modulation of goat monocyte cystatinfrom...
TRANSCRIPT
Modulation of goat monocyte function by HCcyst-2, a secreted
cystatin from Haemonchus contortus
By: Rezaei Riabi Roya
Helminth Immunomodulation in Autoimmune Disease
Helminth
IL-4, IL-5, IL-10, and IL-13
(TGF) β
IL-10 IgE
mastcells, eosinophils, and basophils
promotes Th2 polarization
• Failure of the immune system
o Inflammatory bowel disease (IBD)
o Multiple sclerosis (MS)
o Rheumatoid arthritis (RA)
o Type 1 diabetes (T1D)
Haemonchus contortus
Scientific classification
Kingdom: Animalia
Phylum: Nematoda
Class: Secernentea
Subclass: Rhabditia
Order: Strongylida
Family: Trichostrongylidae
Genus: Haemonchus
Species: H. contortus
• Excretory/secretory (ES)
products are the primary interface between parasitic worms and their hosts
• Cysteine protease inhibitor (cystatin)
• Cysteine protease inhibitor (cystatin)
• Migration inhibitory factor (MIF)
protease activity
monocyte
phagocytosis
Life cycle
Haemonchus contortus
Sheep, goats, deer, exotic ruminants
Blood-sucking parasite
– highly pathogenic
– causes severe anemia
– causes low blood protein -- “bottle jaw”
Most important parasite in sheep/goats raised in warm/wet environments
Haemonchus contortus
• Haemonchus contortus – barberpole worm/ wire worm
– 1” (25 mm)
– Abomasum of
small ruminants
– feeds on blood
– Clinical signs
• anemia, bottle jaw, chronic weight loss
• death
larvae (L3) cultured
2500 larvae/ml at 4 °C.
male goats were fed
fecal samples infected goats
• Expression and purification of rHCcyst-2 in
Escherichia coli (RT PCR, pMD19-T vector )
• Cloning of HCcyst-2 and bioinformatics
analyses(cleaved by EcoRI)
purified rHCcyst-2 with adjuvant
subcutaneously
Localization of HCcyst-2 by immunohistochemical study (bySPECTRAFLUOR )
cystatins
• Belongs to superfamily papain-like cysteine proteases
• The cystatins family find in invertebrates, vertebrates, plants and even in
protozoa
• four subfamilies
Type 1 cystatins or stefins are cytoplasmic proteins
lacking of signal peptides
Type 2 cystatins are secretory-type proteins(excretory/secretory (ES)
Type 3 called kininogens (circulating plasma protein, blood
coagulation )
Type 4 fetuins and histidine-rich proteins
cystatin gene of H. contortus (rHCcyst-2)143 amino acid & size of 14 kDa
+
goat monocyte
immune modulatory activity
rHCcyst-2 inhibits MHC-II expression on goat monocytes. Monocytes were cultured in the presence of control buffer (PBS/DTT) or different concentrations of rHCcyst-2 for 24 h. MHC-II expression was measured by flow cytometric analysis and calculated as the percentage of mean fluorescence intensity (MFI) of controls. Bars represent the MFI ± SD of controls. (A) MHC-II; (B) MHC-I. The data are representative of three independent experiments
• Detection of cytokine secretion (ELISA)
decreased the production of TNF-α, IL-1β and IL-12
increased the secretion of IL-10 in goat monocytes
• Results showed:
I. rHCcyst-2 immunomodulatory effects on goat monocytes
II. Induced the production of IL-10 in goat monocytes
III. Induce an anti-inflammatory environment
IV. Decreased the production of TNF-α, IL-1β and IL-12
V. Increased the secretion of IL-10 in goat monocytes
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