Download Doctoral thesis from the Department of Immunology,

Doctoral thesis from the Department of Immunology,
Wenner-Gren Institute, Stockholm University, Sweden
Bioactive leishmanicidal alkaloid molecules from Galipea
longiflora Krause with immunomodulatory activity
Jacqueline Calla-Magariños
Stockholm 2012
All previously published papers were reproduced with permission from the publishers
©Jacqueline Calla-Magariños, Stockholm, Sweden 2012
ISBN 978-91-7447-586-9
Printed by Universitetsservice AB, Stockholm 2012
Distributed by Stockholm University Library
2
“Happy are those who dream dreams and are ready to pay the price to make them come
true”
Leon J. Suenes
A Walter, Adriana y Walter (jr), con quienes recorrí esta larga jornada
3
SUMMARY
According to the last report from WHO, leishmaniasis is endemic in 98 countries or
territories, with more than 350 million people at risk. Published figures estimated an
incidence of 2 million new cases per year (0.5 million of visceral leishmaniasis (VL) and l.5
million of cutaneous leishmaniasis (CL). VL causes an estimated more than 50 000 deaths
annually and 2 357 000 disability-adjusted life years lost, placing leishmaniasis ninth in a
global analysis of infectious diseases.
Treatment of leishmaniasis has been based on the use of pentavalent antimonials but problems
of toxicity and developing resistance have been reported. Traditional medicine and scientific
studies have shown that the raw extract of Evanta (Galipea longiflora, Angostura longiflora
(Krause) Kallunki) exhibits antileishmanial activity. We hypothesized that the healing
observed when using this plant might not only be due to the direct action on the Leishmania
parasite, but possibly to a parallel effect on the host immune response to the parasite. We first
determined the toxicity of an alkaloid extract of Evanta (AEE) on eukaryotic cells in vitro and
afterwards analyzed the effect of AEE on parasite growth. At 10 µg/ml we observed
inhibition of Leishmania braziliensis promastigote growth while viability of eukaryotic cells
was practically not affected. The whole extract was also found to be stronger than 2phenylquinoline (2Ph), the most prominent alkaloid in AEE. AEE did not directly stimulate B
or T cells or the mouse J774 macrophage cell line. However, AEE interfered with the
activation of both mouse and human T cells, as revealed by a reduction of in vitro cellular
proliferation and IFN- production. The effect was more evident when the cells were
pretreated with AEE and subsequently stimulated with the polyclonal T-cell activators either
Concanavalin A (ConA) or anti-CD3.
AEE treatment also reduced Leishmania-specific re-stimulation of lymphocytes both in vitro
and in vivo as revealed by the reduced production of IFN-γ, IL-12 and TNF, signature
cytokines of a Th1 immune response and also responsible of inflammatory reactions. More
important, AEE treatment of mice hosting L. braziliensis modified the dynamics of the
infection and showed that AEE is able to control both inflammation and parasite load.
Additionally, the healing process was improved when AEE and the conventional drug
meglumine antimoniate (SbV) were administered simultaneously.
Dendritic cells (DCs) play a pivotal role in T-cell stimulation and polarization of naïve T cells
towards a Th1, Th2, Th17 or regulatory phenotype. Therefore, we investigated if AEE could
alter the maturation/activation of DCs and if allostimulatory DCs properties were altered if
activated in the presence of AEE or 2Ph. Neither AEE nor 2Ph altered the expression of
activation markers on DCs, but the production of IL-12p40 and IL-23 was reduced. When we
analyzed the allostimulatory capacity of AEE or 2Ph-treated DCs, we found that AEE or 2Ph
did not affect the expression of various T-cell activation markers, but allogeneic CD4+ T-cells
secreted lower levels of IFN-γ following co-culture with DCs treated either with AEE or 2Ph.
In conclusion the work presented in this thesis provides valuable insight into the effects of
Evanta derived extract. The dual effect found for AEE, on the parasite and on the immune
response, suggests that AEE may be useful in controlling the parasite burden and preventing
over-production of inflammatory mediators, and subsequently avoiding tissue damage.
4
LIST OF PAPERS
This thesis doctoral is based on the following original papers (manuscripts), which will be
referred to by their Roman numerals:
I.
Calla-Magariños J, Giménez A, Troye-Blomberg M, Fernández C. An alkaloid
extract of Evanta, traditionally used as anti-Leishmania agent in Bolivia, inhibits
cellular proliferation and interferon-gamma production in polyclonally activated
cells. Scand J Immunol. 2009;69(3):251-8.
II.
Calla-Magariños J, Quispe Teddy, Giménez A, Freysdottir J, Troye-Blomberg M,
Fernández C. Quinolinic alkaloids from Galipea longiflora Krause suppress
production of proinflammatory cytokines in vitro and control inflammation in vivo
upon Leishmania infection in mice (Scand J Immunol. 2012. Accepted).
III.
Calla-Magariños J, Fernández C, Troye-Blomberg M, Freysdottir J. Galipea
longiflora Krause alkaloids modify functions of human dendritic cells by
decreasing the production of cytokines (Submitted).
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LIST OF ABBREVIATIONS
AEE
APC
CL
ConA
DAMPs
cDCs
DC
L-BMM
GM-CSF
HIV
IFN-γ
IL-12
imDCs
iNOS
i.p.
kDa
LPS
MCL
mDCs
MHC
NK
NO
PAMPs
PCR
PKDL
SbV
TCR
TLRs
TNF
VL
WHO
Alkaloid extract of Evanta
Antigen-presenting cell
Cutaneous leishmaniasis
Concanavalin A
Damage-associated molecular pattern
Classical or monocyte derived DC
Dendritic cell
Leishmania infected bone-marrow derived macrophages
Granulocyte- macrophage colony-stimulating factor
Human immunodeficiency virus
Interferon gamma
Interleukin 12
Inmature DCs
Inducible nitric oxide synthase
Intraperitoneal
kiloDalton
Lipopolysaccharide
Mucocutaneous leishmaniasis
Mature DCs
Major Histocompatibility complex
Natural-killer cell
Nitric oxide
Pathogen-associated molecular pattern
Polymerase-chain reaction
Post Kala-Azar dermal leishmaniasis
meglumine antimoniate
T-cell receptor
Toll-like receptor
Tumor necrosis factor
Visceral leishmaniasis
World Health Organization
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Table of contents
Summary .................................................................................................................................... 4
List of papers .............................................................................................................................. 5
Abbreviations ............................................................................................................................. 6
Chapter I ................................................................................................................................... 9
1. Traditional Herbal Medicine............................................................................................. 9
1.1 Introduction ................................................................................................................... 9
1.2 Some historical facts ................................................................................................... 10
1.3 Biological background ................................................................................................ 11
2. The Amazonas and Evanta ............................................................................................. 12
2.1 The Amazon rainforest ............................................................................................. 12
2.2 Galipea longiflora Krause (Evanta) ......................................................................... 13
3. Leishmaniasis ................................................................................................................. 15
3.1 Global burden ........................................................................................................... 15
3.2 Etiology of leishmaniasis .......................................................................................... 16
3.3 Taxonomy ................................................................................................................. 17
3.4 Life Cycle ................................................................................................................. 17
3.5 Clinical forms ........................................................................................................... 18
3.6 Current treatment available....................................................................................... 19
3.6.1 Pentavalent antimonials ................................................................................ 19
3.6.2 Amphotericin B ............................................................................................ 20
3.6.3 Paromomycin ................................................................................................ 20
3.6.4 Miltefosine .................................................................................................... 21
Chapter II................................................................................................................................ 22
1. The immune system ........................................................................................................ 22
2. The immune response in leishmaniasis .......................................................................... 23
2.1 The battle of Leishmania with host mechanisms-the complement system .............. 23
7
2.2 Phagocytosis, an important defense mechanism against infection , but Leishmania
parasite take4s advantage of it .................................................................................. 25
2.3 Participation of neutrophils and monocytes ............................................................. 28
2.4 The central role of DCs............................................................................................. 30
3. Induction of adaptive immune responses ....................................................................... 33
4. Regulation of the inflammatory response ....................................................................... 38
5. Experimental models of leishmaniasis ........................................................................... 40
5.1 Experimental infection with L. braziliensis .............................................................. 41
Chapter III .............................................................................................................................. 44
1. The present study ...................................................................................................... 44
2. Materials and methods .............................................................................................. 44
3. Results and discussion .............................................................................................. 45
4. Overall conclusions .................................................................................................. 54
5. Future investigation .................................................................................................. 56
Acknowledgements .............................................................................................................. 57
References ............................................................................................................................ 60
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CHAPTER I
1. Traditional Herbal Medicine
1.1 Introduction
Traditional herbal medicine is the most ancient form of healthcare known to mankind; herbs
have been used in all cultures since history started to be documented. Herbal medicine
sometimes known also as phytotherapy, phytomedicine, herbalism or botanical medicine is
the use of herbs for their therapeutic or medicinal value. A medicinal herb is a plant or a part
of a plant (leaf, stem, root, bark, etc.) that can be used for healing purposes.
The World Health Organization (WHO) estimates that around 4 billion people, 75-80 % of
the world population mainly in the developing countries, use herbal medicine for some aspect
of primary health care. WHO indicates that of 119 plant-derived pharmaceutical medicines
about 74 % are used in modern medicine in ways that correlate directly with their traditional
practices as plant medicines by native cultures (1-4). Now, pharmaceutical companies are
leading research on plant materials collected from the rain forests for their potential medicinal
value.
The use of plant materials as a source of medicines for a wide variety of human diseases have
increased because of better cultural acceptability, better compatibility with the human body
and lesser side effects, and also because of insufficient supply of drugs, unaffordable cost of
treatments and development of resistance to currently used drugs for infectious diseases (1-4).
The major limitation of the incorporation of herbal medicine into modern medical practices is
the lack of scientific and clinical data and better understanding of their efficacy and safety.
9
The research on traditional medicinal plants has to be focused on providing scientific
evidence for the presence of active molecules and assessment of specific effects and
toxicities. To ensure the efficacy, the quality and the safety of herbal products, standardization
is of vital importance, so that the herbal medicines can be safely used to treat the diseases (25).
1.2 Some historical facts
The use of plants as medicines precedes written human history; archaeological data indicate
that humans were using medicinal plants since the Paleolithic era, around 60,000 years ago.
The study of herbs started in ancient times, according to written records available, with the
Sumerian civilization in 4000 B.C., who created stone tablets with lists of hundreds of
medicinal plants. Ayurveda medicine in India has used herbs as early as 1900 B.C. Sanskrit
writings such as the Rig Veda from around 1500 B.C., are some of the earliest available
documents detailing the medical knowledge that formed the basis of the Ayurveda system.
The ancient Egyptians, in 1536 B.C., wrote the Ebers Papyrus (6), with information on over
850 medicinal plants. The first Chinese herbal document , the Pen Tsao, written by the
Chinese emperor Shennong, lists 365 medicinal plants and their use, which includes Ephedra
(ephedrine to modern medicine) and chaulmoogra (plant of the genus Hydnocarpus, one of
the first effective treatments for leprosy). De herbis et curis and Therapeutics are the writings
from Greek and Roman medicinal practice that provided the pattern for later western
medicine (7).
During the Early Middle Ages, Benedictine monasteries were the primary source of medical
knowledge in Europe and England. However, most of their efforts were focused on translating
10
and copying ancient Greco-Roman and Arabic works, rather than creating new information
(7).
Modern medicine has been effective in the treatment of many diseases, resulting in an
increase in the life span of the population; however, negative factors such as side effects and
high cost, among others, have led to the adaptation of herbal medicine as a valuable
alternative.
Currently, many drugs used in modern medicine derive from traditional medicine, e.g. opium,
aspirin, atropine, quinine, etc. There are about 120 active molecules obtained from higher
plants and used by modern medicine today and many of them are used in a way that is very
close to their traditional use (8).
The study of natural products with potential use in medicine as a goal must be done in
association/collaboration with field ethnobotanists in order to develop reasonable laboratory
hypotheses and for a more productive work (9,10).
1.3 Biological background
All plants synthesize chemical substances as part of their normal metabolic activities. These
phytochemicals are of two types: primary metabolites, used by the plant for its basic functions
(e.g. sugars, fats), and secondary metabolites for specific functions, for example, used as a
defense or to attract insects for pollination. It is this secondary type of phytochemicals which
has potential therapeutic actions in humans. Plants synthesize a variety of secondary
metabolites but most are derivatives of a few biochemical structures: polyphenols, glucosides,
terpenes and alkaloids.
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Alkaloids are a class of chemical compounds containing a nitrogen ring and can be purified
from crude extracts by acid-base extraction (Fig.1). Among known alkaloids are caffeine and
nicotine (stimulants); morphine (analgesic); berberine (antibacterial); vincristine (anticancer)
and quinine (antimalarial) (11).
Fig. 1. Basic structure of quinoline alkaloids
2.
The Amazonas and Evanta
2.1 The Amazon rainforest
The Amazon rainforest covers a vast area of Ecuador, Peru, Bolivia and Brazil, around the
Equator. It is a dense, warm, wet forest that treasures an incredible biodiversity. The Amazon
rainforest in South America is the world's greatest natural and the most powerful and bioactively diverse natural phenomenon on the planet (12). Rainforest plants are rich in secondary
metabolites, particularly alkaloids. The National Cancer Institute in USA identified 3000 plants
that are active against cancers. Seventy percent of these plants are found in the rainforest.
Twenty-five percent of the active ingredients in today's cancer-fighting drugs come from
organisms found in the rainforest. Vincristine, extracted from the rainforest plant periwinkle
(Catharanthus roseus), is one of the world's most powerful anticancer drug. It has dramatically
increased the survival rate for acute childhood leukemia since its discovery (13,14).
12
In 1983, there were no pharmaceutical manufacturers in USA involved in research programs to
discover new drugs from plants. Today, over 100 pharmaceutical companies are engaged in
plant research projects for possible drugs to treat different diseases (13,15).
2.2 Galipea longiflora Krause (Evanta)
G. longiflora (Angostura longiflora Krause) is a tree of the family Rutaceae found in the
Amazon area of South America. This genus has around 40 species distributed from
Guatemala to Bolivia and in the south region of Brazil. The tree is 12 m high, has strong smell
and is characterized by the common occurrence of spines and winged petioles and alternate or
opposite, simple or palmately or reduced to spine leaves. In Bolivia this tree is found in the
tropical woods of the last Andean counterforts in the areas of Beni and La Paz and is used as
antiparasitic medicinal plant. G. longiflora is known widely by its vernacular name Evanta.
The information about the traditional uses of G. longiflora is mainly from the ethnicities
Tacana, Mosetene (La Paz) and Tsimane (Beni). An ethnobotanic survey and workshop with
representatives of 16 communities has been published which includes a list of 140 Amazonian
plant species and its medicinal uses. This field work has been published as a book with more
than 30 co-authors and was developed in coordination with the Indigenous council of the
Tacana Communities (Consejo Indígena de Los Pueblos Tacana, CIPTA). The identification
of the specie and the voucher samples of G.longiflora are found in the “Herbario Nacional” of
La Paz (AS49, zona de Caicahuara y SD17, Santa Rosa de Maravillas) (16-18).
The traditional and frequent use of Evanta is in the treatment of diarrhea caused by intestinal
parasites and as fortifying agent for children and adult people. For the treatment of
13
leishmaniasis, fresh or dehydrated bark is smashed and applied directly on the ulcers; in
addition, the infusion of the plant is drunk (19).
During the period 1985-91, a group of French-Bolivian researchers confirmed the
antiparasitic/anti-Leishmania activity of the extracts obtained from Evanta. A total of 12
quinolinic alkaloids were isolated and identified from the leaves, bark and roots. Some of the
alkaloids isolated from Evanta showed to be new structures and due to the efficacy
demonstrated against the parasite Leishmania and also on in vivo models of infection, they
were patented (Chimaninas A, B, C y D, US4209519/15/04/93) (20-23).
In 2006 G. longiflora was selected as one of the most promising plants of more than 800
crude extracts and pure substances evaluated for its antiparasitic activity using in vitro
methods (24). Further studies have been done on the toxico-kinetic behavior and on acute and
chronic toxicity in murine model using crude extracts, total alkaloids and pure substances
obtained from the bark of Evanta (25,26).
In the present study the crude total alkaloid extract of Evanta (AEE) was used. The major
alkaloid component of this extract is 2-phenylquinoline (2Ph). In the following figure the
alkaloids present in the crude extract are shown (21,25,26).
14
OCH3
N
N
N
[MS+1]= 206 (C15 H11 N) [MS+1]= 200 (C14 H17 N) [MS+1]= 236 (C16 H13 N O)
N
O
N
O
[MS+1]= 172 (C12 H13 N)
[MS+1]= 278 (C18 H15 N O2)
OCH3
OCH3
N
N
O
O
[MS+1]= 230 (C15 H19 N O)
[MS+1]= 308 (C19 H17 N O3)
OCH3
N
[MS+1]= 228 (C15 H17 N O)
Fig.2. Alkaloids present in AEE.
3. Leishmaniasis
3.1 Global burden
According to the last report from WHO, leishmaniasis is endemic in 98 countries or
territories, with more than 350 million people at risk. Published figures estimated an
incidence of 2 million new cases per year (0.5 million of visceral leishmaniasis (VL) and l.5
million of cutaneous leishmaniasis (CL). VL causes an estimated of more than 50 000 deaths
15
annually, a rate surpassed among parasitic diseases only by malaria, and 2 357 000 disabilityadjusted life years lost, placing leishmaniasis ninth in a global analysis of infectious diseases
(27).
3.2 Etiology of leishmaniasis
Leishmaniasis is caused by the obligate intracellular parasite Leishmania spp and belongs to
the kinetoplastid family. The parasitic protozoa are transmitted by female sand flies belonging
to the genus Phlebotomus or Lutzomyia.
Leishmania exists in two basic forms: amastigote and promastigote. The amastigote is the
intracellular form present in the vertebrate host, it is a non-motile form and it divides by
longitudinal binary fission at 37 °C. Amastigotes are 3-6 µm in length and 1.5-3.0 µm in
width. Even though amastigotes seem to lack a flagellum, it is simply that the flagellum does
not protrude beyond the body surface and it cannot be seen by light microscopy (Fig. 3a).
Amastigotes are taken up from the blood of an infected host when the female sand fly bites,
and in the sand fly gut they develop into promastigotes where they multiply.
Promastigotes are 15-30 µm in body length and 5 µm in width; it is extracellular, has a
flagellum, is motile, and grows and divides by longitudinal binary fission at 27 °C in the sand
fly. Promastigotes can be grown in vitro at 25 °C (Fig. 3b).
a
b
Fig. 3. A macrophage containing Leishmania amastigotes (a) and Leishmania promastigotes (b).
16
3.3 Taxonomy
The parasites of the genus Leishmania are among the most diverse of human pathogens, both
in terms of geographical distribution and the variety of clinical syndromes that they cause
(24). Over 20 species and subspecies of Leishmania infect humans, each causing a broad
spectrum of clinical manifestations.
The genus Leishmania has been classified into two subgenera: Leishmania, present in both the
old and the new worlds, and Viannia, restricted to the new world. The classification of
Leishmania is shown in further detail in Fig. 4 (27).
Family
Trypanosomatidae
Genus Crithidia Leptomonas Herpetomonas Blastocrithidia Leishmania Sauroleishmania Trypanosoma Phytomonas Endotrypanum
Subgenus
Leishmania
Species
L. donovani
L. chagasi
L. donovani
L. infantum
L. tropica
L. Major
L. aethiopica
L. killicki* L. Major L. aethiopica
L. tropica L.garnhami L. Peruviana
Viannia
L. mexicana
L. amazonensis
L. Pifanoi
L. mexicana
L. venezuelensis
L. braziliensis
L. guyanensis
L. braziliensis Unassigned:
L. Panamensis L.Liansoni
L. guyanensis
Fig. 4. Taxonomy of Leishmania parasite.
3.4 Life cycle
Leishmaniasis is transmitted by the bite of a female phlebotomine sand fly. The sand fly
injects the infective stage, metacyclic promastigotes, during a blood meal. Metacyclic
promastigotes that reach the puncture wound are phagocytozed by macrophages and
transform into amastigotes. Amastigotes multiply in infected cells and affect different tissues,
depending on the Leishmania species. This starts the clinical manifestations of leishmaniasis.
17
Sand flies become infected during blood meals on an infected host when they ingest
macrophages infected with amastigotes. In the midgut of the sand fly, the parasites
differentiate into promastigotes, which multiply, differentiate into metacyclic promastigotes
and migrate to the proboscis (28,29) (Fig. 5).
Fig. 5. Leishmania life cycle (Nat Rev Immunol. 2002 Nov;2(11):845-58). Reprinted with permission from Nature
Publishing Group.
3.5 Clinical forms
The range of manifestations of leishmaniasis is wide and presents in four different clinical
forms: CL, MCL, VL and post Kala-Azar dermal leishmaniasis (PKDL).
CL is mainly caused by Leishmania major and Leishmania tropica in the old world and
Leishmania braziliensis and Leishmania mexicana in the new world. But CL may be caused
by other Leishmania species such as Leishmania guyanensis, Leishmania naiffi, Leishmania
18
shawi, Leishmania lainsoni, Leishmania amazonensis, Leishmania panamensis, and
Leishmania pifanoi. The species involved depend on the geographical distribution, and the
disease may appear as simple or diffuse ulcerations on skin, mainly in the face, causing
sometimes disfiguration of the patient. The disease may regress spontaneously or evolve, thus
requiring treatment (27,30-32).
L. braziliensis is also the principal causative agent for MCL, though additional species have
been described (L. amazonensis, L. panamensis and L. guyanensis). MCL is characterized by
chronic ulcers on the skin, mouth and nose, with destruction of underlying tissue (e.g. nasal
cartilage). Tissue destruction with disfigurement can be very severe (27,31,32).
Leishmania parasites can also produce more severe, life-threatening VL caused by the
Leishmania donovani in the Indian subcontinent, Asia and East Africa or Leishmania
infantum in Europe, North Africa and Latin America and Leishmania infantum/chagasi in
Europe and Latin America (27,28).
3.6 Current treatment available
3.6.1
Pentavalent antimonials
Two pentavalent antimonials
are available:
meglumine antimoniate and sodium
stibogluconate. They are chemically similar, and their toxicity and efficacy are related to their
antimonial (SbV) content. Pentavalent antimonials are usually administered parenterally
(intramuscularly) but can be administered intralesionally for the treatment of CL (33,34).
Pentavalent antimonials have been in use against leishmaniasis for more than six decades.
Although they have an obvious effect on the parasite, their molecular and cellular
19
mechanisms of action are not yet well understood. While there are two forms, SbV and SbIII
(trivalent antimony), it is not clear yet which is the active form (35). Initial studies suggested
that sodium stibogluconate SbV inhibits macromolecular biosynthesis in amastigotes (36),
possibly via perturbation of energy metabolism due to inhibition of glycolysis and fatty acid
beta-oxidation (37). However, the specific targets in these pathways have not been identified.
In humans, pentavalent antimonials can produce severe side effects, such as cardiotoxicity
and hepatotoxicity, when administered systemically (38).
3.6.2
Amphotericin B
Amphotericin B is a polyene antibiotic, administered intravenously. The primary site of action
of amphotericin B on L. donovani promastigotes appears to be membrane sterols that result in
a loss of the permeability barrier to small metabolites (39).
Among the side effects, nephrotoxicity is common, leading to frequent interruption of
treatment in some patients. Other uncommon but serious toxic effects are hypokalemia and
myocarditis. Treatment should always be given in a hospital to allow continuous monitoring
of patients.
3.6.3
Paromomycin
Paromomycin (aminosidine) is an aminoglycoside antibiotic, usually administered by
intravenous infusion. Paromomycin inhibits protein synthesis and modifies membrane fluidity
and permeability of the parasite (40). Other studies have reported that exposure of L.
donovani promastigotes and amastigotes to paromomycin, decreased their mitochondrial
potential, indicating that mitochondria are the targets of this drug (41).
20
Treatment with paromomycin produces reversible ototoxicity in 2 % of the patients, renal
toxicity is rare and some patients may develop hepatotoxicity when administered parenterally.
3.6.4
Miltefosine
This alkyl phospholipid (hexadecylphosphocholine) was originally developed as an oral
anticancer drug but was shown to have antileishmanial activity. It has been demonstrated that
miltefosine induces apoptosis-like death in L. donovani (42).
Miltefosine commonly induces gastrointestinal side effects, such as anorexia, nausea,
vomiting (38 %) and diarrhea (20 %). Most episodes are brief and resolve as treatment is
continued. Occasionally, the side-effects can be severe and require interruption of treatment.
Miltefosine is potentially teratogenic and should not be administrated to pregnant women.
21
CHAPTER II
1. The immune system
Humans, as other mammals, live in an environment that is inhabited by a vast range of
microorganisms, pathogenic and non-pathogenic, and has huge numbers of substances that are
a potential threat to their survival. The immune system has as a primary function to protect
the host against microbial invasion, but it also has to maintain homeostasis.
The immune function has been theoretically divided into innate and adaptive immunity,
which both have to work together. Innate immunity is the first line of defense, characterized
by rapid response to a large but limited number of stimuli. Its components include physical,
chemical and biological barriers, specialized cells and soluble molecules. These components
are present in all individuals, regardless of previous contact with the antigen. Among the
mechanisms of the innate immunity are phagocytosis, activation of the complement system,
activation of natural-killer (NK) cells, secretion of preformed molecules, such as
antimicrobial agents (e.g. defensines), proteins of the complement system, newly synthesized
molecules, such as acute phase proteins and release of cytokines and chemokines. Innate
immunity is activated by specific stimuli; the well-known pathogen associated molecular
patterns (PAMPs), and endogenous damage-associated molecular patterns (DAMPs). PAMPs
and DAMPs interact with receptors on the surface of the innate immune cells, the pattern
recognition receptors (PRR), such as Toll-like receptors (TLRs), receptors that are already
encoded in the germline.
22
The second line of defense constitutes the adaptive (or acquired) immune response that is
characterized by a late response. The mechanisms of the adaptive responses are activation of
T and B cells, production of cytokines and antibodies. The main characteristics of the
adaptive responses are: high specificity of recognition for any foreign molecule, memory
(long-lived cells that persist and can be restimulated rapidly after encounter with the same
specific antigen) and tolerance to self components. Adaptive responses are activated by
interaction with the antigen-specific receptors expressed on the surface of T and B-cells and
encoded by genes that are assembled by somatic rearrangement of germ-line-gene elements to
form a T-cell receptor (TCR) and immunoglobulin B-cell receptor (BCR) genes.
2. The immune response in leishmaniasis
2.1 The battle of Leishmania with host defense mechanisms-the complement
system
The complement system is an efficient mechanism of the innate immune response that helps
to clear pathogens from the organism by disrupting the membrane of the target cell, and
consists of a tightly regulated network of more than 30 proteins (43). Apart from the lysis of
target cells, the central effect of complement activation, opsonization of pathogens and
cellular activation (chemotaxis) are also important. The complement system can be activated
through different pathways that are summarized in Fig. 6. Most of the complement
components circulate as soluble proteins in the blood and other body fluids in an inactive
form while others are present as membrane-associated proteins.
23
C4bC2b
C3 convertase
C4bC2bC3b
C5 convertase
Fig. 6. The three different pathways of complement activation: alternative, classical and lectin pathways; factors
that can inhibit the pathways are indicated in boxes ( Cell Tissue Res. 2011 Jan;343(1):227-35). Reprinted with
permission from Springer.
When an infected female sand fly inoculates metacyclic promastigotes into the skin dermis of
the host during blood feeding, the promastigotes interact immediately with serum
components. Metacyclic promastigotes of Leishmania have been shown to activate
complement components of both the lectin and the alternative pathways. Opsonization of
Leishmania metacyclic promastigotes with complement is fast and lysis via the membrane
attack complex (C5b–C9 complex) (MAC) begins 60 s after serum contact. Complement
activation results in efficient killing of 90 % of all inoculated parasites within 3 min (44,45).
Leishmania parasites must evade complement-mediated lysis until they are engulfed by a
24
macrophage. The resistance to the complement lysis depends on the morphological stage of
the parasite, L. major procyclic promastigotes, for example, cannot resist complement action,
while the metacyclic form can fully avoid complement lysis (46). This difference in
complement resistance has been shown to depend upon lipophosphoglycan (LPG), the main
surface molecule on the promastigote parasite surface. The LPG structure differs between
Leishmania species and also on the morphological stage of the parasite (47). LPG found on
the surface of metacyclic promastigotes is longer and that seems to be the reason why CAM
can not attach, so lysis is eluded. However, L. donovani promastigotes prevent C5 convertase
formation by adhering the inactive C3bi subunit on their surfaces (48). Another important
molecule that contributes to parasite virulence is the surface glycoprotein gp63. This is a
metalloprotease, also called leishmanolysin, that is abundant on the membrane surface of all
Leishmania species. Gp63 may contribute to parasite virulence by controlling complement
fixation while taking advantage of the opsonic properties of complement (49,50).
2.2 Phagocytosis, an important defense mechanism against infection, but
Leishmania parasite takes advantage of it
The major phagocytic cells are neutrophils, macrophages, monocytes and dendritic cells
(DCs). Phagocytic cells use different Fc receptors and complement receptors to improve
uptake of particles that have been marked by the adaptive and innate immune responses for
elimination (51). These cells engulf microbes and localize them in intracellular vacuoles
(phagosomes) where they are exposed to toxic effector molecules when lysosomes fuse with
the phagosomes. Among the toxic molecules are radical oxygen intermediates (ROI)
(superoxide, hydroxyl radicals) and radical nitrogen intermediates (RNI) such as nitric oxide
(NO), and degradative enzymes. Phagolysosomes have an acidic pH and their content of
25
hydrolases and degradative enzymes are capable of breaking down the internal components of
the phagocytized particle.
Several macrophage subsets with distinct functions have been described. Two main subsets
are the classically activated macrophages (M1 macrophages), which mediate defense and
inflammatory reactions and the alternatively activated macrophages (M2 macrophages),
which have anti-inflammatory function and regulate wound healing (52).
Leishmania parasites are obligate intracellular protozoan microorganisms, contributing to
their own phagocytosis by favoring the opsonization with C3bi interacting with the
macrophage complement receptor 3 (CR3), rather than with C3b interacting with CR1. When
the parasite attaches to the macrophage using CR3 instead of CR1, it eludes oxidative burst
during phagocytosis (53).
Once inside of the macrophage, Leishmania promastigotes acquire different tactics to evade
toxic lysosomal compounds found in phagolysosomes. It has been shown that Leishmania
parasites delay normal phagosome formation/maturation allowing time for the parasite to
develop into the more acid hydrolase resistant amastigote form (54). The delay in phagosome
maturation results in delay of maturation by failure to recruit lysosomes that contain cytotoxic
chemicals. This mechanism depends on the promastigote cell surface molecule LPG that
prevents the formation or disrupts lipid microdomains on the phagosome membrane by
modifying the molecular structure of lipid bilayers (55-57). Once the promastigote has
entered the phagosome, LPG inserts into lipid rafts and inhibits phagosome–lysosome fusion.
L. donovani accumulates a ring of F-actin around the phagosome providing a physical barrier
preventing interaction with late endosomes. It is believed that the enlarged F-actin ring
26
surrounding the phagosome stops the fusion of membrane markers with the phagosome. LPG
also plays an active role by inhibiting lysosomal enzymes, too. Additional metacyclic
promastigote-derived virulence factors may cross the phagosome membrane and thereby
reach their cytosolic targets (55). (Fig. 7).
Fig. 7. Mechanisms of evasion of Leishmania parasite (Nat Rev Microbiol. 2011 Jul 11;9(8):604-15). Reporinted with
permission from Nature Prublishing Group.
When the phagolysosome is formed, the acidic enzymes present in the lysosomes, get in
contact with Leishmania parasites, but the parasites can maintain its intracellular pH near to
neutral due to a proton pump that is found on its surface. The acid phosphatases also found on
the surface of Leishmania parasites inactivate the oxidative activity of superoxide and
hydroxyl radicals produced by macrophages (58-60). It has been shown that some Leishmania
species possess mechanisms to resist the action of ROI or RNI. One of the mechanisms is the
recognized ROI-scavenging function of phosphoglycans and the expression of superoxide
dismutase, catalase and/or peroxiredoxins most of which are shown to promote the
persistence of Leishmania within macrophages (61-64). Phagolysosomes, with their
microbicidal mechanisms, represent an aggressive environment that Leishmania parasites
27
must subvert and finally evade. Amastigotes, then, replicate (up to 1,000- fold), and
eventually the macrophage is lysed and the infectious amastigotes are released into the milieu,
where they infect neighboring macrophages (60).
2.3 Participation of neutrophils and monocytes
The tissue damage produced by the mosquito bite induces the release of endothelial
chemotactic cytokines that recruit neutrophils, eosinophils and macrophages and initiates a
strong inflammatory reaction. In addition, promastigotes induce macrophages to secrete
CCL2 (MCP-1) and CXCL1 (IL-8, in humans) that act as chemoattractants for monocytes and
neutrophils, respectively. Development of clinically evident lesions occurs coincident with the
influx of these inflammatory cells (65,66). In Fig. 8 the monocyte-to-macrophage recruitment
and initiation of the inflammatory response processes are shown.
28
Fig. 8. Iniciation of inflammatory response in Leishmania infection. During infection and tissue stress,
monocyte recruitment has a key role in providing the damaged tissues with adequate numbers of macrophages.
Leishmania major parasites that have infiltrated the skin after a sandfly bite elicit a local macrophage response.
Extravasation of the monocytes is followed by differentiation into macrophages that phagocytose the parasites
and present their antigens to T cells. Interferon-γ (IFNγ) production from T cells drives an M1 response that
contains parasite growth. TH1, T helper 1. (Nat Rev Immunol. 2011 Oct 14;11(11):723-37). Reprinted with
permission from Nature Publishing Group.
29
2.4 The central role of DCs
DCs are the most potent antigen-presenting cells (APCs), capturing, processing and
presenting antigens to initiate an acquired immune response or induce tolerance. DCs are
considered to be the link between innate and adaptive immunity because they are recruited
and activated by effectors of innate immunity and can stimulate the adaptive immune
response by presenting antigens to T cells, both CD4+ and CD8+, and B cells (67).
DCs are a heterogeneous cell population with different subsets identified. In mice and humans
there are two types of DCs: classical or myeloid DCs (cDCs) and plasmacytoid DCs (pDCs),
both containing distinct subsets. To date, many different classifications of the cDCs exist, e.g.
based on location and/or expression of surface markers, and they are often not the same for
mice and humans. One such classification is the division of DCs into immature DCs (imDCs)
and mature DCs (mDCs).
ImDCs reside in tissues where they patrol their surroundings for harmful signals. They
express many receptors on their surface that aid in phagocytosis of antigens, such as Fc
receptors, complement receptors and PRRs. Activation through PRRs leads to stimulation of
the imDCs where they undergo a process called maturation. They lose their phagocytizing
capacity and upregulate the surface expression of MHC class I and class II peptide complexes
and co-stimulatory molecules (CD40, CD54, CD80 and CD86), and start to secrete
proinflammatory cytokines (Fig. 9) (71,72). Mature DCs (mDCs) also upregulate chemokine
receptors enabling migration into draining lymph nodes. All these phenotypic changes in DCs
allow them to prime T-cell mediated immune response (72).
30
Fig. 9 ImDCs are present in the tissue where they are act as “sentinels” and capture any antigen they encounter.
DCs recognize microbes, and secrete cytokines. Cytokines secreted by DCs in turn activate effector cells of
innate immunity such as eosinophils, macrophages and NK cells. Activation/maturation triggers DCs migration
towards secondary lymphoid organs. These migratory mDCs display antigens in the context of classical MHC
class I and class II or non-classical CD1 molecules, which allow selection antigen-specific T lymphocytes.
Activated T lymphocytes reach the injured tissue, where they eliminate microbes and/or microbe-infected cells.
B-cells, activated by DCs and T- cells differentiate into plasma cells that produce antibodies against the initial
pathogen. (Immunity. 2010 Oct 29;33(4):464-78). Reprinted with permission from Elsevier.
Activated DC produce IL-12, especially important because its expression during infection
regulates innate responses and determines the type and duration of adaptive immune response
mainly attributed to its involvement in the development of Th1 cells. IL-12 promotes the
differentiation of naïve CD4+ T-cells into T helper 1 (Th1) cells that produce IFN-γ and aid in
cell-mediated immunity. IL-12 induces IFN-γ production by NK cells, T-cells, DCs, and
macrophages (73).
31
Approximately six weeks post Leishmania infection, the number of cDC increases at the site
of infection, as does the percentage of infected DCs. DCs are attracted through mechanisms of
the innate immune response, e.g. mast cell-derived mediators, cytokine and chemokine
release by neutrophils (74,75).
The imDCs phagocytose the amastigote form of the parasite mainly through the Fc receptor
(FcR) I and FcRIII. In the case of L. major, imDCs are activated and they upregulate MHC
class I and II expression as well as co-stimulatory molecules (76). At the same time infected
DCs release proinflammatory cytokines including IL-12.
Many studies have shown that IL-12 is necessary not only in the initial defense of Leishmania
infections, but also to maintain an established response against this parasite (77-80).
In addition, it has been suggested that IL-12 is indispensable in the development of a
protective immune response and that in the absence of IL-12, susceptibility to L. major is due
to the failure to induce a Th1 response but not because of the development of a Th2 response
(81). Conversely, recent studies have demonstrated that under certain circumstances Th1
development can be achieved in the absence of IL-12 (82).
Besides IL-12, there are other cytokines that contribute to induce a Th1 response, such as IL23, IL-27 and IL-1β, which are also secreted in the early stages of infection. IL-18 is another
proinflammatory cytokine that can help in evoking Th1 immune responses particularly in
collaboration with IL-12. A very important fact is that the sustained release of IL-12 allows
the perpetuation of Th1 immunity (83).
32
L. amazonensis infection induces relatively low levels of DC maturation/activation (in terms
of expression of surface markers as well as the production of IL-12p40), while L. braziliensis
infection induces high levels of activation in both infected DCs and bystander DCs. Both
infected and bystander DCs are capable of priming naïve CD4+ T cells in vitro; therefore,
strong DC activation is a characteristic for L. braziliensis infections (84).
IL-10 is another cytokine involved in the immune response to Leishmania; this cytokine can
suppress Th1 responses with the consequent effect on the activation of macrophages. IL-10
was not considered to be important in L. major infection, because treatment of BALB/c mice
with an anti-IL-10 monoclonal antibody had almost no effect on disease progression (85). On
the contrary, IL-10-deficient mice with a BALB/c background were markedly more resistant
to L. major infection than wild-type BALB/c mice (86,87). IL-10-receptor blockade has been
shown to confer partial resistance to L. major in wild-type BALB/c mice. Finally, in humans
overproduction of IL-10 in lesional tissue from VL patients and in plasma from patients with
PKDL has been found (86-89).
3. Induction of adaptive immune responses
When DCs present antigens to naïve CD4+ T cells, they stimulate them to proliferation and
promote the polarization towards a different immune response, mainly Th1, Th2, Th17 and T
regulatory (Treg) response. The interaction of co-stimulatory molecules with their respective
ligands and the local cytokine environment directs the differentiation. It has been proposed
that there are two distinct subsets of dendritic cells, known as DC1 and DC2, which in turn,
direct a Th1 or Th2 differentiation pathways, respectively. In Th1 polarization, certain
pathogens or PAMPs trigger APCs, through TLRs, to secrete IL-12, which promotes the
33
differentiation of naive T cells into Th1 cells (29). In this model Th2 polarization might be
due to an inability of antigen to activate DCs to produce IL-12 (Fig.10). Th1 immune
responses are characterized by the production of IFN- and tumor necrosis factor (TNF),
among others (28,29).
Fig. 10. Polarization of Th1 and Th2 cells by DCs. The interaction of co-stimulatory molecules with their
respective ligands and the local cytokine environment stimulates the differentiation Th1/Th2. It has been
proposed that there are two distinct subsets of dendritic cells, known as DC1 and DC2, which in turn, direct to a
Th1 or Th2 differentiation pathways (Nat Rev Immunol. 2002 Nov;2(11):845-58). Rerpinted with permission
from Nature Publishing Group.
IFN-γ is the signature cytokine of a Th1 immune response, mainly acting on macrophages,
NK cells and B cells. Macrophages stimulated with IFN-γ induce direct antimicrobial activity
and production of TNF as well as up-regulating antigen processing and presentation
34
pathways. In addition IFN-γenhances NK cell activity and regulates B-cell functions, such as
immunoglobulin (Ig) production and Ig class switching (90). TNF is a pleiotropic cytokine
linked to Th1 immune responses and to inflammation. TNF is a powerful pro-inflammatory
agent that regulates many facets of macrophage function and induces the production of other
cytokines, e.g. IL-1, IL-8, IL-6 and granulocyte-macrophage colony-stimulating factor (GMCSF) (91).
Th1 immune responses are critical for the control of Leishmania infection, mainly because of
the production of IFN-γ, which activates macrophages and DCs, leading to parasite death.
Most studies on leishmaniasis have been done in experimental models with L. major and the
importance of the Th1/Th2 paradigm in resistance or susceptibility to infection has been
demonstrated. According to this paradigm, some strains of mice such as C57BL/6 (resistant
strain) develop a self-limiting infection when infected with L. major, mediated by Th1immune response while BALB/c mice (susceptible strain) develop uncontrolled infection
mediated by Th2 immune response (29,92,93). However, the Th1/Th2 paradigm usually
applies in the case of L. major but it does not apply to mice infected by other Leishmania
species such as L. braziliensis or L. amazonensis (94). In the view of new discoveries on
CD4+ T cell populations among others, it seems that this paradigm may not be relevant
anymore (Fig. 11) (95).
35
Fig.11. The mechanisms that influence the expansion of different CD4+ T cell populations as part of the
adaptive immune response following Leishmania major infection, and their role in determining the outcome of
disease (Front Immunol. 2012;3:80).
The ability of DCs to induce Th1 or Th2 immunity may depend on the characteristics of the
microenvironment present when DCs first interact with the antigen. However, in Leishmania
cutaneous infections, DCs preferentially induce Th1/Tc1 immunity, priming both CD4+ as
well as CD8+ T cells (96-100). Activated CD4+ T cells can interact with macrophages through
CD40 and CD40L and induce IL-12 expression and production of NO from iNOS. CD40L
deficient mice are susceptible to Leishmania infection and produce less IL-12, IFN-and NO
compared with its wild type counterparts suggesting that CD40-CD40L interaction is required
for the clearance of the infection (96).
In Leishmania infection IFN-γis critical for macrophage activation as demonstrated by the
fact that knock out resistant mouse strains (either for IFN-γor IFN-γreceptor genes), were
36
unable to restrict growth of L. major in vivo and suffered fatal infection. It has also been
shown that, recombinant IFN-γwas capable of activating infected macrophages from both
resistant and susceptible mice to clear L. major in vitro (81,101).
The importance of IFN-γin the induction of NO was demonstrated by the finding that IFN-γ,
among a number of cytokines, was capable of independently enhance iNOS transcription and
NO release from stimulated mouse peritoneal macrophages (102). These results are consistent
with the impaired production of NO by macrophages from IFN-γor IFN-γreceptor gene
knock-out mice and the crucial role of IFN-in macrophage activation (74).
The role of TNF as anti-leishmanial cytokine has been extensively studied. The participation
of TNF in the development of a protective immune response to L. major infection has been
analysed in experimental CL and the results were contradictory. Administration of
recombinant murine or human TNF to resistant or susceptible strains of mice infected with L.
major ameliorated the course of disease by reducing lesion size and parasitic burden.
However, neutralizing with anti-TNF antibody transiently exacerbated disease in resistant
mice, although the course of infection in BALB/c mice was unaltered or affected minimally
(103-108).
Th17 cells have recently been described as a third subset of Th cells, and have provided new
insights into the mechanisms important in immune responses essential for effective
antimicrobial host defence (109). Th17 responses are critical for mucosal and epithelial host
defence against extracellular bacteria and fungi. However, recent studies have reported that
Th17 responses can also contribute to viral persistence and chronic inflammation associated
37
with parasitic infection. IL-17 has long been implicated in several inflammatory diseases
(110).
The role of Th17 cells in leishmaniasis is still not well understood. Some studies have
associated elevated levels of IL-17 together with IFN-γ production with healing in L.
donovani model (111). In humans, sub-clinical infection due to L. braziliensis correlated with
high levels of IL-17 (112), conversely the neutrophil infiltrate induced by IL-17 cause tissue
destruction in patients with mucosal leishmaniasis (113-115).
The role of CD8+ T cells in cutaneous leishmaniasis is not clear, some studies associated these
cells with control of the infection, such as in the case of VL, but studies of CL indicate that
CD8+ T cells were not important for the control of a primary infection but for the resistance to
reinfection (116). Other studies showed that recruitment of CD8+ T cells expressing the
granule-associated serine protease granzyme B is correlated with lesion progression in
patients infected with L. braziliensis (117).
3.1 Regulation of the inflammatory response.
Although the importance of a Th1 immune response in leishmaniasis has been demonstrated,
in humans the resulting inflammatory reaction produced might be associated with disease
exacerbation. The immunologic characteristics of disease vs. protection in VL and control vs.
chronicity in CL and MCL are very different and not so well understood.
In VL, many studies have shown that IFN-γ and IL-12 are decreased during the acute phase of
the disease, showing that immunosuppression occurs during VL (78,118-121). However, data
from these studies must be analyzed carefully because the immune system is in fact highly
38
activated during acute phase of VL, producing IFN-γ and TNF as well as IL-10 and TGF-β.
But these processes are expected to occur mainly in the spleen and liver and not in peripheral
blood. In this way peripheral blood mononuclear cells (PBMCs) respond poorly to
Leishmania antigens because reactive lymphocytes are trapped in the lymphoid organs (120).
Recently, high levels of IL-10 are suggested to play a role in counterbalancing the
exacerbated polarized response that may develop following a cure (122,123).
MCL is associated with a strong Th1-type immune response to Leishmania antigens with high
levels of TNF and IFN-γ. Larger lesions correlated with a higher frequency of the IFN-γ- and
TNF-producing lymphocytes, even if parasites were often absent or rarely found in lesions.
Exacerbated immune responses are not so favorable to the patient since it leads to an
uncontrolled inflammatory reaction causing tissue destruction (123-130).
The association of Leishmania-induced pathology and parasite persistence and production of
TNF has lead attempts to block TNF. Indeed, TNF blockade with pentoxyfyllin has some
remarkable clinical effects in drug resistant cases of CL (129-130). Although Th1-type
immune responses protect against most forms of leishmaniasis, it is likely that some types of
immune responses directed against Leishmania can lead to more severe clinical forms, while
others lead to resolution of the infection with little pathology. It seems as if a balance between
Th1 and Th2 type of immune responses against Leishmania is critical for the establishment of
effective control of the disease (131-134).
In summary it can be concluded that uncontrolled inflammatory reaction, as part of the
immune response, may lead to tissue damage, which means that inflammation must be
strongly regulated to avoid its destructive effects.
39
4. Experimental models of leishmaniasis
Murine experimental models of leishmaniasis have been developed to contribute to the
understanding of the pathogenesis of this disease. These studies have provided a great amount
of information about the host-parasite relationship and the factors involved in the
development of susceptibility or resistance and have defined mouse strains as being either
resistant or susceptible to leishmaniasis (135).
Genetic studies in mice can give valuable information about the complex interactions between
the parasite and the host, which indicate host genes that might control the outcome of the
infection by the induction of distinct components of the immune response. The existing
information about genetics of leishmaniasis is based mainly on mouse models with L. major
and L. donovani. These studies have shown a multigenic basis of susceptibility which
correlates well with the complexity of the immune response to infection (93,136).
Susceptibility and resistance data of various Leishmania species on some mouse strains are
summarized in Table 1. In mice, different forms of the disease depend on the species of
Leishmania and on the genetic background of the mouse (82,137). For example BALB/c mice
are susceptible to L. amazonensis infection and develop non-healing skin lesions with high
parasite loads and have a predominantly Th2-type of response (137). On the other, hand L.
braziliensis causes only a transitory disease, in mice, which depends on the production of a
Th1-type immune response. Studies have shown that the difference on the behavior is due to
immunoregulatory virulence factors produced by L. amazonensis, but not by L. braziliensis
(e.g. a parasite-derived serine protease activity) apart from the genetic background (138).
40
Table 1. Suceptibility of mice strains to Leishmania infection.
Leishmania specie
Susceptible mouse strain
Resistant mouse strain
L. braziliensis
----
BALB/c, C57BL/6
L. amazonensis
BALB/c
C3H/HePas
L. major
BALB/c, P/J, SWR/J and CcS16
AKR, B10.D2, CBA, C57BL/6, C3H,
DBA/2J
L. donovani
BALB/c, C57BL/6, C57BL/10,
CE/J, DBA/1J, SWV
A, AKR/J, 129/Sv, C3H/HeJ, CBA,
DBA/2
L. panamensis
The BALB/c and DBA/2J
C57BL/6
Hamsters and guinea pigs are also used as experimental models to study leishmaniasis;
hamsters provide an excellent susceptible host model which can be used for histopathological,
drug efficiency and vaccine studies. One of the problems is the lack of high quality
immunoreagents that limit immunological studies. Guinea pigs have been a traditional model
for studies of delayed type hypersensitivity and also to test potential antigens for use in
diagnostic tests.
4.1 Experimental infection with l. braziliensis.
Several studies with murine models of L. braziliensis have demonstrated that all mouse strains
tested are resistant to infection by this parasite (138,139). It has been shown that the weak
infectivity of L. braziliensis, in mice, is mainly dependent on IFN-γ activity. Many authors
have indicated that the resistance of mice to infection is due to the inability of the parasite to
elicit strong and sustained IL-4 production or production of other cytokines that inhibit the
41
development of a Th1-type immune response, but data are controversial. For example, IL-10
inhibits Th1 development; however, BALB/c mice produce equivalent amounts of IL-10
following infection with either L. braziliensis or L. major (140-144).
A study carried out in Brazil showed different behavior of two strains of L. braziliensis
isolated from different regions in the country. One of them (H3227) could induce detectable
lesions in BALB/c mice whereas the other strain (BA788) could not do so. The cells
recovered from the draining lymph nodes of BA788-parasitized mice produced higher levels
of IFN-γ, showing different immunological characteristics of the parasite strain. The authors
attributed the different behavior to their different genomic profiles (142).
As indicated before, infection with L. braziliensis in humans might lead to a self healing
infection, but it may also progress, later on, to a destructive, disfiguring disease. Efforts to
develop suitable experimental models that allow mimicking the biology of natural
transmission of L. braziliensis led to the use of peptides found in the saliva of the mosquito.
The saliva peptides are injected in conjunction with the parasite during blood feeding.
Lutzomyia longipalpis saliva has been shown to affect the course of the infection caused by L.
braziliensis which has been associated with an elevated IL-4 production and a reduction in the
IFN-γ/IL-4 ratio (145,146). It has been shown that mice inoculated with L. braziliensis
together with sand fly saliva developed lesions with heavily parasitized macrophages,
neutrophil and eosinophil infiltration, demonstrating that sand fly saliva changes the
inflammatory response. These effects are attributed at least in part to maxadilan, a polypeptide
that induces vasodilation and presents a range of immunomodulatory activities (147). It has
been found that maxadilan can inhibit T-cell proliferation and delayed-type hypersensitivity,
decrease TNF release by macrophages and increase LPS-induced IL-6 and IL-10 production,
42
both in vitro and in vivo (147). In addition, maxadilan inhibits the intracellular killing of
Leishmania by macrophages suggesting that the ability of the sand fly saliva to reduce
nitrogen oxidation in response to IFN-γ may be responsible for the inhibitory effect of the
saliva on the intracellular killing (148). In summary, maxadilan allows the parasite to
establish a more long lasting infection (149,150).
Although a great number of studies have been trying to elucidate the biology of Leishmania
infections and have helped to understand many aspects of the immune response to
intracellular pathogens, the immunopathogenesis of human leishmaniasis is still not fully
understood (95).
43
CHAPTER III
1. The present study
Detection of new active molecules for treatment of leishmaniasis has received considerable
attention over the past few decades (4,5,151,152). Evanta, a tree found in Bolivia, has been
associated with traditional medicine in the treatment of leishmaniasis and has been selected
among the most promising medicinal plants of Bolivia (24). Alkaloids purified from the plant
Evanta, have demonstrated leishmanicidal effect in vitro on various species of Leishmania
parasite, e.g. L. amazonensis, L. infantum and L. donovani (21,22). In in vivo studies some of
the alkaloids showed activity against Leishmania infected mice (23). In the present study we
hypothesized that the total alkaloid extract of Evanta (AEE) might have immunomodulatory
activity.
Specifically we examined the effect of AEE on:
-
Viability and proliferation of L. braziliensis promastigotes.
-
Activation of T-cell, including two different activation pathways: a) polyclonal and b)
Leishmania-specific activation.
-
Infection caused by L. braziliensis in mice.
-
Maturation and function of DCs.
2. Materials and Methods
The materials and methods for these studies are described in the separate papers.
44
3. Results and discussion
3.1 Paper I
In Latin America leishmaniasis is a major public health problem, with CL, mostly caused by
L. braziliensis in Bolivia, being the most frequent form of the disease (153,154).
Therapy of leishmaniasis is usually based on pentavalent antimonial drugs, but due to their
toxicity and increased resistance new therapeutic alternatives are needed (35). Additionally,
anti-leishmanial drugs are unaffordable in most of the affected countries and as there is no
effective vaccine available yet, chemotherapy remains the only alternative. In this sense
Evanta could have a great potential because of its leishmanicidal effects either used singly or
in combination with conventional anti-leishmanial drugs.
Plant-derived-drugs are mostly used by modern medicine in a way that is similar to their
traditional use by native cultures, which is why we studied the effects of a total alkaloid
extract of Evanta (AEE) rather than isolated molecules. To optimize our procedures we first
determined the toxicity of AEE on eukaryotic cells in vitro. This first step allowed us to find
the extract dose that would not affect the viability of the cells. We found that at 50 µg/ml
AEE was already toxic, so lower doses were used in the following experiments with
eukaryotic cells.
The second step was to analyze the effect of AEE on parasite growth. We found, as expected,
that doses of 50 µg/ml and higher caused a total elimination of the parasite. However, at 25
µg/ml a strong effect (97 %) was still detected while at 10 µg/ml a 69 % inhibition of parasite
45
growth was observed. It is important to underline that at this dose (10 µg/ml) the viability of
eukaryotic cells was practically not affected.
The most frequent alkaloid in AEE, 2Ph, could be an interesting candidate for the
development of an alternative treatment used as a single compound. In order to find out if the
isolated 2Ph was as efficient as AEE, we compared their effects on in vitro culture of L.
braziliensis. We found that AEE had stronger inhibitory effect than 2Ph on the parasite
growth, indicating possible additive anti-leishmanial effects among the alkaloids found in
AEE.
Many studies have demonstrated the importance of the immune response on the control of
Leishmania infections. These studies have indicated a beneficial effect of a Th1 type of
immune response to control the infection properly. Several mechanisms are triggered during
T-
and
B-cell
activation:
proliferation/expansion,
differentiation/maturation,
production/secretion of active molecules (e.g. cytokines, chemokines, Igs) and memory
formation. To examine this we investigated the effects of AEE on T and B cells. As a measure
of T and B-cell activation we studied proliferation and production of cytokines and Igs. We
also tested the direct effect of AEE on macrophages. Our results indicated that AEE was not
directly stimulating B-cells, T-cells or macrophages.
Thereafter, we analyzed the effects of AEE on activated murine spleen cells and human
PBMCs and observed that AEE affected proliferation of spleen cells at 25 µg/ml, while
PBMCs were affected already at 10 µg/ml. 2Ph showed similar effect compared with AEE.
The effect of AEE was shown to be specific and not due to an effect on the viability of the
cells in contrast to what has been seen for curcumine (alkaloid molecule).
46
According to the well-known Th1/Th2 paradigm of Leishmania infection, IFN-γ is one of the
most important cytokines for an effective immune response. We tested the effect of AEE on
polyclonally induced of IFN-γ production of murine spleen cells and human PBMCs. We
used two different polyclonal T-cell activators, Concanavalin A (ConA) and monoclonal antiCD3 antibody, to evaluate different activation pathways.
IFN-γ production by murine T cells was decreased by AEE in a dose dependent manner,
regardless of the polyclonal activator used (ConA or anti-CD3) (P=0.0005 or 0.001,
respectively). We also observed that the effect was more evident when the cells were
pretreated with AEE and subsequently stimulated with ConA or anti-CD3 as compared with
cells treated with AEE and simultaneously stimulated with polyclonal activators. IFN-γ
production by ConA-activated human PBMCs was reduced at lower concentrations of AEE
than was seen with murine spleen cells, which may due to the different cellular composition
of murine spleen cells as compared with PBMCs.
Our results showed an unexpected effect of AEE on the immune response, parameters that,
according to the literature, are decisive to control Leishmania infection and to eliminate the
parasite: polarization to a Th1-type immune response with the production of IFN-γ, TNF and
NO (102-104,106,118,121). Although the development of a Th1-type immune response is
important in the control of leishmaniasis, many studies have shown that the maintenance of a
chronic inflammatory response could potentially lead to pathological manifestations and
tissue damage (126-132).
47
Our results suggest that AEE has direct leishmanicidal effect as well as inhibitory effects on
T- cell activation, as measured by proliferation and IFN-γ production, and might contribute to
the control of the chronic inflammatory reaction seen in Leishmania infection.
3.2 Paper II
In this paper we studied how AEE affected Leishmania specific reactivation of lymphocytes.
More importantly, we analysed the capacity of AEE to control Leishmania infection in mice.
Our previous studies showed that AEE, in addition to its leishmanicidal activity on L.
braziliensis, could reduce polyclonal induced cellular proliferation and IFN-γ production. In
order to determine if AEE could similarly interfere with a Leishmania-specific activation of
lymphocytes we examined whether AEE could affect the antigen specific reactivation of
lymphocytes in vitro using different mechanisms of activation.
Therefore, mice were immunized with total Leishmania lysate (TLL), TLL-primed spleen
cells were restimulated in vitro either with TLL or bone marrow derived macrophages
infected with L. braziliensis (L-BMM).
We first tested the effect of AEE in vitro on spleen cells from TLL-immunized mice. We
found that Leishmania-specific restimulation was affected by in vitro pre-treatment with AEE,
similarly to that observed for polyclonally activated cells.
To test the effect of AEE in vivo, mice immunized with TLL were treated with AEE two
weeks post infection and then the spleen cells were restimulated in vitro. We found that
restimulation of spleen cells with TLL and L-BMM had different effect. In AEE-treated mice,
restimulation with L-BMM showed a clear reduction in IFN-γ production, whereas IFN-γ
48
production after restimulation with TLL did not differ between treated and untreated mice.
The reasons for the differences observed using these two ways of stimulation are not known
but could be due to different antigenic epitopes being presented by the BMMs that has
actively phagocytized and processed a microorganism (a more natural process) as compared
with the splenic APCs (155).
We further showed that AEE considerably inhibited the production of IL-12 and TNF while
no effects were seen on other cytokines, e.g. IL-10.
Pentavalent antimonial has remained the first choice to treat leishmaniasis for decades, so it
was of interest to investigate the effect of meglumine antimoniate (SbV) in our models. We
found that SbV had no effect on antigen-specific reactivation of cells, and that the combined
treatment AEE/SbV did not alter the effect seen using AEE alone. The results indicated that
the combination of AEE plus SbV did not synergize or antagonize the effects on the
Leishmania-specific reactivation of lymphocytes in vitro.
An important question to address was whether the administration of AEE might be able to
control an in vivo infection. We tested the effect of AEE on BALB/c mice infected with L.
braziliensis. Similarly to humans, BALB/c mice are relatively resistant to infection with
Leishmania. It has been reported that in L. braziliensis-infected BALB/c mice there is an
intense inflammatory reaction due to T-cell sensitization and enhanced production of IFN-γ
and TNF (the major cytokines involved in the pathogenesis) (86,134). The development L.
braziliensis skin lesions is also accompanied by the expression of a broad spectrum of
chemokines that are known to attract neutrophils, monocytes/macrophages, NK cells, and
CD4+ and CD8+ T-cells (156).
49
When BALB/c mice were injected with L. braziliensis, a transient inflammation that resolved
in few days was obtained. Therefore, we co-injected maxadilan with the parasite to resemble a
natural infection by means of a sand fly bite. It has been reported that the salivary gland
polypeptide maxadilan enhances Leishmania infections.
Inoculation of L braziliensis in combination with maxadilan modified the inflammatory
response induced by the infection in BALB/c mice, increased the infectivity and exacerbated
the infection (both lesion size and parasite survival) as shown previously (145,146). Other
studies have reported that the observed effects of maxadilan are modulation of the host
immune response, e.g. inhibited TNF release and increased IL-6 production, which leads to
extensive accumulations of heavily parasitized epithelioid macrophages, with persistent
neutrophilia and eosinophilia in the lesions (147-151).
We showed that i.p. treatment with AEE, SbV and AEE/SbV differently affected the kinetics
of infection. AEE treated mice controlled efficiently the inflammation during the first weeks
post infection (p.i.) compared with PBS-treated mice. In contrast, SbV-treated mice did not
seem to control inflammation as efficiently as AEE treated mice (p<0.1).
The parasite load, was also affected differently, with. AEE-treated mice being able to control
the parasite load more efficiently compared with PBS treated mice. When we compared the
effect of AEE with SbV we found that SbV could control the parasite load more efficiently.
Apparently, AEE could control the inflammatory reaction more efficiently than the parasite
load but SbV reduced more efficiently the parasite load, but it could not control the
inflammation in the same effective way as AEE during the first weeks p.i.
50
When we combined the treatment with AEE and SbV, the overall inflammatory process
seemed to be controlled more efficiently; both the footpad thickness and the parasite load
compared with when AEE and SbV were used separately. Thus, it looks like the inflammatory
process is caused both by the parasite itself and also by the immune response developed in
response to the infection. SbV controlled the parasite burden, while AEE controlled the
parasite burden as well as the inflammatory reaction. This is in agreement with other studies
that have shown the beneficial effects of combining the use of SbV with other drugs
(129,130).
In summary, the data presented here suggested that AEE, in addition to its leishmanicidal
effect, also could be considered as an immunomodulatory agent. We can conclude that the
effect on the inflammatory reaction seen in mice treated with AEE most likely is due to the
capacity of AEE to suppress the production of proinflammatory cytokines. Moreover, the
combined use of AEE and SbV may be a more efficient alternative for the treatment of
leishmaniasis.
3.3 Paper III
DCs are considered the bridge between innate and adaptive immunity, they can stimulate the
adaptive immune response, both humoral and cellular. DCs play an important role in T-cell
stimulation and polarization of naïve T-cells towards a Th1, Th2, Th17 or regulatory
phenotype. The ability of DCs to induce Th1 or Th2 immunity may depend on the
characteristics of the microenvironment present when DCs first interact with the antigen
(157,158). In leishmaniasis, the interactions between the parasites and DCs are complex and
involve contradictory functions that can stimulate or suppress T-cell responses, leading to the
51
control of infection or progression of disease (159,160). Thus, since DCs are pivotal cells in
eliciting an immune response, it was of importance to find out whether AEE might induce or
suppress the maturation/activation of DCs and/or affect their functions.
We first examined the effects of AEE and 2Ph on DCs maturation by examining the
expression of maturation markers and production of cytokines. We found that neither AEE
nor 2Ph altered the expression surface activation markers on the DCs, but both AEE and 2Ph
were able to reduce the production of IL-12p40, an important Th1 signature cytokine (78),
and IL-23, a Th17 promoting cytokine (110,161). On the other hand IL-10 and IL-6 were not
significantly reduced by AEE or 2Ph treatments. These results are consistent with our
previous results and indicate that AEE and 2Ph may have an anti-inflammatory effect.
Therefore, we analyzed the allostimulatory capacity of AEE- or 2Ph-treated DCs and we
found that AEE or 2Ph-treated DCs did not affect the expression of various T-cell activation
markers, such as CD54, CD62L, CD69 and CD40L on co-cultured allogeneic CD4+ T cells.
This shows that CD4+ T-cells were fully activated in the presence of DCs, regardless of the
treatment DCs received during their stimulation. In contrast, the allogeneic CD4+ T cells
secreted lower levels of IFN-γ following co-culture with the AEE-DCs and 2Ph-DCs. As IL12 is the main inducer of Th1 polarization, the reduction in IFN-γ secretion by the CD4+ Tcells observed in this study is most likely resulting from a reduced ability of the DCs
stimulated in the presence of AEE or 2Ph to secrete IL-12.
DCs treated with AEE or 2Ph secreted reduced levels of IL-6 and IL-23, both cytokines that
affect Th17 polarization and hence IL-17 secretion of Th cells (162). However, the levels of
IL-17 secretion by the allogeneic CD4+ T-cells were not significantly affected by co-culture
52
with the AEE-DCs or 2Ph-DCs. Th17 differentiation is affected by other cytokines which
may be counteracting the effect of IL-6 and IL-23, resulting in unchanged levels of IL-17.
The fact that IFN-γ, IL-12p40, IL-6 and IL-23 were significantly decreased following
treatment with AEE or 2Ph suggests an effect of the drugs in the control of chronic
inflammatory reaction.
In addition, several studies have reported that extracts from plants are able to affect DC
stimulation (163-167). Our results suggest that AEE and 2Ph interfere with LPS-induced
stimulation of DCs, possible affecting any of a number of signaling pathways that are
involved in LPS-induced DC activation and maturation, including MAP kinase, PI3 kinase,
P38 SAP kinase, and NF-κB pathways (168,169). Whether the NF-κB signaling pathway or
other pathways are affected in this case remains to be determined and will be the next part of
this project.
In conclusion, our results showed that AEE and 2Ph affect the LPS-induced maturation of
DCs and their ability to stimulate allogeneic CD4+ T-cells, indicating an immunomodulatory,
predominantly anti-inflammatory, capacity, in addition to their leishmanicidal activity.
53
4. Overall conclusions
Treatment of leishmaniasis remains controversial and the need to develop new alternatives to
the conventional used drugs is clear. Generally, the treatment of leishmaniasis has been based
on the use of pentavalent antimonials but problems of toxicity and developing resistance have
been reported. In this scenario many investigations started to look into the plant kingdom that
could offer an unlimited source of new drugs, which in the past have demonstrated to be
effective.
In contrast to the idea of using drugs to treat infectious diseases and only considering the
effects on the microorganism, without taking into account the host, new interesting
approaches are being developed in which the host also provides an environment that is
dynamic and plays an important role in the outcome of the disease.
We studied the effects of a total alkaloid extract obtained from the plant known as Evanta
(AEE) and postulated that due to its capacity to reduce cellular proliferation and production of
the proinflammatory cytokines IFN-γ, IL-12 and, TNF (important mediators of inflammation)
it might control the inflammatory response. This observation correlated with the effects seen
on an in vivo infection with L. braziliensis, where AEE controlled inflammation at the site of
the infection more efficiently than SbV. We could also show that AEE reduced the production
of cytokines in mature DCs as well as their capacity to activate T lymphocytes. The most
relevant results showed the reduction of proinflammatory cytokines IL-12p40 and IL-23 of
AEE-treated and 2Ph-treated DCs. We also found that activation of DCs in the presence of
AEE or 2Ph affected the capacity of DCs to stimulate CD4+ T cells observed by a reduction in
54
the production of IFN-γ. All these results, in line with our previous findings, suggest that AEE
may be useful in controlling inflammatory responses at many levels (Fig. 12).
Fig. 12. Suggested model of the effects of AEE on Leishmania infection. AEE inhibits the parasite growth (1).
At the same time AEE affects the production of IFN-γ by T-cells (2). AEE also affects the stimulation of DCs
(3), causing a decrease in various cytokines (IL-12p40, IL-23) (4), which in turn affects the secretion of IFN-γ,
IL-6 and also IL-10. The decrease in the production of proinflammatory cytokines produces indirectly the
decrease in the secretion of acute phase proteins and chemtotaxis of neutrophils (5), controlling inflammation. In
this way AEE might control the parasite burden and the inflammatory response, in summary the overall infection
process.
The analysis of the activity of the purified molecule 2Ph showed that due to the weaker effect
on the parasite (albeit its similar effects on the immune response), it would not be beneficial
to use it as compared with AEE. This is in line with our main suggestion that natural products
should be used as close to its traditional use as possible.
55
Although we could not find any effect of SbV on the immune responses studied, we found
that in vivo combined AEE/SbV treatment was extremely effective in the control of both
inflammation and parasite load. This suggests that combined treatment might be an interesting
approach that must be investigated further in the future. Using SbV at lower concentrations
when used in combination with AEE might also be the solution to problems of toxicity as well
as resistance.
5. Future investigation
We consider that two important questions need to be addressed in the future, first is to find
out which signaling activation pathways are affected by AEE. Similar alkaloids have
demonstrated to have immunomodulatorty effects in the control of inflammatory reactions
and have shown to affect different signaling activation pathways. Some of these studies
pointed out that the activity on some signalling molecules and transcription factors such as
MAP kinase phosphorylation and activation of NF-κB, Stat and others, all involved in the
production of proinflammatory cytokines, were blocked (158-160). Whether AEE can also
affect these signaling pathways remains to be determined.
Secondly, it is very important to find out if the complementary in vivo effects seen for the
combined treatment could lead to a reduction in the dose of SbV which would mean less
toxicity/costs for the patients. This would initially be investigated by combined treatment in
mice infected with a resistant strain of the parasite.
Answering these questions will
demonstrate the real potential of the AEE in the treatment of leishmaniasis.
56
ACKNOWLEDGEMENTS
This work was carried out at the Department of Immunology, Wenner Gren Institute,
Stockholm University, Sweden, at the Department of Immunology and Center for
Rheumatology Research, Landspitali - The National University Hospital of Iceland,
Iceland and at the Laboratory of Immunology, SELADIS Institute, Universidad Mayor de
San Andrés, Bolivia.
I want to express my sincere gratitude to all those who contributed in many ways to make
this thesis possible, and an unforgettable experience for me. I especially want to
acknowledge:
My supervisors Carmen Fernandez and Marita Troye-Blomberg, for the guidance,
encouragement and friendship you gave me during all these years, for your valuable
advice and constructive criticism; with your concern and generous support you made this
thesis possible, forever, thank you!
Jona Freysdottir, words fail me to express my sincere gratitude for your insightful
suggestions and contributions to this thesis; my deep appreciation for accepting me in
your lab and for giving me the opportunity to learn. You have been great to me; I will
always remember you and the time I spent in Reykjavik.
The “seniors” at the Department: Klavs Berzins, Eva Sverremark-Ekström and Eva
Severinson, for sharing your knowledge with all the students and for your kindness and
sympathy.
My sincere thanks to the colleagues and students (past and present) at the department of
immunology, Stockholm University, for the friendship and support.
I am thankful to John Arko-Mensah, for your friendship, for all the useful and helpful
assistance and for many perceptive comments. To Esther Julian for the interesting
suggestions and help, and to Magdi Ali for all your support and for having time to listen.
You were among those who kept me going at the beginning.
The staff at the Department, especially to Margareta Hagstedt (“Maggan”), Gelana Yadeta
and Anna-Leena Jarva for your kind assistance.
The staff at the animal house, especially to Eva Nygren, for taking care of my mice and
for making the animal house a pleasant place to work, my sincere gratefulness.
57
To my wonderful friends in Sweden, Annette Kolb and Ana Sola-Gomez, for the special,
true and great friendship, for the support you gave me in hard times, forever thank you. I
treasured all precious moments we shared.
I owe gratitude to Swechha Mainali Pokharel, my great friend in Iceland, who willingly
dedicated so much time in giving assistance to me, for sharing your expertise with me. I
will always remember the afternoons walking downtown Reykjavik, you are such a great
person! I also want to thank Sigrún Þórleifsdóttir, I appreciate your kindness and
warmth.
To the students and staff at the Department of Immunology and Center for
Rheumatology Research, Landspitali - The National University Hospital of Iceland, my
gratitude for your kind assistance and sympathy.
To Alberto Giménez for providing me with the extract of “our plant” and all related work,
thank you!
For the people in Bolivia,
A mi familia,
Walter, mi amado esposo, por tu incansable solicitud y amor, por tu apoyo constante y por
ser la fuerza que me hace seguir adelante en la vida. A mis adorados hijos Adriana y
Walter, por ser los mejores hijos que alguien pueda desear, por haber estado dispuestos para
charlar y escucharme. Por alentarme y darme las fuerzas que necesite en momentos
difíciles. A ese pedacito de vida que apenas empieza y que nos ilumina y nos hacer sonreír
aun antes de haber nacido.
A mis queridos papás, Rolando y Maria Luisa, por su amor, por ser el modelo que seguí
toda mi vida. A mis hermanos por su apoyo durante todo este tiempo, especialmente a José,
imprescindible en mis temporadas en Suecia, sin ti no lo habría logrado.
A mi querida familia “Magariños” muchas gracias por todo el apoyo, en especial a mi
“papa David”, se que a pesar de no estar conmigo, me miras desde el cielo y compartes
conmigo estos momentos, gracias por darme ese ejemplo de amor.
A la Rectora de la Universidad Mayor de San Andrés, Dra. Teresa Rescala, por su apoyo
durante el desarrollo de mis estudios y por su linda amistad.
A los colegas y estudiantes del laboratorio de Inmunología del Instituto SELADIS de la
Universidad Mayor de San Andrés, Teddy Quispe (mi co-autor), Fabiola Montes, Ruth
58
Guzmán, Gladys Pérez, Wendy Villarreal, Carla Díaz, por el trabajo eficiente durante mis
temporadas de ausencia del “labo”, por el cariño y amistad; muy especialmente a Karina
Delgado por ser una persona íntegra, humana y profesionalmente, gracias por su cariño y
apoyo constantes.
A mis queridas amigas y compañeras de trabajo, Raquel Calderón, Mercedes Morales y
Maryluz Soto.
I want to thank the support of the Swedish International Development Cooperation Agency
(SIDA) for the funding provided.
Finally, I want to thank God for EVERYTHING (“If God brings you to it, He will bring
you through it”. Unknown).
59
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