Contributions For The Improved Control Of

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Contributions For The Improved Control Of

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CONTRIBUTIONS FOR THE IMPROVED CONTROL OF Leishmania infantum INFECTION: ROLES OF IRON,
REACTIVE OXYGEN SPECIES AND AMINOQUINOLINES
SÍLVIA MARISA NOGUEIRA DO VALE COSTA
Tese de doutoramento em Ciências Biomédicas
2012

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SÍLVIA MARISA NOGUEIRA DO VALE COSTA
CONTRIBUTIONS FOR THE IMPROVED CONTROL OF Leishmania infantum INFECTION: ROLES OF IRON, REACTIVE OXYGEN SPECIES AND AMINOQUINOLINES
Tese de Candidatura ao grau de Doutor em Ciências Biomédicas, submetida ao Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto.
Orientadora – Doutora Maria Salomé Custódio Gomes Categoria – Professora Associada Afiliação – Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto Co-orientadora – Doutora Ana Maria Luís Ramos Tomás Categoria – Professora Associada Afiliação – Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto
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This work was financed by the doctoral fellowship SFRH/BD/36661/2007 from the Fundação para a Ciência e a Tecnologia (FCT, Portugal).
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Acknowledgements
Apart from one’s efforts, the success of any work depends largely on the encouragement and guidance of many others. I take this opportunity to express my gratitude to those who have been instrumental in the completion of my PhD project.
My first words of appreciation go to my supervisor Professor Salomé Gomes from the Iron and Innate Immunity Group at IBMC, who encouraged me to engage in doctoral research and mentored my project. Without her critical sense, great patience and excellent writing skills this dissertation would not have materialized. I am also grateful to my co-supervisor Professor Ana Tomás from the Molecular Parasitology Group at IBMC for the constructive discussions and comments on the work, for permitting me to freely circulate in her laboratory and interact with her students. Likewise, I am very thankful to Professor Rui Appelberg from the Laboratory of Microbiology and Immunology of Infection at IBMC and Professor Pedro Rodrigues from the Iron and Innate Immunity Group at IBMC, who have welcomed me into their groups in distinct phases of my PhD and allowed me to conduct my research without restrictions.
I would like to acknowledge Professor Paula Gomes and Dr Nuno Vale from the Centro de Investigação em Química at FCUP for introducing me into the field of medicinal chemistry and for the enthusiasm demonstrated throughout the course of our collaboration.
Lastly, it was a great pleasure and privilege to have worked with Carolina Caldas, Tânia Silva, João Neves, Sandro Gomes and Tiago Duarte from the Iron and Innate Immunity Group, António Barroso and Ana Rita Gomes from the Laboratory of Microbiology and Immunology of Infection, as well as Sandra Carvalho and Tânia Cruz from the Molecular Parasitology Group. These were excellent bench companions that aided me to conduct my experiments in a very pleasant environment. In particular, I am indebted to my “mycobacterial” PhD fellows Sandro Gomes, António Barroso and Ana Rita Gomes for passing on to me their knowledge and expertise, for their ready availability, patience and companionship in and out of the laboratory. To Sandra Carvalho and Tânia Cruz I would like to acknowledge the sharing of reagents, protocols and thoughts on the intricacies of working with iron and Leishmania, as well as to thank for the amusing moments outside the laboratory.
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Table of Contents
LIST OF ABBREVIATIONS ................................................................................................ 9 ABSTRACT ....................................................................................................................... 13 RESUMO ........................................................................................................................... 15 CHAPTER I | GENERAL INTRODUCTION........................................................................... 19
1. LEISHMANIASIS...........................................................................................................................19 1.1. General considerations.....................................................................................................19 1.2. Clinical presentations .......................................................................................................20 1.3. Diagnosis..........................................................................................................................21 1.4. Treatment .........................................................................................................................22 1.4.1. Current therapeutic modalities ..................................................................................22 1.4.2. Developing new chemotherapeutics for VL: primaquine derivatives.........................24 1.5. Control and prevention .....................................................................................................26
2. Leishmania-HOST INTERACTIONS ...............................................................................................27 2.1. Life cycle of Leishmania ...................................................................................................27 2.2. Host immune response to Leishmania .............................................................................29 2.2.1. Organ-specific immunity in VL ..................................................................................30 2.3. Host antimicrobial oxidative mechanisms.........................................................................31 2.3.1. Leishmania susceptibility to host derived oxidants ...................................................34 2.3.2. Leishmania resistance to host derived oxidants .......................................................35
3. IRON AND Leishmania-HOST INTERPLAY......................................................................................37 3.1. Iron properties and functions ............................................................................................37 3.2. Iron and infection ..............................................................................................................37 3.2.1. Host iron homeostasis...............................................................................................37 3.2.2. Host mechanisms of iron withholding........................................................................39 3.2.3. Leishmania iron acquisition .......................................................................................40 3.2.4. Host iron status and Leishmania infection ................................................................41
CHAPTER II | DEFINITION OF OBJECTIVES ...................................................................... 45
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CHAPTER III | IRON OVERLOAD FAVORS THE ELIMINATION OF Leishmania infantum FROM MOUSE TISSUES THROUGH INTERACTION WITH REACTIVE OXYGEN AND NITROGEN SPECIES ............................................................................................................................ 49 CHAPTER IV | PHAGOCYTE NADPH OXIDASE, BUT NOT NITRIC OXIDE SYNTHASE 2, IS ESSENTIAL FOR THE CONTROL OF Leishmania infantum IN THE MOUSE TISSUES ............ 71 CHAPTER V | PEPTIDOMIMETIC AND ORGANOMETALLIC DERIVATIVES OF PRIMAQUINE ACTIVE AGAINST Leishmania infantum ................................................................................ 81 CHAPTER VI | GENERAL DISCUSSION ............................................................................. 97
1. RELATIONSHIP BETWEEN THE HOST IRON STATUS AND Leishmania INFECTION...............................97 1.1. Iron mediates host resistance to Leishmania: a role for ROS and RNS...........................97 1.2. Influence of iron overload on the host immune response to Leishmania .......................100 1.3. Host iron overload offers resistance to re-infection with Leishmania .............................102 1.4. Host iron deficiency does not correlate with susceptibility to Leishmania ......................102 1.5. Iron exerts direct toxicity towards Leishmania................................................................104 1.6. Iron potential as a therapeutic tool to fight Leishmania infections ..................................105
2. ROLE OF ROS AND RNS IN THE CONTROL OF Leishmania INFECTION.........................................106 2.1. ROS, but not RNS, mediate the in vivo control of L.infantum infection ..........................106 2.2. In vivo control of other Leishmania species by ROS and RNS ......................................108 2.3. Factors influencing the in vivo control of Leishmania by ROS and RNS........................109
3. VL TREATMENT: POTENTIAL OF PQ-DERIVED COMPOUNDS AS THERAPEUTIC AGENTS ...................109 3.1. Modification of PQ side chain as a strategy to improve its anti-leishmanial activity: imidazolidin-4-one and ferrocene moieties............................................................................110 3.2. Screening for anti-leishmanial drugs: intramacrophagic amastigote assay....................112 3.3. In vivo studies on the efficacy, stability and toxicity of PQ derivatives ...........................113 3.4. Mechanism of action of PQ and its analogues ...............................................................114 3.5. Improvement of PQ peptidomimetic and organometallic derivatives..............................115
CHAPTER VII | CONCLUDING REMARKS........................................................................ 119 REFERENCES ................................................................................................................ 123 APPENDIX | PUBLICATIONS AND COMMUNICATIONS IN SCIENTIFIC MEETINGS........... 161
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List of abbreviations
AmB: amphotericin B ABC: ATP-binding cassette 8-AQ: 8-aminoquinoline BSA: bovine serum albumin CarboxyPQ: carboxyprimaquine CD163: hemoglobin scavenger receptor / hemoglobin-haptoglobin receptor CD91: low-density lipoprotein receptor-related protein / heme-hemopexin receptor CL: cutaneous leishmaniasis CYBRD1: cytochrome b reductase 1 DC: dendritic cell DCL: diffuse cutaneous leishmaniasis DCT1: divalent cation transporter 1 DCYTB: duodenal cytochrome b DFO: desferrioxamine dLN: draining lymph node DMEM: Dulbecco's modified eagle medium DMT: divalent metal transporter FAD: flavin adenine dinucleotide FBS: fetal bovine serum Fc: ferrocene FLVCR: feline leukemia virus subgroup C cellular receptor FPN: ferroportin FT: ferritin HB: hemoglobin HBSS: Hank's balanced salt solution
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HEPES: hydroxyethyl piperazineethanesulfonic acid HIV: human immunodeficiency virus Ig: Immunoglobulin IFN: interferon IL: interleukin IRE: iron responsive element IREG1: iron-regulated transporter 1 IRP: iron regulatory protein KC: Kupffer cell LABCG5: Leishmania ATP-binding cassette, sub-family G, member 5 LCCM: L929 cell conditioned medium LF: lactoferrin LFR: Leishmania ferric iron reductase LHR: Leishmania heme response LIT: Leishmania iron transporter LPG: lipophosphoglycan LPS: lipopolysaccharide MAA: medium for axenic amastigotes MCL: mucocutaneous leishmaniasis MHC: major histocompatibility complex MTP: metal transporter protein MZ: marginal zone NADPH: nicotinamide adenine dinucleotide phosphate NOS: nitric oxide synthase NRAMP: natural resistance associated macrophage protein ON: overnight PALS: periarteriolar lymphoid sheaths PBS: phosphate buffered saline
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IronLaboratoryControlRosIbmc