight: 85,000 Daltons
▪ Ability to increase vascular permeability. ▪ Concentration in serum: 50 µg/mL
▪ Recruit monocytes and neutrophils to the ▪ A serine protease enzyme, initially an
area of antigen concentration. inactive proenzyme—becomes activated
▪ Trigger the secretion of immunoregulatory once C1q is bound to the antibody
molecules that amplify the immune molecules.
response. ▪ C1s subunit has a limited specificity, with
its only substrates being C4 and C2.
▪ Chronic activation can lead to inflammation and ▪ Once the C1s subunit is activated, the
tissue damage to the host. recognition stage ends.
2. The Activation Unit: ▪ The half-life is estimated to be
Phase two, the formation of the activation unit, between 15 seconds and 3 minutes,
begins when C1s cleaves C4 and ends with the so C3 must be bound quickly.
production of the enzyme C5 convertase. ▪ If the binding does occur, C3 is
cleaved by the C3 convertase into
a. C4 two fragments, C3a and C3b.
▪ Second most abundant complement protein
▪ Molecular weight: 205,000 Daltons c. C3
▪ Concentration in serum: 300-600 µg/mL ▪ The major and central constituent of the
▪ The cleavage of C4 represents the first complement system—the key intermediate
amplification step in the cascade in all three pathways of complement
because, for every C1 attached, activation.
approximately 30 molecules of C4 are split ▪ People with C3 deficiency are susceptible to
and attached. bacterial infection.
▪ Cleaved by C1s into 2 fragments: ▪ Molecular weight: 190,000 Daltons
▪ Concentration in serum: 1,200 µg/mL
o C4b – bigger C4 fragment ▪ The cleavage of C3 to C3b represents the
▪ MW: 190,000 Daltons most significant step in the entire process
▪ Interacts with the C2a fragment to of complement activation.
form the C3 convertase (C4b2a) of ▪ The cleavage of C3 represents a second
the classical pathway. and major amplification step because
▪ Must bind to protein or about 200 molecules of C3 are split for
carbohydrate within a few seconds every molecule of C3 convertase (C4b2a).
or it will react with water ▪ Cleaved by the C3 convertase into 2
molecules to form iC4b, which is fragments:
rapidly degraded.
o C3b – bigger C3 fragment
o C4a – smaller C4 fragment ▪ MW: 180,000 Daltons
▪ MW: 9,000 Daltons ▪ C3b is potent in opsonization:
▪ Consists of 77 amino acids. tagging pathogens, immune
▪ An anaphylatoxin—is a small complexes (antigen-antibody), and
peptide that causes increased apoptotic cells for clearance or
vascular permeability, contraction phagocytosis.
of smooth muscle, and release of ▪ Macrophages have specific
histamine from basophils and mast receptors for it.
cells. ▪ Glycoprotein composed of the
modified C3-α chain (α′) (MW:
b. C2 105,000) and the intact C3-β chain
▪ Closely related to Factor B of the (MW: 75,000).
alternative pathway. ▪ If C3b is bound within 40-nm of the
▪ Molecular weight: 102,000 Daltons C4b2a, this creates a new enzyme
▪ Concentration in serum: 25 µg/mL known as the C5 convertase
▪ When combined with C4b in the presence (C4b2a3b).
of magnesium ions (Mg2+), C2 is cleaved
by C1s into two (2) fragments; an o C3a – smaller C3 fragment
exception to the rule of complement ▪ MW: 10,000 Daltons
protein nomenclature—C2a and C2b—the ▪ An anaphylatoxin.
bigger and smaller C2 fragments, ▪ C3a has a regulatory process and a
respectively. structure homologous to that of
complement component C5a.
o C2b – smaller C2 fragment ▪ C3a formation is triggered by
▪ MW: 34,000 Daltons pathogenic infection.
▪ Accelerates decay-dissociation of
the C3 convertase. 3. The Membrane Attack Complex (MAC):
▪ Acts as a feedback inhibitor on the The membrane attack complex (MAC) “punches” a
activation of the classical pathway hole through the plasma membrane of the target
of the complement system. cell, killing the pathogen in the process. It is
composed of the following complement components:
o C2a – bigger C2 fragment
▪ MW: 70,000 Daltons a. C5
▪ C2a interacts with the C4b ▪ Molecular weight: 190,000 Daltons
fragment to form the C3 convertase ▪ Concentration in serum: 80 µg/mL
(C4b2a) of the classical pathway. ▪ Function: initiates the membrane attack
▪ Provides the catalytic activity to complex (MAC).
the C3 and C5 convertases of o The cleaving of C5 with deposition of
classical and lectin pathways. C5b at another site on the cell
membrane constitutes the beginning of
▪ C3 Convertase (C4b4a): the MAC.
▪ The C3 convertase complex ▪ Structure: C5 consists of two polypeptide
(C4b2a) is not very stable. chains, α and β, which are linked by
disulfide bonds.
▪ C5 convertase (C4b2a3b) cleaves C5 into ▪ It takes one IgM molecule attached to two
two (2) fragments: adjacent antigenic determinants to initiate
the classical cascade of activation.
o C5b – bigger C3 fragment o Immunoglobulin G (IgG):
▪ MW: 75,000 Daltons ▪ IgG1, IgG2, and IgG3, but not IgG4.
▪ C5b attaches to the cell membrane, ▪ Two (2) IgG molecules must attach to
forming the beginning of the MAC. antigen within 30 nm to 40 nm of each
▪ C5b is extremely labile and rapidly other before complement can bind; it may
inactivated unless binding to C6 take at least 1,000 IgG molecules to ensure
occurs. that there are two, close enough to initiate
▪ Once C6 is bound to C5b, such binding.
subsequent binding involves C7, ▪ Some epitopes, notably the Rh group, are
C8, and C9. None of these proteins too far apart on the cell for this to occur;
has enzymatic activity. therefore, they are unable to fix
complement.
o C5a – smaller C2 fragment ▪ Within the IgG group, IgG3 is the most
▪ MW: 10,400 Daltons effective, followed by IgG1 and then IgG2.
▪ Released into the circulation (as an
anaphylatoxin – most potent). ▪ Triggering substances: In addition to antibodies,
a few substances can bind complement directly to
b. C6 initiate the classical cascade. These include C-
▪ Molecular weight: 110,000 Daltons reactive protein (CRP), several viruses,
▪ Concentration in serum: 45 µg/mL mycoplasmas, some protozoa, and certain Gram-
▪ Function: binds to C5b in MAC. negative bacteria such as Escherichia coli.

c. C7 Classical Pathway of Complement Activation:


▪ Molecular weight: 100,000 Daltons
▪ Concentration in serum: 90 µg/mL (1) IgM or (2) IgG
▪ Function: binds to C5bC6 in MAC. C1qrs complex C1qrs complex

d. C8
▪ Molecular weight: 150,000 Daltons
C1qrs
▪ Concentration in serum: 55 µg/mL C4 C4a + C4b
▪ Function: binds to C5bC67 in MAC; starts
the pore formation on the membrane.
C1qrs
e. C9 C2 C2a + C2b
▪ Molecular weight: 70,000 Daltons
▪ Concentration in serum: 60 µg/mL
▪ Function: multiple C9 molecules bind to C4b + C2a C4b2a (C3 convertase)
the C5bC678 complex and polymerize to
form a transmembrane channel, the
membrane attack complex (MAC), which
C4b2a
causes lysis of the cell. C3 C3a + C3b
▪ If the complex is soluble in circulation, it is
known as sC5b–9.
▪ Measurement of the level of sC5b–9 is an C4b2a + C3b C4b2a3b (C5 convertase)
indicator of the amount of terminal
pathway activation that is occurring.
C4b2a3b
C5 C5a + C5b
PATHWAYS OF THE COMPLEMENT SYSTEM:

The complement system can be activated in three (3) C5b + C6 + C7 + C8 C5bC678


different ways:
1. Classical pathway
2. Alternative pathway C5bC678 + C9 molecules
3. Lectin pathway

CLASSICAL PATHWAY: C5b–9 (Membrane Attack Complex)


▪ Antibody-directed mechanism for triggering the
complement activation.
▪ The classical pathway, so-called because it was
discovered first, uses a plasma protein called C1q
to detect antibodies bound to the surface of a
microbe or other structure.
▪ However, not all immunoglobulins are able to
activate this pathway:
o Immunoglobulin M (IgM):
▪ Most efficient in complement fixation
because of its multiple binding sites.
ALTERNATIVE PATHWAY: 5. C5 Convertase (C3bBb3bP)
▪ First described by Pillemer and his associates in ▪ If, however, some of the C3b produced remains
the early 1950s. bound to the C3 convertase, the enzyme is
▪ It was originally named for the protein properdin, altered to form C3bBb3bP, which has a high
a constituent of normal serum with a concentration affinity for C5 and exhibits C5 convertase
of approximately 5 to 15 μg/mL. activity.
▪ In addition to properdin, the serum proteins
Factor B and Factor D are unique to this Alternative Pathway of Complement Activation:
pathway. 1. C3 is hydrolyzed by water to produce C3b, which
▪ C3 is a key component of this pathway as well as binds Factor B and together they attach to the
the two other pathways (classical and lectin). The target cell surface.
conversion of C3 is the first step in this pathway. 2. B is cleaved by Factor D into the fragments Ba and
▪ Triggering substances for the alternative Bb. Bb combines with C3b to form C3bBb, an
pathway include bacterial cell walls, especially enzyme with C3 convertase activity.
those containing teichoic acid, zymosan, and 3. More C3 is cleaved, forming more C3bBb. This
lipopolysaccharide (LPS); fungal cell walls, yeast; enzyme is stabilized by properdin, and it continues
viruses, virally infected cells; tumor cell lines; and to cleave additional C3.
some parasites, especially trypanosomes. 4. If a molecule of C3 remains attached to the C3bBbP
enzyme, the convertase now has the capability to
Proteins of the ALTERNATIVE PATHWAY: cleave C5. The C5 convertase thus consists of
C3bBbP3b. After C5 is cleaved, the pathway is
1. Factor B identical to the classical pathway.
▪ Molecular weight: 93,000 Daltons
▪ Concentration in serum: 200 μg/mL
▪ Function: Bb fragment binds to C3b to form
the C3 convertase (C3bBb).
▪ Once bound to C3b, Factor B can be cleaved by
Factor D.
▪ The role of Factor B is thus analogous to that
of C2 in the classical pathway because it forms
an integral part of a C3 convertase.

2. Factor D
▪ Molecular weight: 24,000 Daltons
▪ Concentration in serum: 2 μg/mL (lowest
concentration in the plasma)
▪ Function: a serine protease that cleaves its
only substrate, Factor B, into two (2)
fragments—Ba and Bb.

o Bb
▪ MW: 60,000 Daltons
▪ Bb remains attached to C3b, forming
the initial C3 convertase of the
alternative pathway (C3bBb).
▪ Bb is rapidly inactivated unless it
becomes bound to a site on one of the
triggering cellular antigens.
▪ Some C3b attaches to cellular surfaces
and acts as a binding site for more
Factor B, resulting in an amplification
loop. LECTIN PATHWAY:
▪ Instead of activation through antibody binding, the
o Ba lectin pathway is activated by recognition of surface
▪ MW: 33,000 Daltons moieties that are found on pathogens.
o This pathway is initiated by the binding of
3. Properdin mannose-binding lectin (MBL), collectin 11
▪ Molecular weight: 55,000 Daltons (CL-K1), and ficolins (Ficolin-1, Ficolin-2, and
▪ Concentration in serum: 15-25 μg/mL Ficolin-3) to microbial surface oligosaccharides
▪ Function: Properdin (P) stabilizes the C3 and acetylated residues, respectively.
convertase. The binding of properdin ▪ It involves non-specific (non-selective) recognition of
increases the half-life of C3bBb from 90 carbohydrates that are common constituents of
seconds to several minutes. microbial cell walls and that are distinct from those
found on human cell surfaces.
4. C3 Convertase (C3bBb) ▪ The lectin pathway plays an important role as a
▪ As the alternative pathway convertase, defense mechanism in infancy (innate immune
C3bBb is then capable of cleaving response), during the interval between the loss of
additional C3 into C3a and C3b. maternal antibody and the acquisition of a full-
▪ C3bBb can also cleave C5, but it is much fledged antibody response to pathogens.
more efficient at cleaving C3. ▪ Once C4 and C2 are cleaved, the rest of the pathway
is identical to the classical pathway.
Proteins of the LECTIN PATHWAY: Further formation of C3 convertase in the classical
and lectin pathways is inhibited by four main
1. Mannose-Binding Lectin (MBL) regulators: soluble C4-binding protein (C4BP) and
▪ Another term: mannan-binding lectin three cell-bound receptors, complement receptor type 1
▪ Molecular weight: 200,000 to 60,000 Daltons (CR1), membrane cofactor protein (MCP), and decay-
▪ Concentration in serum: 0.0002 to 10 μg/mL accelerating factor (DAF).
▪ Function:
o As the name implies, it binds to mannose 2. Factor I
or related sugars in a calcium-dependent ▪ Molecular weight: 88,000 Daltons
manner to initiate this pathway. ▪ Concentration in serum: 35 μg/mL
o It is considered an acute-phase protein ▪ Function:
because it is produced in the liver and is o Cleaves C3b and C4b
normally present in the serum but o For the regulation of the classical and
increases during the initial inflammatory lectin pathways
response. o All of the C3 convertase regulators
mentioned above act in concert with
The enzymatic role played by C1r and C1s in the Factor I, a serine protease that inactivates
classical pathway is played in the lectin pathway by C3b and C4b when bound to one of these
serine proteases called MBL-associated serine regulators.
proteases (MASPs).
3. Factor H
2. MASP-1 ▪ Molecular weight: 150,000 Daltons
▪ Molecular weight: 93,000 Daltons ▪ Concentration in serum: 300 to 450 μg/mL
▪ Concentration in serum: 1.5 to 1.2 μg/mL ▪ Function:
▪ Function: o Principal soluble regulator of the
o MASP-1 is synthesized as a zymogen (an alternative pathway.
inactive form of the enzyme) and is o It acts by binding to C3b, preventing the
activated when it complexes with the binding of Factor B.
pathogen recognition molecules of the o Factor H also accelerates the dissociation of
lectin pathway, the mannose-binding the C3bBb complex on cell surfaces.
lectin, and the ficolins. o When Factor H binds to C3bBb, Bb
o This protein is not directly involved in becomes displaced.
complement activation but may play a role o Additionally, Factor H acts as a cofactor
as an amplifier of complement activation by that allows Factor I to break down C3b.
cleaving complement C2 or by activating
another complement serine protease, 4. C4-Binding Protein (C4BP)
MASP-2. ▪ Molecular weight: 520,000 Daltons
▪ Concentration in serum: 250 μg/mL
3. MASP-2 ▪ It is abundant in plasma
▪ Molecular weight: 76,000 Daltons ▪ Function:
▪ Concentration in serum: unknown o Acts as a cofactor with Factor I to
▪ Function: inactivate C4b.
o MASP-2 is very similar to the C1s o If C4BP attaches to cell-bound C4b, it can
molecule, of the classical complement dissociate it from C4b2a complexes
pathway—it cleaves C4 and C2 (classical pathway C3 convertase), causing
o When the carbohydrate-recognizing heads the cessation of the classical pathway.
of MBL bind to specifically arranged
mannose residues on the surface of a 5. S Protein (Vitronectin)
pathogen, MASP-2 is activated to cleave ▪ Molecular weight: 84,000 Daltons
complement components C4 and C2 into ▪ Concentration in serum: 500 μg/mL
C4a, C4b, C2a, and C2b, respectively. ▪ Function:
o S protein is a soluble control protein that
acts at a deeper level of complement
SYSTEM CONTROLS: activation (the terminal components).
Activation of complement could cause tissue damage o Also known as vitronectin, S protein
and have devastating systemic effects if it were allowed interacts with the C5b-7 complex as it
to proceed uncontrolled. forms in the fluid phase and prevents it
from binding to cell membranes.
1. C1 Inhibitor (C1-INH) o Binding of C8 and C9 still proceeds, but
▪ Molecular weight: 105,000 Daltons polymerization of C9 does not occur;
▪ Concentration in serum: 240 μg/mL therefore, the complex is unable to insert
▪ Function: itself into the cell membrane or to produce
o C1 inhibitor (C1-INH) inhibits activation lysis.
at the first stages of both the classical and
lectin pathways.
o Its main role is to inactivate C1 by binding
to the active sites of C1r and C1s.
o C1-INH also inactivates MASP-2 binding
to the MBL-MASP complex, thus halting
the lectin pathway.
Other Regulatory Proteins or Receptors: COMPLEMENT COMPONENT
ABNORMALITIES
6. Complement Receptor 1 (CR1)
▪ Also known as CD35 Deficiencies of complement components can place an
▪ Ligand: C3b, iC3b, and C4b individual at risk for certain infections. Missing or
▪ Molecular weight: 165,000–280,000 Daltons deficient regulators are the cause of diseases such as
▪ It is found mainly on peripheral blood cells, paroxysmal nocturnal hemoglobinuria and hereditary
including neutrophils, monocytes, angioedema.
macrophages, erythrocytes, eosinophils, B
lymphocytes, some T lymphocytes, and
follicular dendritic cells.
▪ Function:
o A main function of CR1 is as a receptor on
platelets and red blood cells (RBCs), which
helps to mediate transport of C3b-coated
immune complexes to the liver and spleen.
o It binds C3b and C4b but has the greatest
affinity for C3b.
o Once bound to CR1, both C4b and C3b can
then be degraded by Factor I.

7. Membrane Cofactor Protein (MCP)


▪ Also known as CD46
▪ Ligand: C3b and C4b
▪ Molecular weight: 50,000–70,000 Daltons
▪ It is found on virtually all epithelial and
endothelial cells, except erythrocytes (RBCs).
▪ MCP is the most efficient cofactor for Factor I-
mediated cleavage of C3b.
▪ It can serve as a cofactor for cleavage of C4b,
but it is not as effective as C4BP.
▪ MCP also helps to control the alternative
pathway because binding of Factor B to C3b is
inhibited

8. Decay Accelerating Factor (DAF)


▪ Also known as CD55
▪ Molecular weight: 70,000 Daltons
▪ Third main receptor and has a wide tissue
distribution.
▪ It is found on peripheral blood cells, on
endothelial cells and fibroblasts, and on
numerous types of epithelial cells.
▪ Ligand: C3b and C4b
▪ Function:
o DAF is capable of dissociating both
classical and alternative pathway C3
convertases.
o It can bind to both C3b and C4b in a
manner similar to CR1.
o The presence of DAF on host cells protects
them from bystander lysis. It is one of the
main mechanisms used in discrimination of
self from non-self because foreign cells do
not possess this substance.

9. Membrane Inhibitor of Reactive Lysis (MIRL)


▪ Also known as CD59
▪ Ligand: C8
▪ MIRL also acts to block formation of the MAC.
▪ MIRL is widely distributed on the cell
membranes of all circulating blood cells,
including RBCs, and on endothelial, epithelial,
and many other types of cells.
LABORATORY DETECTION OF ASSAY FOR THE ALTERNATIVE PATHWAY:
COMPLEMENT ABNORMALITIES
Hemolytic Titration (AH50) Assay:
Techniques to determine complement abnormalities ▪ An AH50 can be performed in the same manner
generally fall into two (2) categories: as the CH50, except magnesium chloride and
ethylene glycol tetraacetic acid (EGTA) are
(1) Measurement of components as antigens in serum added to the buffer and calcium is left out.
▪ Measurement of C3 and C4 levels – routinely ▪ This buffer chelates calcium, which blocks
measured by nephelometry or turbidimetry classical pathway activation.
▪ Measurement of C1-INH, C3bBbP, or C3bP – ▪ RABBIT RBCs are used as the indicator
measured by enzyme-linked immunosorbent because they provide an ideal surface for
assay (ELISA) alternative pathway activation.
▪ Radial immunodiffusion (RID)—uses
agarose gel into which specific antibody is Enzyme-Linked Immunosorbent Assay:
incorporated. Serum serves as the antigen and ▪ An additional means of testing for alternative
is placed in wells that are cut in the gel. pathway function is via ELISA.
Diffusion of the antigen from the well occurs in ▪ One such test can detect C3bBbP or C3bP
a circular pattern. The radius of the resulting complexes in very small quantities.
circle can be related to antigen concentration. ▪ Microtiter wells are typically coated with
bacterial polysaccharide to trigger activation of
the alternative pathway.

(2) Measurement of functional activity

The hemolytic titration or CH50 assay is a


measure of lysis, the end point of complement
activation in the classical pathway. The AH50
assay is a similar test for measuring the activity of
the alternative pathway

ASSAY FOR THE CLASSICAL PATHWAY:

Hemolytic Titration (CH50) Assay:


▪ This assay measures the amount of patient
serum required to lyse 50% of a standardized
concentration of antibody-sensitized SHEEP
erythrocytes.
▪ Because all proteins from C1 to C9 are
necessary for this to occur, absence of any one
component will result in an abnormal CH50,
essentially reducing this number to zero
▪ The titer is expressed in CH50 units, which is
the reciprocal of the dilution that is able to lyse
50% of the sensitized cells.
▪ The 50% point is used because this is when the
change in lytic activity per unit change in
complement is at maximum.

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