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Chagas' disease affects 16-18 million people in Latin America, ... 5 mCi [14C]-oleic acid. 16 h, at 28 or 37 C. Metabolic labeling and lipid analysis. Washed ... – PowerPoint PPT presentation

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Title: Apresentao do PowerPoint


1
New Chemo- and Immunotherapeutic Approaches in
Chagas Disease
Rosa A. Maldonado Medina Federal University of
São Paulo (UNIFESP)
2
Chagas Disease (American trypanosomiasis)
  • Chagas disease affects 16-18 million people in
    Latin America, from Mexico to Argentina.
  • The ethiological agent is the protozoan
    parasite
  • Trypanosoma cruzi
  • Current treatment (benznidazole, Rochagan) is
    partially effective (60 cure).
  • Recently, drug-resistant strains have been
    found.

.

3
Chemotherapeutic Target Properties
a) The molecule must perform essential functions
in the parasite.
Basic Metabolism
Life or death


b) It can be selectively inhibited
Present in T. cruzi, but not in mammals

4
Oleate (D-12) desaturase (ODTc ) A New
Chemotherapeutic Target in Chagas Disease
5
X CoA or ACP (acyl carrier protein)
or phospholipid.
  • OD is absent in humans, then it can be
    selectively inhibited.
  • Linoleic acid is a major component of GPI
    anchor of T. cruzi trypomastigote mucins and
    phospholipids. So, the product of OD seems to
    perform essential functions for the parasite.

OD fulfills all the requirements as a potential
chemotherapeutic target.
6
Cloning of the T. cruzi Oleate Desaturase (ODTc)
7
Chromosomal localization
Y CL
CL-Brener
Mb
Mb
Mb
1.03
0.95
0.85
0.85
0.45
Strain
Chrom. Mb Dm28c Sylvio CL Y
CL Band X10/7 Brener IX 1.03 VII 0.95 VI 0.
85 I 0.45
Phylogenetic groups I (sylvatic cycle) Dm28c,
Sylvio X10/7 II (domestic cycle) Y, CL Hybrid
CL Brener
8
Alignment of Trypanosomatid ODs vs. Plant OD
T.cruzi L.major T.brucei B.officinalis
T.cruzi L.major T.brucei B.officinalis
T.cruzi L.major T.brucei B.officinalis
T.cruzi L.major T.brucei B.officinalis
T.cruzi L.major T.brucei B.officinalis
T.cruzi L.major T.brucei B.officinalis
T.cruzi L.major T.brucei B.officinalis
Putative transmembrane motifs
His-rich motifs possibly participating in
catalysis
PSORT II - k-nearest neighbor (k-NN) algorithm
44.4 endoplasmic reticulum
9
Homologous expression of ODTc fused to GFP
5-Xba I
gDNA
Xba I -5
PCR
GFP
ODTc
1 x 106 epimastigotes/ 200 mL
Eco RV
Xba I
Xba I
Neor
Electroporation
pTEX
50 mg plasmid
AMPr
Selection
LIT G418 (100 mg/mL) 1 blood
Transfected parasites
10
Co-localization of ODtc in the Endoplasmic
Reticulum
OD-GFP
ER-Tracker Blue-White DPX
Phase
Merge
11
Molecular characterization of OD in other
trypanosomatids
12
Biochemical studies of the native OD activity in
different trypanosomatids
13
Metabolic labeling and lipid analysis
1010 parasites 5 mCi 14C-oleic acid 16 h, at
28 or 37ºC
Washed
Freeze-dried
Lipid extraction (CM, 21 CMW, 120.8)
Total lipids
HPTLC Autoradiography
14
Fatty acid hydrogenation of Trypanosomatid
phospholipids
Phospholipids (PC, PE, PI)
Alkaline hydrolysis
Methylation
/-Hydrogenation
RP-HPTLC Autoradiography
C183
C183
C182
C182
C181
C181
C180
C180
Hydrogenation -
- - -
- -
Tc epi Tc tryp Cf
La Hm Ls
15
CONCLUSIONS
  • ODTc gene has 2 copies in tandem array and is
    transcribed in all T. cruzi stages.
  • The enzyme is localized in the endoplasmic
    reticulum.
  • The enzymes ?-12 and ?-15 desaturases are active
    in trypanosomatids.
  • OD seems to be promising chemotherapeutic target
    in Chagas disease, leishmaniasis and African
    trypanosomiasis, as well as in fungal infections.

16
The Spider Antimicrobial Peptide Gomesin
as a New
Adjuvant in the Immunotherapy against T. cruzi
17
Antimicrobial peptides (AMP)
  • Involved in innate immunity found from bacteria
    to mammals
  • Cationic molecules composed of 12 to 50 amino
    acids
  • Frequently rich in cysteine residues
  • Positively charged in physiological pH, and with
    amphypathic structure in ?-helix, ?-sheet or both
  • Primary action mechanism is the permeabilization
    of the microbial membrane

18
Gomesin
  • Isolated from hemocytes of the Brazilian
    tarantula spider Acanthoscurria gomesiana (Silva
    Jr. et al, JBC, 2000)
  • Mass of 2270 Da, 18 amino acids
    (ZCRRLCYKQRCVTYCRGRa), two disulfide bonds
    (Cys6-11 e Cys2-15)
  • Antibacterial (Gram-negative and Gram-positive)
    and antifungal activity

19
Gomesin kills T. cruzi epimastigotes in vitro
DL50 6.5 mM
Survival ()
Concentration (mM)
20
In vivo activity of gomesin in the experimental
infection of mice BALB/c by T. cruzi
1.5107
1.0107
Parasitemia (No. of parasites/mL)
0.5107
0
0
1
2
3
4
5
6
7
8
9
10
Time (days after infection)
21
In vivo activity of gomesin in the experimental
infection of mice BALB/c by T. cruzi
22
How gomesin was protecting the animals if it was
not by directly killing the parasites?
  • One possibility was that gomesin like defensins
    (Biragyn et al., Science, 2002) was acting on the
    mouse innate immune system, inducing
    pro-inflammatory cytokines, through the
    activation of Toll-like receptors.
  • TLRs are primary sensors of the innate immunity.
    They recognize Pathogen-Associated Molecular
    Patterns (PAMPs)(e.g. LPS, GPIs, etc), initiating
    signaling pathways that stimulate the host
    defense against microorganisms.

23
METHODOLOGY
  • Transfected CHO/CD14 cells CHO/CD14/TLR2
  • CHO/CD14/TLR4
  • CHO/CD14/mMD2
  • CHO/CD14/mTLR4
  • Gomesin stock solution was cleaned-up of
    endotoxins using an affinity column of polimyxin
    B (depletion of LPS)
  • The stimulation of the TLR was measured by the
    expression of CD25 on the surface of the cells
    by FACS

mutants not able to signal via TLR4
24
Activation of TLR4 by Gomesin
Gomesin activates TLR4 at low nanomolar to
picomolar range
25
Gomesin activates Toll-like receptor 4
26
CONCLUSIONS
  • Gomesin stimulates TLR4, so it is a new PAMP.
  • Gomesin could be used as an adjuvant of the
    innate immune response against Chagas infection
    and, probably, other infectious diseases.

27
ACKNOWLEDGMENTS
  • University of São Paulo (USP)
  • ICB
  • Lab. Parasite Glycobiology
  • Igor C. Almeida
  • Renata K. Kuniyoshi
  • Lab. of Biochem. and Immunol. of Arthropodes
  • Sirlei Daffre
  • Marcello R. Burgierman
  • IQ
  • Maria Terêsa Miranda
  • Iolanda Cuccovia
  • Federal University of São Paulo (UNIFESP)
  • Antônio Miranda
  • Marcos Fazio

University of Dundee Alan Fairlamb Financial
support FAPESP/ CNPq WHO/TDR The Wellcome
Trust-UK
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