Fabio T M Costa, PhD. University of Campinas (UNICAMP); Campinas, SP, Brazil. Supported by:
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1 Fabio T M Costa, PhD University of Campinas (UNICAMP); Campinas, SP, Brazil Supported by:
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3 P. falciparum associated pathologies In falciparum malaria cytoadhesion of mature forms are related to fatalities!
4 Number of articles and Budget 95% =
5 million cases annually 2.85 billion people at risk (50%) - 66% Southeastern and eat Asia, 14% America Africa Few cases Mostly individuals Duffy (Fy) negative More widely distributed than P. falciparum
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7 Absence of in vitro continuous culture, Ethical considerations blood collected from patients Need to establish functional and reproducible assays, Limit knowledge of parasite biology High genetic Carência diversity, de low tratamento parasitemias, belief alternativo as a benign form of the disease Need new approaches and strategies for drug discovery
8 Chloroquine resistance cases since 1999!
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11 Could this parasite cytoadhere? If so, mature forms disappear from peripheral circulation? Are the mechanisms involved similar than P. falciparum? Is still vivax malaria benign? What can we learn for helping drug tests?
12 Marcus VG Lacerda, MD/PhD Tropical Medicine Foundation Hospital 150 beds Referral Hospital for complicated cases Treats 30% of the malarial cases reported in Manaus Patients treated within 48 h of presentation of symptoms 4-hour flight Manaus, AM Population: 2,000,000 Annual Parasite Index (2008): 40
13 Field study design: cytoadhesion in P. vivax 1.Blood collection in sodium citrate tubes (5-10 ml from uncomplicated malaria-infected patients ) 4. Leukocyte depletion by CF11 columns 2. Diagnosis Giemsastained thick smears (Parasitemia %) 6. Ex-vivo cytoadhesion of Pv-iEs to HLEC, SBEC, Placenta cryosections, CHO-transfected cells 3. Diagnosis Confirmation of P. vivax mono-infection by Nested-PCR 5. Parasite enrichment by Percoll 45%
14 P. vivax trophozoites Phase DAPI FITC Naive Mab -MSP-1 19 (3F8) Mab -MSP-1 19 (K23)
15 P. vivax-ie adhesion 15 fold lower than P. falciparum-ie
16 Phase DAPI α-vir-e5 α-msp-1 19 Merge Phase DAPI α-vir-a4 α-msp-1 19 Merge 80 Inhibition (% of control) * * -GST -Vir-E5 -Vir-A4 * 0 Pv isolate 095 Pv isolate 096 (1:5) (1:10)
17 Isotype control Non stimulated LPS 0.1 mg/ml LPS 1 mg/ml CD54 (ICAM-1) - PE *** Bound Pv-iE (%of control) Pv-iE non-stimulated HLEC Pv-iE on LPS-stimulated HLEC * * * Shear stress (Pa)
18 Does P. vivax cytoadhere in vivo? Immunohistofluorescence from a tissue section of the lung Negative peripheral parasitemia Positive nested-pcr for P. vivax in the lung tissue
19 B
20 B % schizonts % 49.3% ** Pv-IE/mm R S =0.47 * Pv-IE/mm 2 80 ** Before Maturation After Maturation schizonts (%) 0 Before Maturation After Maturation All asexual blood forms available and synchronized for drug tests
21 Rosetting Association with clinical complications (mostly CM) Related to high parasitemia Blood group O Protection Blood group A Giant rosettes FCR3S1.2 (P. falciparum rosetting model) High rosetting rate High multiplication rate Binds to non-immune IgM PfEMP-1 mediates bindings Published articles Udomsanpetch et al. mature parasites Chotivanich et al. Heparin inhibition Russell et al. Trypsin sensitive Chotivanich et al. 93% isolates form rosettes 2013 Marin-Menendez et al. Anemia association
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25 P. vivax gametocyte: Enrichment to allow functional analysis 1.Blood collection in sodium citrate tubes (5-10 ml from uncomplicated malaria-infected patients ) 2. Diagnosis Giemsastained thick smears (Gametocytes in blood smear from 0 to 65% of total parasites (0 to 4,060 gametocytes/μl) 3. Diagnosis Confirmation of P. vivax mono-infection by Nested-PCR 4. Leukocyte depletion by CF11 columns 5.1. Parasite enrichment by Percoll 45% (Trophozoites, schizonts, Gametocytes) 5.2. Parasite enrichment by Percoll 60% (young trophozoites, Gametocytes) Gametocytes (%) LD columns (MACs) for parasite purification (Only parasites with large amount of hemozoin) 7. Gametocytes purification P45 P45 +LD P60 P60 +LD Total parasitemia increases from % to % * Before After
26 P. vivax gametocyte: Enrichment to allow functional analysis Viability evaluation: Trypan Blue and exflagellation stimulation Gametocyte dilution for mosquito feeding assay
27 P. vivax gametocyte: Enrichment to allow functional analysis 1. Mosquito Feeding assay 200 mosquitoes/group 100 mosquitoes on control group 2h of feeding Temperature 37.5 o C Dissection on 7 th day 100X 2. P. vivax sporozoites P. vivax sporozoites formed in An. aquasalis mosquitoes salivary glands. Light microscopy. P. vivax oocysts formed in An. aquasalis mosquitoes midgut. Light microscopy.
28 TRANSCRIPTOME DATA Plasmodium vivax: genetic diversity Multiple-clonal infections Higher microsatellite diversity Orjuela-Sánchez, P., et al., Experimental Parasitology, 2013
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30 Unveiling Plasmodium vivax Biology: Lab Team
31 Unveiling Plasmodium vivax Biology: Collaborative Network
32 Thank you!!!
33 Isolated from Chromobacterium violaceum Potential immunomodulatory, analgesic and antipyretic activities Anti-tumourogenic Anti-trypanocydal, anti-leishmanial and anti-malarial activity Mechanism of action: oxidative stress, apoptosis?
34 P. c. chabaudi AS(30CQ) is a clone resistant to 30 mg/kg chloroquine and 10mg/kg of pyrimetamine, P. c. chabaudi AS (ATNMF1) is a clone resistant to 40mg/kg of artesunate and 4 mg/kg of mefloquine
35 Pool 6000 heterozygous barcoded yeast strains Grow in the presence of Violacein Extract DNA and detect barcodes by chip hybridization Identify strains depleted in Violacein treated cultures
36 Identification of strains in the pool Barcoded heterozygous pool 6000 different (barcoded) heterozygous deletion strains
37 Chemogenomic profiling of heterozygous strains grown for 24h in 40μM Violacein
38 The CCT chaperonin complex Essential for actin and tubulin folding Conserved from Archeae to Humans Ring structure with 8 sub-units
39 Transient disruption of yeast actin by violacein Validate the mode of action of Violacein in Plasmodium; Test the anti-plasmodial potential of violacein intermediates (deoxyviolacein, deoxyviolaceinic acid, proto deoxyviolaceinic acid); Test the effect of violacein and intermediates against drug resistant Plasmodium strains; Investigate the effect of violacein in parasite adhesion and pathogenesis (intense reorganization of the cytoskeleton is essential for adhesion); Solve the structure of Violacein with the CCT complex to allow the optimization of Violacein derived compounds (collaboration with Marcio Dias).
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41 MRPs are ABC transporters that can confer resistance to multiple drugs; We constructed a synthetic GFP tagged P. vivax - MRP1 for expression in yeast; This transporter is functional in the heterologous host; PvMRP1-GFP will be used in drug screens to identify inhibitors of pleiotropic drug export.
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