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Chemokine and complement
Chemokine classification Chemokine function Chemokine receptor Colony stimulating factor (CSF) Complement
Chemokines participate in leukocyte recruitment during inflammation
chemokine
Chemokines Chemotactic cytokine A large family of structurally
homologous cytokines stimulate transendothelial leukocyte
movement from the blood to tissue site of infection (Extravasation)
Cause rapid shape changes in leukocytes(pseudopod formation)
Classification of chemokine at least 43 members, posses 4 conserved
cysteine residules, and based on the position of two of the four invariant cyctein residuels, almost all fall into one of two subgroup, CC and CXC chemokines
One receptor bind more than one chemokines and one chemokine binds to more than one receptor (redundancy)
Chemokine and chemokine receptor CC chemokine: first two of the four
cysteine residues are adjacent. CXC chemokine: a single amino acid
residue interposed between the first two cysteines. ELR+: chemotactic for neutrophil, angiogenesis ELR-: chemotactic for lymphocyte, restrain
angiogenesis
CX3C chemokine: fractalkine(CX3CL1) C chemokine: lymphotactin(XCL1)
Chemokines signal through receptors coupled with heterotrimeric large G proteins
Chemokine inhibits HIV entry to cells
Colony-Stimulating Factors CSFs are cytokines that stimulate
stem cells or their progeny to produce large number of blood cells
Variety of CSFs are made such as G-CSF and GM-CSF
CSFs are potentially important in the treatment of human disease(GM-CSF 用在一些 immunocompromised condition)
CSFs GM-CSF G-CSF M-CSF Erythropoietin
(EPO)
IL-3(multi-CSF) IL-7 IL-9 IL-11
CSFs GM-CSF
lymphocytes,monocytes,fibroblast,endothelial cells to stimulate granulocytes and macrophage precursors
to mature and proliferate G-CSF
G-CSF,GM-CSF have been injected into patients to increase stem cell (CD34+)for peripheral blood stem cell (PBSC)transplantation
M-CSF lymphocytes,monocytes,fibroblast,endothelial cells to stimulate macrophage precursors to mature and
proliferate
Erythropoietin(EPO) produced by kidney to stimulate RBC to
mature and proliferate. EPO have been injected into patients with
anemia
Complement outline Complement activation pathways Complement receptors Complement regulation Complement functions Complement abnormality Complement detection Complement fixation assay
The complement system Sequential activation (cascade) of
zymogens to generate proteolytic enzymes
Activated complement components bind to surface or be degraded.
Complement activation is inhibited by regulatory proteins on host cell surface.
Overview of complement system
The classical pathway is initiated by activation of the C1 complex
C1q hexamer bindingC1r cleaves C1sC1s cleaves C4, C2
classical pathway generates C3b
The mannan-binding lectin pathway is homologous to the classical pathway
Mannan binding protein associated serine protease (MASP) like C1r, C1s
Complement activation is largely confined to the surface on which it is initiated
C3 is the same
Hydrolysis of C3 causes initiation of the alternative pathway of complement
C3b bind to microbial surface but not host cell can activate factor B
Classical and lectin pathways can also activate alternative pathway
Surface-bound C3 convertase deposits large numbers of C3b fragments on pathogen surfaces and generates C5 convertase activity
Complement receptors CR1 promote phagocytosis of C3b, C4b
coated immunocomplex CR2 bind C3d or EBV stimulate B cells and
FDC CR3 (Mac 1 or CD11b/CD18), integrin
bind iC3b, ICAM-1, cause phagocytosis CR4 (CD11c/CD18) like Mac-1 with
different chain
Complement receptors
Small fragments of some complement proteins can initiated a local inflammatory response
The terminal complement proteins polymerize to form pores in membranes that can kill certain pathogens
Complement control proteins regulate all three pathways of complement activation and protect the host from its destructive effects
Factor I cut C3b on host surface
Complement–mediated functions
Cell lysis Chemotaxis(C5a>C3a>C4a) Opsonization(C3b)
Elevated complement levels
Inflammatory condition Trauma Acute illness(C3)
Decreased complement levels
Excessively activated recently Currently being consumed A single complement component is
absent because of a genetic defect
C2 -- the most common complement deficiency
C3 -- an acute phase protein C4 -- the most sensitive indicator of
disease activity
C1 inhibitor (C1INH) deficiencty causes angioedema
Hereditary angioedma (HAE): genetic defect cause C1 inhibitor (C1INH) deficiencty
Autosomal dominant, occurs in 1 in 106.
C4 and C2 degrade to produce C4a anaphylatoxin and C2a-kinin like fragment.
Treat with steroid to induce liver to produce C1INH and other proteins
Hereditary and acquired C1 inhibitor (C1INH) deficiencty
CD59 inhibit C9 bind to C5b, 6, 7, 8
Decay-accelerating factor (DAF) displace Bb from C3b and C2b from C4b
Both CD59 and DAF are defect in PNH because enzyme to synthesize phosphoinositol glycolipid is missing in PNH
paroxysmal nocturnal hemoglobinuria (PNH)
Complement Assay
Component Assays
Functional Assays
Component AssaysRadial immunodiffusion ( 免疫擴散法 ,RID)NephelometryRadioimmunoassayTurbidimetryLatex agglutination photometric assay
Functional assay: CH50 目的:藉由測病人血清使 50%50%紅血球溶血紅血球溶血 所需
的體積,可得血清中的補體在典型途徑典型途徑中的功能。
原理: Patient’s serum (含補體) sensitized SRBC (含 Ab-Ag )
SRBC hemolysis (典型途徑活化)
CH50 方法:病人血清( 1:50 or 1:60 )+ SRBC
(EA) 37℃ 水浴槽 離心 O.D添加物 1 2 3 4 100%
TBS-G 0.9 0.7 0.6 0.5
血清 ml 0.2 0.3 0.4
EA ml 0.6 0.6 0.6 0.6 0.6
H2O ml 0.9
O.D 0.008 0.102 0.185 0.322 0.664
CH50
CH50Von-Krogh transformation :
X 軸: log (y/1-y) Y 軸: log (milliliter of serum) y= 各管校正過的吸光值/完全溶
血的校正吸光值 溶血百分比溶血百分比
當 y/1-y=1 時 有 50% SRBC 溶血
CH50
CH50缺點:
SRBC 脆性不一
SRBC 上免子的 Ab Affinity 不同 黏上 SRBC 量不同
檢體的採集和保存
Complement Fixation 目的:測定待測血清 ( 需去補體 ) 中是否含
有特定抗體或抗原。 原理: test system 和 indicator system
會互相競爭 complement 。Test system:未知 Ab 和已知 Ag or 未知 Ag 和已知 Ab
Indicator system : sensitized SRBC
Complement Fixation
Complement Fixation
Complement Fixation
Complement Fixation: the importance of different controls
Complement Fixation