Laboratorio de Biologia Molecular IMT-AvH-UPCH
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Transcript of Laboratorio de Biologia Molecular IMT-AvH-UPCH
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Presented by
Dr. Jorge Arévalo Zelada
Laboratorio de Biología Molecular y Celular de TripanosomátidosIMTAvH-UPCH
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THE LEISHMANIASES REPRESENT AMAJOR HEALTH PROBLEM IN PERU
AND THE AMERICAS
Incidence in Peru: 32.4/100,000 (1996)
With the exception of Chile, all countries havereported cases of Leishmaniases.
All the clinical forms of Leishmaniasis arepresent in the Americas, including visceralLeishmaniasis.
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GENERAL AIM OF THE LEISHMANIASIS RESEARCH GROUP
To understand parasite and environment contributions to disease transmission and disease evolution using molecular tools to track down phenotype polymorphism and parasite population heterogeneity. These features might be associated to disease manifestations.
To develop molecular tools for better diagnostic procedures, specially under conditions where they show clear cost/benefit comparative advantages over conventional laboratory diagnosis.
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SPECIFIC OBJECTIVES WITHIN DGIS FRAMEWORK
•Formal assessment of diagnostic performance of Leishmania DNA detection procedures within a hospital service attending skin ulcers.•Development of new DNA targets and methodologies to improve parasite detection and to be capable of Leishmania identification in skin biopsies without the need of culture.•To transfer know how to other research groups at the IMTAvH.•To develop human resources with skills and material facilities to carry out gene expression studies. •To develop an animal model that allows to study the possible links between Vitamin A, immunological response and disease course infection.
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Diagnostic performance of Leishmania DNA detection procedures within a hospital service
Joint work with the Clinical component through the skin ulcer project.
Laboratory responsible: Leyda Cabrera
Clinical responsible: Tine Verdonck
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1 2 3 4 5 6 7 8 9 10 11 12 13 14
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Patients attending the IMTAvH
Samples provided by the Clinical component
DNA extraction and PCR detection procedures
Hybridization and non radioactive detection procedures
Diagnostic results delivered according health service needs
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New targets and methodologies For detection and identification of leishmania in skin biopsies
Joint work with the Protozoology unit.
Laboratory responsible: Kathleen Victoir & Jean Claude Dujardin
•Clinical responsible: Alejandro LLanos
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•Gp63 gene organization and DNA sequencing at the IMT (Antwerp)
•Former tests with cultures parasites at the IMT (Antwerp)
•Assays with biopsies from experimentally infected animals at the IMTAvH (Lima)
•Assays with biopsies from patients (Lima and Antwerp)
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5 kb
B B B B B B B B B B B B B B S E S E S E S S E S
B B B B B B B B B B B B B B B B B B B B B B
E S E S E S S E SBS ES ES ESBS BSS ESS BS SBEBSB** **
B9211
A811
C711/D9311
EBS E B B B B B BS EBS EBSEEBSEEBS EBS
BB BB BB BS E SB B BEB BS B B B E BSE B
C4121
C1211
EEBS EBS B B BE BSE BS
G3411
Gp63 gene locus (partial) in L.(V.)braziliensis
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N C S
L.(V.) peruvianaL.(V.) braziliensis
15.8 kb
10.9 kb9.8 kb
3.7 kb
3 kb
Gp63- RFLP analysis
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= amplification product 1.3kb= gp63 probe (pLb134-Sp)
coding region 1.8 kb untranslated region 1.3 kb
3 kb gp63 repetition unit
primers
most variable region
TDM1 TDM2
The gp63 unit amplification :
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Sensitivity of gp63 PCR detection
• 22 samples analysed by kDNA PCR (all +), gp63 PCR and parasitology
• gp63 PCR: 91%
• parasitology: 27%
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Sensitivity and clinical form
• 7 cutaneous samples (kDNA+):
gp63 PCR: 100%
parasitology: 57%
• 15 mucosal samples (kDNA+):
gp63 PCR: 86%
parasitology: 13%
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SalI ApaLI ApaI
x x b p g b p l x x x g x b p
PCR-RFLP (x = biopsy)
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Conclusions
• Gp63 PCR-RFLP: complementary tool
• plus: discrimination of species and intra-specific populations (NW and OW)
• applications: prognosis, epidemiological surveillance, imported pathologies...
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Prospects
• Increasing sensitivity of detection (nested et al.)
• Simplification of the assay
• Alternative genomic targets
• Link with biological features
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Thanks
• K.Victoir, S.De Doncker, M.Boelaert & D.Le Ray: ITG Antwerpen
• L.Cabrera, E.Alvarez, A.Llanos-Cuentas & J.Arevalo: IMTAvH Lima
• S.Rijal: BPKIHS, Dharan, Nepal
• S.Guerbouj, IPT Tunis
• N.Nuwayri-Salti, AUB Beyruth
EC, DGIS, FWO
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OTHER TARGETS
The following are not sequences developed with DGIS direct support but they will be used to design DNA primers that will be used under the same concept of gp63 genes.
rDNA InteGenic Sequences (IGS)Cistein proteinase b (cpb) like genes
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IGS Restriction Map of Leishmania peruviana, HB44 strain
Figure 1.. IGS from HB44 was amplified and cloned into TOPO XL. A recombinant clone , N44 containing a 6500pb insert. The Figure also shows the place of primers annealing sites (arrows) It also depicts the beta and delta sequences (blue bars underlined)
Promotora ETS
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IGS Restriction Map of Leishmania peruviana, LCA04 strain
Figure 2.. IGS from LCA 04 was amplified and cloned into TOPO XL. A recombinant clone , S04 containing a 5500pb insert. The Figure also shows the place of primers annealing sites (arrows) It also depicts the beta and delta sequences (blue bars underlined)
2000
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SINTESIS RNA LC 2177
0
20
40
60
80
100
120
140
1 2 3 4 5
TIEMPO
CP
M
EXP. 1
EXP. 2
EXP.3
SINTESIS RNA LCA 01
0
10
20
30
40
50
60
70
1 2 3 4 5
TIEMPO
CP
M
EXP.1
EXP.2
EXP.3
DEGRADACION RNA LC 2177
0
200
400
600
800
1000
1200
1400
1600
1800
1 2 3 4 5
TIEMPO
CP
M
EXP. 1
EXP.2
EXP.3
DEGRADACION RNA LCA 01
0
500
1000
1500
2000
2500
3000
3500
4000
1 2 3 4 5
TIEMPO
CP
M EXP.1
EXP.2
EXP.3
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Cla I Cla I Cla IXhoIXhoI
XhoI XhoIXhoI XhoIEcoRI EcoRI
1800 bp 2300 bp 3000 bp
PROPOSED MODEL WITH A THREE CP GENES TANDEM ARRAY
The 5´end gene (1800 bp) was cloned in AZ41 and the central gene in AZ13
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az41 CDEGYSGGLQGFGSSETCAFFRCWGTRWAISLSEQELVS 171az13 TDQGMCGSCWAFSAIGNIESQWYLATHSLISLSEQELVS 180 *:* .*. .*.: . .*: **********
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IntramuRal know how transfer
Joint work with the Mycology unit of the IMTAvH.Laboratory responsible: whole unit.
Edgar Neyra, who was member of the Leishmaniasis research unit, onset the setting up of the molecular biology lab at the Mycology unit. In addition, Livia Santibañez has been also recruited recently.
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Gene expression studies.
Joint work with the Protozoology unit.Laboratory responsible: Dionicia Gamboa & Jean-Claude Dujardin
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Training of Dionicia Gamboa in basic molecular biology techniques (Antwerp)
Training of Dionisia Gamboa in Differential Display (Maastrich)
Set up Differential Display at Antwerp
Initial training on DNA cloning techniques (Lima)
Identification of target sequence candidates, cloning and sequencing (Antwerp)
Set up Differential Display in Lima
Identification of other target sequence candidates and working with other Leishmania isolates (Lima)
PhD Thesis
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M L AmastigotesML
M= MetacyclicsL=Logaritmic
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Clones obtained by Differential Dislay Analysis
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Animal model for Vitamin A defficiencies, immunological response and disease course
infection
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D a ys p os t in fe c tion
Lesi
on
dia
met
er
(mm
)
10 15 20 25 30 35 40 45 50 55 600 .00
0 .25
0 .50
0 .75
1 .00
A + (C on tro l)
A + (In fe c te d )
A - (C on tro l)
A - (In fe c te d )
F ig u re 2 . C o u rse o f in fe c tio n in B A L B /c m ice in o cu la te d w ith 1 0 6 p ro m a s tig o te s o fL . m e xica n a M 3 7 9 . A + : v ita m in A -su ffic ien t d ie t; A -: v ita m in A -d e fic ien t d ie t.
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Leuk
ocyt
es
/ mm
3
0
5 0 0 0
1 0 0 0 0
1 5 0 0 0
2 0 0 0 0
2 5 0 0 0
A + (C o n tro l)
A + ( In fe c te d )
A - (C o n tro l)A - ( In fe c te d )
B e fo rein fe c t io n
6 0 d a y sp o s t in fe c t io n
F ig u re 7 . E f fe c t o f v i ta m in A s ta tu s o n th e to ta l n u m b e r o f le u k o c y te s in p e r ip h e ra lb lo o d f r o m B A L B /c m ic e . A + : v i ta m in A -s u f f ic ie n t d ie t ; A -: v i ta m in A -d e f ic ie n t d ie t .
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In f.
D a y s p o s t in fe c tio n
Lym
phoc
ytes
(%
)
-2 0 -1 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0In f.0
1 5
3 0
4 5
6 0
7 5
9 0
A + (C o n tro l)
A + ( In fe c te d )
A - (C o n tro l)
A - ( In fe c te d )
F ig u re 9 . E ffe c t o f v ita m in A s ta tu s o n th e p e rc e n ta g e o f p e r ip h e ra l b lo o dly m p h o c y te s fro m B A L B /c m ic e . A + : v ita m in A -s u ffic ie n t d ie t; A -: v ita m in A -d e fic ie n td ie t. In f: In fe c tio n .
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I n f .
D a y s p o s t in f e c t io n
Neu
trop
hils
(%
)
- 2 0 - 1 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0In f .0
1 0
2 0
3 0
4 0
5 0
6 0
7 0
A + ( C o n t r o l )
A + ( I n f e c t e d )
A - ( C o n t r o l )
A - ( I n f e c t e d )
F i g u r e 1 0 . E f f e c t o f v i t a m i n A s t a t u s o n t h e p e r c e n t a g e o f p e r i p h e r a l b l o o dn e u t r o p h i l s f r o m B A L B / c m i c e . A + : v i t a m i n A - s u f f i c i e n t d i e t ; A - : v i t a m i n A - d e f i c i e n td i e t . I n f : I n f e c t i o n ..
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Respuesta Oxidativa de Macrófagos Peritoneales Deficientes de Vitamina A e infectados con
Leishmania mexicana
0.00
5.00
10.00
15.00
20.00
25.00
30.00
control deficienteDieta de Vitamina A
Lu
min
isc
en
cia
(m
ac
rófa
go
s+
es
tim
ulo
) /
Ma
cro
fag
os
co
ntr
ol
Control
Infectado
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THE NEXT STEP
We aim to become a Molecular Epidemiology Unit that will extend our expertise gained with Leishmaniasis to other infectious diseases relevant to the public health of Peru and other countries of Latinamerica. Two approaches will be undertaken:Starting new research lines with other pathogens (for example drug resistance)Supporting molecular research of other groups at the IMTAvH