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Iron and Infections by Protozoa*

João Pedro Oliveira Morgado Madaíl1,2

1Faculdade de Medicina da Universidade de Coimbra

2Médico Interno de Patologia Clínica no Hospital Curry Cabral (Lisboa)

E-mail address: madailjoaop@gmail.com

Resumo

O ferro, um elemento essencial para quase todos os organismos vivos, constitui um desafio

nas interacções entre o hospedeiro e os patogénios infecciosos. Esta revisão sumariza as

características mais importantes no que diz respeito ao metabolismo do ferro e à sua regulação

no corpo humano e analisa como o ferro pode afectar a interacção do hospedeiro humano com

alguns protozoários patogénicos.

O metabolismo do ferro deve ser eficazmente regulado para alcançar um equilíbrio

necessário para a saúde, inclusive para uma imunidade eficiente. A contínua descoberta e

caracterização de várias vias envolvidas no metabolismo do ferro e na sua regulação têm

contribuído para a nossa compreensão desta área. A recente descoberta da hepcidina, o

regulador-chave do metabolismo do ferro num contexto sistémico, é um exemplo deste facto.

Alguns protozoários são causas importantes de morbidade e de mortalidade e uma

estratégia de defesa importante do nosso organismo apoia-se na redução da disponibilidade do

ferro, a nível intra e extracelular, para estes parasitas. Assim, a aquisição de ferro pode

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constituir um desafio para os protozoários patogénicos, que serão tanto mais virulentos quanto

mais capazes forem de superar os obstáculos e satisfazer as suas necessidades em relação a

este elemento. A investigação nesta área está a revelar alvos possíveis e estratégias para

desenvolver novos tratamentos / medicamentos.

É necessária mais investigação para completar o “puzzle” do metabolismo do ferro no

organismo humano e para caracterizar melhor a importância do ferro nas infecções por

protozoários.

Palavras-chave: ferro, protozoário, infecção, macrófago, anemia

* Trabalho Final no âmbito do Ciclo de Estudos de Mestrado Integrado em Medicina com

vista a atribuição do grau de Mestre em Medicina

Abstract

Iron, an essential element for almost all living organisms, constitutes a challenge in host –

infectious pathogen interactions. This review summarizes the most important features

concerning iron metabolism and its regulation in the human body and analyzes how iron can

affect the interaction of the human host with some protozoan pathogens.

. Iron metabolism must be tightly regulated to achieve an equilibrium necessary for the health,

including for an efficient immunity. The continued discovery and characterization of several

pathways involved in iron metabolism and in its regulation have contributed to our

understanding of this field. The recent discovery of hepcidin, the key regulator of iron

metabolism in a systemic context, is an example of this.

Some protozoans are important causes of morbidity and mortality and an important defense

strategy of our organism rely on the reduction of iron availability, at intra and extracellular

levels, to these parasites. So, iron acquisition can be a challenge to protozoan pathogens that

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will be the more virulent the more able they are to overcome obstacles and satisfy their needs

regarding this element. Research on this area is revealing possible targets and strategies to

develop new treatments / drugs.

Further research is needed to complete the puzzle of iron metabolism in the human body

and to better characterize the importance of iron in protozoan infections.

Keywords: iron, protozoan, infection, macrophage, anemia

Introduction

Iron is an essential element for virtually all living organisms, being responsible for several

functions in the human body (Table 1).

Table 1 – Main Functions of Iron in the Human Body

Energy Metabolism (as a constituent of oxygen transporters and of enzymes involved in the

respiratory chain)

Immunity (affects proliferation and differentiation of immune cells and modulates their anti-

microbial effector pathways)

DNA synthesis and regulation of transcription (as a central component of ribonucleotide

reductase or by modulating the binding affinities of central transcription factors via its

catalyzing role for radical processes)

Myelin and neurotransmitter synthesis

Collagen formation

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The metabolism of this element in the human body is complex. Because of inherent

technical difficulties and ethic concerns in studying the subject in the human organism a

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significant part of present knowledge is inferred from intensive research in vitro and in

murine models. The same is applicable when protozoan infections are studied.

Protozoan infections, such as malaria or visceral leishmaniasis, are important causes of

morbidity and mortality. Research on iron has contributed to the understanding of the

pathology of at least some of these infections and has suggested possible targets and strategies

for their treatment.

Iron Metabolism in the Human Body

The Iron Cycle

Intake

Iron (Fe) is usually acquired from the diet through intestinal absorption (mainly in

duodenum) (Fauci et al., 2008; Friedman et al., 2009). It is estimated that, in healthy

individuals with a balanced diet, iron is absorbed mainly in its hemic form (West and Oates,

2008). This is because hemic iron is more bioavailable than non-heme iron (Nadadur et al.,

2008; Edison et al., 2008). However, in certain pathologies such as iron deficiency anemia,

iron can also be acquired through transfusions and / or through iron supplementation either

oral or intravenous (Andrews 2008; Fauci et al., 2008). In contrast, iron losses occur passively

through the bile and small blood losses as well as through removal of epidermic cells and

enterocytes (Knutson and Wessling-Resnick, 2003).

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In normal circumstances, the daily iron needs are satisfied by the reuse of this metal in the

organism, dietary iron contributing only in a small proportion (usually 1-2mg) to compensate

passive losses (Swinkels et al., 2006; Kemna et al., 2008).

Non-heme iron enters to the enterocyte through the divalent metal transporter 1 (DMT1)

(Rhodes and Ritz, 2008). For this, ferric iron (Fe3+

) must be first reduced to ferrous iron (Fe2+

)

either chemically or through the activity of a brush border ferrireductase probably the

duodenal cytochrome B (DcytB) or members of the six-transmembrane epithelial antigen of

the prostate (STEAP) family (Andrews, 2008; Rhodes and Ritz, 2008). Once in the

enterocyte, iron becomes part of the intracellular calcein-chelatable iron pool (ICIP) from

where it can be utilized for cellular metabolism, be stored as ferritin, or be transported to the

plasma by the 62 kDa exporter ferroportin (FPN) (Nadadur et al., 2008; Friedman et al.,

2009). Plasma iron is bound by the 80 kDa serum glycoprotein transferrin (Tf), after oxidation

of Fe2+

to Fe3+

by the ferroxidase hephaestin (Swinkles et al., 2006; Nadadur et al., 2008).

There is an additional mucin-integrin mobilferrin pathway for iron uptake, which is less

understood (Edison et al., 2008; Nadadur et al., 2008).

Heme iron absorption is poorly understood and there are two main accepted hypothesis on

this subject, namely via receptor mediated endocytosis and through heme transporters (West

and Oates, 2008). The last hypothesis is more recent and suggests that heme is internalized

through a proton-coupled folate transporter / heme carrier protein 1 (PCFT/HCP1) directly

into the cytoplasm of the enterocyte. From there it can be transported across the basolateral

membrane by the feline leukemia virus subgroup C cellular receptor (FLVCR) to bind the

plasma glycoprotein hemopexin, or be catabolised to non-heme iron, carbon monoxide (CO)

and biliverdin by heme oxygenase 1 (HO-1) located on the endoplasmic reticulum or even by

HO-2. The other hypothesis suggests that heme iron is internalized via receptor mediated

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endocytosis with subsequent degradation by HO-2 inside vesicles, being the resulting metal

released to the cytosol by DMT-1. It is also hypothesized that the heme iron absorption rate is

limited by heme degradation through HO which activity is highest in duodenum (West and

Oates, 2008). Any iron released from heme inside the enterocyte can be transported by FPN to

the bloodstream in the same way as non-heme iron (Andrews, 2008; West and Oates, 2008;

Edison et al., 2008).

Iron transport and its uptake and utilization by the cells

In our organism, the majority of iron does not exist as free iron. Extracellular iron exists

mainly as Tf and lactoferrin (Lf) in serum or at mucosal surfaces respectively (Wilson and

Britigan, 1998). As occurs with Lf, one mol of Tf binds two mol of iron (Wilson and Britigan,

1998; Edison et al., 2008). In fact, Tf has two binding sites for Fe3+

, at the C-terminal and N-

terminal of the protein, but iron binding in serum occurs mainly in the latter because it is more

acid-labile (Wilson and Britigan, 1998). Production of Tf occurs mainly in hepatocytes but it

can also be generated in other cells such as Sertoli cells (Andrews, 2008). The Tf-Fe complex

(holotransferrin) is endocytosised by cells expressing transferrin receptors (TfR) at their

surface, diferric Tf possessing the highest affinity for these receptors (Andrews, 2008; Skikne,

2008). There are two types of TfR: TfR1 and TfR2. The latter, discovered more recently, is

highly expressed in the liver and in some proliferating cells and has lower affinity for Tf than

TfR1, which is the main Tf-Fe3+

receptor and is expressed in almost all cells (except in mature

erythrocytes) (Taketani, 2005; Rhodes and Ritz, 2008; Nadadur et al., 2008).

Endocytosis of holotransferrin, via clathrin-coated pits to an acidic endosome, is the usual

pathway for iron acquisition by many cells, specially by erythroblasts and other rapidly

dividing cells (Figure 1) which express a great number of TfR1 (Andrews, 2008; Friedman et

al., 2009). At the low pH of the endosome, Fe3+

is released and reduced to Fe2+

by a Steap

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family member (STEAP3) or by another ferric reductase (such as cytochrome b reductase 1)

so that it can be transported to the cytosol through DMT1 (Knutson and Wessling-Resnick,

2003; Taketani, 2005; Rhodes and Ritz, 2008). This bioavailable iron will be used in cellular

metabolism (where the mitochondria plays an important role), for example in the synthesis of

hemoglobin (Hb) and other proteins, while the endocytosis complex (apoTf plus TfR) is

recycled to the cell surface (Rhodes and Ritz, 2008; Sackman et al., 2009; Friedman et al.,

2009).

Figure 1. Main iron uptake pathway in cells (such as the erythroblast). TfR1 binds holo-Tf and

the resulting complex suffers endocytosis. In the acidic environment of the endosome, Fe3+

is released

and subsequently reduced to be transported to the cytosol by DMT1 while Tf-TfR is recycled to the

cell surface. In the cytosol, Fe2+

will be used in cellular metabolism (for example: Hb synthesis).

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Excess of iron, that is, iron not used for cellular metabolism, can be stored in the cytosol,

as ferritin (Weinberg, 2008; Fauci et al., 2008). Ferritin is a water soluble protein comprised

of 24 light (L) and heavy (H) chain subunits in varying ratios, resulting in a hollow ball that

can hold up to 4500 Fe3+

(Andrews, 2008; Edison et a., 2008) . Liver and spleen are rich in L

subunits in contrast to the heart and the kidney that are rich in H subunits (Northrop-Clewes,

2008). H-ferritin presents ferroxidase activity and is slightly larger than L-type (Andrews,

2008; Northrop-Clewes, 2008). In order to be stored inside ferritin, Fe2+

must reach the

ferroxidase centers in the protein cavity where Fe3+

is formed. This may occur by facilitated

diffusion through 3-fold hydrophilic channels (Bou-Abdallah et al., 2008). Whenever iron is

required, ferritin may undergo hydrolysis. This is a characteristic present, for example, in the

liver, which contributes to recirculation of iron (Edison et al., 2008).

Mitochondria is an important cellular organelle. It is essential for energy metabolism,

participates in heme and iron-sulfur cluster biosynthesis and also in iron metabolism, a

process still not fully understood. In contrast to lysosomes, the amount of iron in

mitochondria does not seem to respond to the cell iron in status (Taketani, 2005).

Nevertheless, the enzyme ferrochelatase, that enters in the final step of heme biosynthesis, is

referred to as being an iron sensor in mitochondria (Taketani, 2005).

Iron import from the cytosol to mitochondria is facilitated by endosomes that come in

contact with this organelle and also through mitochondrial iron importers, mitoferrins (Edison

et al., 2008; Paradkar et al., 2009). Pump proteins ABC7 (ATP-binding cassette 7) and

MTABC3 (mammalian mitochondrial ABC protein 3) are involved in mitochondrial iron

export; ABC7, as Taketani S. (2005) concluded, “plays a role in the maturation of

mitochondrial iron-sulfur-containing proteins, in addition to cytosolic iron-sulfur-containing

proteins” and the mitochondrial transporter ABC-me (ABC-mitochondrial erythroid) also

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promotes Hb synthesis and erythroid differentiation (Taketani, 2005). The mitochondrial

matrix protein frataxin increases the bioavailability of Fe essential for Fe-S cluster and heme

biosynthesis and mitochondrial ferritin (Mt-ferritin), which is homologous to the H chain of

the cellular ferritin, acts as the organelle iron storage site playing a protective role against iron

mediated toxicity inside this cell compartment (Taketani, 2005; Edison et al., 2008). GTP may

also participate in mitochondrial iron homeostasis (Edison et al., 2008).

Cells of the reticuloendothelial system (RES) are of major importance in iron metabolism.

They phagocyte senescent red blood cells and, together with the hepatic parenchyma, are the

main storage sites for iron (Weiss, 2005; Theurl et al., 2005; Edison et al., 2008). When the

erythrocyte is phagocytized, Hb undergoes proteolysis in the phagolysosome, originating

heme that probably migrates to the endoplasmic reticulum (ER) to be catabolysed by HO-1

(Knutson and Wessling-Resnick, 2003). Nevertheless, the exact site for heme catabolism and

the existence and characterization of relevant heme transporters need to be clarified (Knutson

and Wessling-Resnick, 2003). Fe resulting from red blood cells catabolism is either stored in

the cell or exported to plasma via FPN (which is also present in other iron exporting cells such

as the already referred enterocytes as well as placental syncytiotrophoblast and hepatocytes),

although 10-20% will remain as part of the labile iron pool, which is important for cellular

functions as well for regulation of cellular iron homeostasis (Theurl et al., 2005; Schaer et al.,

2008; Fleming, 2008). The recycling process resulting from erythrophagocytosis will satisfy

the majority of daily iron needs (Knutson and Wessling-Resnick, 2003; Fauci et al., 2008).

Cells from the RES can also acquire iron through other ways. The receptor CD163, which

is present in monocytes and macrophages, participates in the clearance of Hb-haptoglobin

complexes from the circulation (Taketani, 2005; Theurl et al., 2005; Edison et al., 2008). This

receptor might also remove free Hb, resulting from normal or increased intravascular

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hemolysis or from erythrocyte disruption during erythrophagocytosis (Knutson and Wessling-

Resnick, 2003; Schaer et al., 2006; Schaer et al., 2008). CD163 is recycled to the cell surface

after internalization, which improves Hb clearance (Schaer et al., 2006). Genetic

polymorphisms in haptoglobin seem to have a role in this uptake process because it was found

that the multimeric Hp2-2 phenotype has the highest functional affinity for CD163 (Knutson

and Wessling-Resnick, 2003). RES cells also possess specific hemopexin receptors which

may constitute a way for clearance of heme resulting from intravascular degradation of Hb

(and exported by the enterocytes through FLVCR?) (Knutson and Wessling-Resnick, 2003;

West and Oates, 2008). Inside the cell, Hb and/or heme will be catabolized (the last one by

HO-1) with subsequent release of iron (Schaer et al.. 2008). In a study with human

macrophages, Schaer et al. (2008) detected HCP-1 within early endosomes of the CD63 Hb

uptake pathway, which suggests that it can transport some of the heme released from Hb in

these vesicles to the cytosol; it was also observed traffic of Hb-haptoglobin complexes

through the endosomal compartment to lysosomes where a possible heme exporter remains

unknown. Macrophages also express on their surface TfR1 and receptors for Lf. In addition

there is also experimental evidence for Lf re-circulation (Theurl et al., 2005; Das et al., 2009).

Glyceraldehyde-3-phosphate dehydrogenase is present on the macrophage cell surface where

it act as a TfR and which expression is increased by iron depletion (Edison et al., 2008).

As indicated above, the RES is of vital importance in iron storage. In this process, ferritin

is of fundamental importance as is its insoluble and partially digested form, hemosiderin,

which concentration in these cells is the highest in the body thus facilitating larger deposits of

intracellular iron (Knutson and Wessling-Resnick, 2003).

Iron release from RES cells occurs in a poorly understood circadian rhythm and it seems

that neither Tf nor its iron-binding capacity are essential for this release, in contrast to the

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multicopper ferroxidase ceruloplasmin, which oxidizes Fe2+

to Fe3+

enabling iron binding to

apoTf (Knutson and Wessling-Resnick, 2003). Although most of the iron released into the

plasma will bind Tf, macrophages can also release iron in the form of Hb, heme and ferritin

(Knutson and Wessling-Resnick, 2003).

Like macrophages, hepatocytes can also acquire iron via multiple pathways, for example

through hemopexin receptors and TfR, but they must be better elucidated (Knutson and

Wessling-Resnick, 2003; Swinkels et al., 2006; Edison et al., 2008).

Lymphocytes, key cells in specific immunity, require iron in order to differentiate and

proliferate. These cells are more dependent on the Tf/TfR pathway than monocytes /

macrophages (Weiss, 2005). B cells seem to be less sensitive than T cells to modifications in

iron homeostasis and a similar comparison can be drawn between Th1 and Th2 cells, where

the first are more sensitive (Weiss, 2005; Theurl, 2005). H-ferritin receptors as well as Lf

receptors can also be expressed by lymphocytes, but their exact functions need to be better

elucidated (Weiss, 2005).

In what concerns the central nervous system (CNS), transport of iron across the blood

brain barrier is mediated either by Tf or by Lf-, melanotransferrin- and H-ferritin endo- and/or

transcytosis (Rhodes and Ritz, 2008). In this system, ferritin is expressed mainly in

oligodendrocytes, astrocytes and, specially, in microglia (Rhodes and Ritz, 2008).

Nevertheless, some particular aspects of iron metabolism in CNS are an open field and need

further research.

During pregnancy, the growing fetus requires large amounts of iron, which are acquired

from maternal blood. So, Fe is actively transported against a gradient across the

syncytiotrophoblast that expresses TfR1 on the maternal surface and FPN on the fetal surface

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(Friedman et al., 2009). Fe will eventually be incorporated in fetal Tf but these mechanisms

are still poorly understood (Friedman et al., 2009).

Regulation of Iron Metabolism

In order to keep our organism healthy, iron homeostasis must be ensured. This permits the

necessary stores of iron required for life to be maintained in a form that avoids the inherent

toxicity of Fe2+

. This arises from the formation of oxygen free radicals by the Fenton reaction

and can damage cell membranes, proteins and nucleic acids (Andrews, 2008; Edison et al.,

2008). One effective way to maintain iron non-reactive is through its chelation to proteins,

mainly Tf and ferritin (as discussed before). Heme can also damage cell structures because it

is a pro-oxidant and a strategy to prevent its toxicity is the induction of HO-1 expression, the

rate-limiting enzyme in heme catabolism, by various stimuli (including Hb-CD163 in

monocytes and macrophages) (Schaer et al., 2006; West and Oates, 2008).

As referred before, our organism has no regulated pathway to eliminate iron, so, its balance

is primarily controlled at duodenal absorption and through storage and recycling mechanisms

(Nadadur et al., 2008; Subramaniam, 2009).

Iron requirements in bone marrow are an important factor in the physiological regulation

of iron release from the RES (Knutson and Wessling-Resnick, 2003).

In adverse circumstances leading to iron deficiency, such as hemorrhage, dietary

deficiency or inadequate duodenal absorption, a significative quantity of iron can be

mobilized from the stores (Fauci et al, 2008). Iron deficiency also leads to an increase in heme

and non-heme iron absorption as well in HO activity in duodenum, but heme absorption

cannot be upregulated to the same extent as non-heme iron (West and Oates, 2008).

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Cellular level

Intracellular iron homeostasis is controlled through several mechanisms. An important one

occurs at the post-transcriptional level through the interaction between the iron regulatory

proteins (IRP) 1 and 2 with iron responsive elements (IRE) present on mRNAs of genes

involved in cellular iron metabolism (Knutson and Wessling-Resnick, 2003; Edison et al.,

2008). In vivo studies reported that, apparently, only inactivation of the IRP2 gene produced

phenotypic abnormalities (such as microcytic hypochromic anemia); however inactivation of

both genes is incompatible with life, which demonstrates the vital importance of this system

(Andrews, 2008). Nevertheless IRP-IRE interactions need to be better elucidated in vivo

(Nadadur et al., 2008).

At high Fe levels, IRP-1 displays aconitase activity and, in contrast, at low Fe levels, it

binds IREs and loses its 4Fe-4S cluster, which leads to the loss of aconitase activity

(Andrews, 2008; Dos Santos et al., 2008). Reactive oxygen species, serine phosphorylation,

nitric oxide (NO) and hypoxia can also affect the regulation of this protein (Edison et al.,

2008). Additionally, IRP-1 is quickly degraded when binds heme irreversibly (Taketani,

2005).

IRP-2 does not show aconitase activity and has a 73 amino acids (aa) iron-degradation

domain (IDD). This IRP is degraded in proteosomes, except when Fe levels are low in which

situation it binds to IREs, in an IDD-dependent fashion (Taketani, 2005). NO and hypoxia

also affect IRP2 (Edison et al., 2008)

IREs are present at the 5’UTRs of ferritin, FPN and e-ALAS (erythroid amino levulinic

acid synthase, the enzyme responsible for the first step of heme biosynthetic pathway)

mRNAs (Theurl et al., 2005). The 3’UTR of TfR1 mRNA possesses five IREs and the 3’UTR

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of DMT1 a single IRE (Dos Santos et al., 2008). IRE-like structures were also found in other

mRNAs not directly related to iron metabolism such as the case of alpha Hb stabilizing

protein (AHSP) mRNA (Dos Santos et al., 2008).

When cytoplasmic Fe is low, IRPs bind IREs, increasing the stability of TfR1 and DMT1

mRNAs and decreasing the translation of ferritin, FPN and e-ALAS genes, which will

enhance iron uptake by the cell and reduce its storage and exportation (Rhodes and Ritz,

2008; Nadadur et al., 2008). The inverse occurs when Fe levels are high: IRPs separate from

IREs, which facilitates TfR and DMT1 mRNAs degradation by nucleases and iron storage in

ferritin (Taketani, 2005; Rhodes and Ritz, 2008).

Other mechanisms than the IRE/IRP interactions have been reported and hypothesized. For

example, in the case of TfR2, its mRNA has no IRE (Taketani, 2005) and, so, other factors

may be involved. TfR1 expression at the cell surfaces is also regulated at the transcriptional

level through the status of cellular proliferation and oxygen saturation (Sackmann et al.,

2009). HFE may also modulate the transferrin-dependent iron uptake by forming a

stoichiometric complex with TfR1, thus reducing the affinity of the receptor for Tf (Taketani,

2005; Theurl et al., 2005). In inflammatory conditions, cellular iron homeostasis can be

modified by several cytokines (Edison et al., 2008; Northrop-Clewes, 2008).

The vital role of Hepcidin

Hepcidin, the key regulator of systemic iron metabolism, is a 25aa cationic peptide

hormone, produced mainly by the liver, but also by macrophages, monocytes, fat cells,

kidney, spinal cord, myeloid cells and cardiomyocytes (Collins et al., 2008; Schulze et al.,

2008; Huang et al., 2008). After its secretion into the circulation, hepcidin is filtered by the

kidney and detected in urine (Collins et al., 2008; Fleming, 2008).

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The hepcidin gene (HAMP) is located on chromosome 19q13.1 and encodes the 84 aa pre-

prohepcidin, which undergoes a first enzymatic cleavage of a 24aa amino-terminal

endoplasmic reticulum - targeting signal peptide originating the 60aa prohepcidin (Swinkels

et al., 2006; Collins et al., 2008). Remotion of the 35aa proregion of prohepcidin by a furin-

like proprotein convertase leads to mature hepcidin (Huang et al., 2008). Pre-prohepcidin can

also be secreted into the circulation, where its stepwise conversion into mature hepcidin can

also occur (Huang et al., 2008).

Hepcidin decreases ferremia by interaction with FPN and some studies also indicate that

this hormone negatively regulates DMT1 and DcytB (Howard et al., 2007; Nadadur et al.,

2008). When hepcidin binds to aa 324-343 on an extracellular loop of FPN, it triggers FPN

tyrosine phosphorylation [mediated by Janus kinase 2 (JAK2)] with consequent

internalization through clathrin-coated pits and ubiquitin-mediated degradation in lysosomes

(Collins et al., 2008; Friedman et al., 2009; Subramaniam, 2009). This inhibits duodenal and

macrophage iron export and iron mobilization from hepatic stores (Collins et al., 2008; Huang

et al; 2008). Hepcidin is also a negative regulator of placental transport of iron to the fetus,

with an accumulation of iron in trophoblast cells (Edison et al., 2008; Friedman et al., 2009).

Iron status is an important factor for the expression of hepcidin, even in pregnancy

(Schulze et al., 2008). The expression of this hormone is enhanced in iron overload conditions

and is suppressed in iron deficiency (Rhodes and Ritz, 2008; Schulze et al., 2008). In

addition, its expression is also decreased by hypoxia and erythropoiesis and is elevated by

inflammation (Andrews, 2008; Collins et al., 2008).

There are a number of pathways that regulate hepcidin expression. The bone

morphogenetic protein / sons of mothers against decapentaplegic homologue (BMP/SMAD)

pathway is a main activator of hepcidin transcription not completely understood, specially in

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response to body iron stores (Fleming, 2008; Kemna et al., 2008).

In this pathway,

hemojuvelin (HJV), a glycosylphosphoinositide (GPI) - linked cell surface protein, that is

expressed in hepatocytes and in skeletal and cardiac muscles, acts as a BMP coreceptor and it

seems that SMADs will bind directly to the hepcidin promoter (Andrews, 2008; Fleming,

2008). The competition between HFE and holoTf in binding to TfR1 promotes the formation

of the TfR2/HFE complex which induces hepcidin production (Kemna et al., 2008; Edision et

al., 2008). Also, hepcidin transcription is induced by IL-6, a cytokine involved in the acute

phase response, through binding of the signal transducer and activator of transcription 3

(STAT 3) protein to the promoter region of this hormone (Fleming, 2008; Rhodes and Ritz,

2008). This modulation on hepcidin transcription in inflammatory conditions needs SMAD4

to STAT3 activation and may be strong enough to induce hepcidin release even in a context

of anemia, although this subject must be better elucidated when all the regulatory pathways of

hepcidin and their interactions are well understood (Nadadur et al., 2008; Oliveras-Vergés and

Espel-Masferrer, 2008; Sackmann et al., 2009). Recently it was also found that the

transmembrane serine protease 6 (TMPRSS6) gene is important for the detection of iron

deficiency and for blocking HAMP transcription (Huang et al., 2008). There may exist other

pathways which regulate hepcidin and that are being investigated (Fleming, 2008; Nadadur et

al., 2008).

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Iron and Infectious Diseases: the case of the Protozoa

In the same way that it is essential to humans, iron is fundamental to almost all living

organisms, and infectious agents, such as some protozoans, are no exception. An infectious

disease can be a very stressing event influencing the general homeostasis of the body

(including iron metabolism) by multiple mechanisms. These can be triggered both by the host

and by the infectious agent(s). In this context, defense strategies of our organism may consist

in modifications leading to the reduction of iron availability to invader pathogens. Therefore,

these can be as more virulent as the more capable they are of developing ways to acquire

necessary iron (León-Sicairos et al., 2005; Weinberg, 2009).

An adequate iron balance is relevant for all cells including those that participate in the

defense of our organism, and its impairment, particularly in iron-overloaded situations, may

compromise immunological pathways directed to the clearance of the pathogens (Weiss,

2005).

Monocytes and macrophages, key cells in iron metabolism (as discussed before), have an

important paper in fighting these threats. Their natural resistance associated macrophage

protein 1 (Nramp1) is associated to resistance against infection by certain intracellular

pathogens. Nramp1 belongs to the same family as DMT1 sharing 64% of the amino acid

sequence, is present in lysosomes and late endosomes but may be quickly recruited to

membranes of maturing phagosomes (Knutson and Wessling-Resnick, 2003; Weiss, 2005;

Huynh et al., 2006). Together with other strategies to keep off iron from intracellular

pathogens (particularly those mediated by INF-γ), Nramp1 (as a pH dependent divalent cation

efflux pump) may work to clear iron from infected phagolysosomes (Marquis and Gros, 2007;

Huynh and Andrews, 2008; Weinberg, 2009).

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Although iron-mediated generation of toxic radicals can be harmful to human cells, they

can be used by neutrophils and macrophages / monocytes to kill pathogens (Weiss, 2005).

However, in excess, iron can be harmful to defense strategies of these cells (Weiss, 2005). As

an example, iron-loaded macrophages can experience impaired capacity to destroy

intracellular pathogens by INF-γ-mediated pathways (Theurl, 2005; Weiss, 2005). In this

case, iron may decrease generation of NO, an important molecule in macrophage defense

strategy (Theurl, 2005; Weiss, 2005).

An infectious process is generally accompanied by inflammation, which severity depends

on various factors both of the host and of the infectious agent. Inflammatory processes,

especially chronic ones, frequently lead to mild / moderate anemia that is, in part, due to

alterations in iron metabolism (Andrews, 2008; Friedman et al., 2009). These alterations

consist in hypoferremia (deficient iron supply to erythroid precursors) and, in contrast to iron

deficiency anemia, iron-binding capacity may be decreased (Weiss, 2005; Northrop-Clewes,

2008). Iron stores are usually normal or even elevated due, in part, to increased

erythrophagocytosis (Weiss, 2005; Northrop-Clewes, 2008). Mechanisms that explain

inflammatory-mediated changes in iron metabolism depend on a complex interaction of

cytokines, acute-phase proteins and radicals (Weiss, 2005; Northrop-Clewes, 2008). In this

process, it is important to highlight hepcidin, which expression is enhanced by inflammation

(as referred before), and α1-antitrypsin (α1-AT), another acute phase protein that competitively

blocks the binding of Tf to its receptor, affecting iron uptake by erythroid progenitor cells

(Theurl 2005; Weiss, 2005; Howard et al., 2007). Hence, inflammatory-mediated changes

may also limit iron availability to pathogens and strengthen cell-mediated immune effector

pathways (Theurl 2005; Weiss, 2005).

19

The iron-binding protein Lf is also an acute phase molecule and it is very important

because it has many immunoregulatory functions (Wilson and Britigan, 1998; Weiss, 2005).

As Tf or ferritin do (by chelating iron), Lf limits iron availability to infectious pathogens,

even in low pH sites, that can occur in an infectious process, which contrast to Tf that

liberates iron as soon as pH starts to decrease (Wilson and Britigan, 1998; Weinberg, 2009).

So, acquisition of iron in our organism is a real challenge to invader pathogens with many

barriers to be overcome.

In order to exemplify the importance of iron (and changes in its metabolism) in what

concern to infectious diseases in the human body, some aspects currently known or under

discussion will be now presented for some protozoan pathogens.

Entamoeba histolytica

This protozoan is an extracellular parasite that causes an intestinal and hepatic disease

usually denominated amoebiasis, which incidence is higher in most developing countries in

the tropics (Fauci et al., 2008). Its successful adaptation in the human body is partly related to

its capacity to acquire iron from several sources.

Entamoeba histolytica can internalize ferritin via clathrin-coated vesicles and holo-Lf

through a specific receptor-mediated endocytosis involving caveolae-like filipin-sensitive

vesicles which subsequently release iron in the acidic environment of amoebic vesicles with

participation of cysteine proteases (León-Sicairos et al., 2005; López-Soto et al., 2009). This

parasite has also the ability to acquire ionic iron, holo-Tf and Hb (Léon-Sicairos et al., 2005;

Cruz-Castañeda and Olivares-Trejo, 2008). In what concerns to Hb, two recent discovered

20

parasitic Hb-binding proteins – Ehhmbp45 and Ehhmbp26 – may play a role in its acquisition

(Cruz-Castañeda and Olivares-Trejo, 2008; Cruz-Castañeda et al., 2009). (Figure 2)

Figure 2. Iron sources for Entamoeba histolytica during its infection in the human body. This

range of iron sources seem to be advantageous to Entamoeba histolytica. This parasite may use holo-

Lf and Hb as iron sources during the infection in intestinal mucosa, while the same is true for holo-Tf

(and Hb) when the parasite reaches the blood and, in the liver, ferritin may become of higher

importance to its adaptation.

One of the reasons why iron is indispensable for Entamoeba histolytica survival is because

the enzyme EhADH2 (alcohol dehydrogenase 2), a key on the parasite’s energy metabolism,

requires Fe2+

as a cofactor (Espinosa et al., 2009). It was suggested that iron may play an

important role in virulence of this protozoan, by modulation of the expression of genes related

to adherence and cytotoxicity (Lee et al., 2008). Not surprisingly, it was reported that low-

21

iron-content diets may confer a certain degree of resistance to amoebiasis, what suggests that

iron-starvation-based therapies like, for example, iron chelation, may become important in

amoebiasis treatment in the future (Espinosa et al., 2009).

Leishmania

Several species of Leishmania, spread in many countries worldwide, cause a wide

spectrum of clinical disease that range from “benign” cutaneous lesions to potential life-

threatening visceralization (Huynh et al., 2006; Marquis and Gros, 2007; Fauci et al., 2008).

In severe visceral leishmaniasis, anemia usually develops and bleeding can also occur (Fauci

et al., 2008). There are two forms of this parasite: the promastigote, transmitted by the bite of

phlebotomine, and the amastigote, the main form in the human host, found inside macrophage

phagolysosomes (Fauci et al., 2008; Das et al., 2009).

Iron is vital for Leishmania, being necessary for its basic metabolic activity and essential

for the function of important enzymes such as the metalloenzyme superoxide dismutase

(SOD) and ribonucleotide reductase (RR) (Sen et al., 2008; Huynh and Andrews, 2008; Das et

al., 2009). SOD has a protective role by detoxifying O2-, which is important for resistance to

oxidative stress, while RR is critical for DNA synthesis (Sen et al., 2008; Huynh and

Andrews, 2008).

It was found that Leishmania can acquire iron from several sources, but Fe2+

acquisition

strategies seem to be of particular importance, at least, for the intracellular amastigote form

(Huynh et al., 2006; Sutak et al., 2008). These parasites can take up holoTf and holoLf,

although the respective mechanisms need to be better elucidated (Wilson and Britigan, 1998;

Huynh et al., 2006). It was also detected in Leishmania donovani promastigotes and in

22

Leishmania infantum amastigostes a specific receptor involved in Hb endocytosis

(Krishnamurthy et al., 2005; Carvalho et al., 2009). Evidence for receptor-mediated uptake of

heme also exists, both for Leishmania mexicana promastigotes and Leishmania infantum

amastigotes (Wilson and Britigan, 1998; Carvalho et al., 2009). A recently identified ferrous

iron transporter, LIT-1, present in the membrane of Leishmania amazonensis was reported to

participate in uptake of Fe2+

by intracellular amastigotes, being expressed particularly in iron

starving conditions (Huynh et al., 2006). Nevertheless, LIT-1 is essential for Leishmania

amazonensis virulence but not for its survival (Huynh et al, 2006). It also seems that a single

integral membrane ferric reductase exists in Leishmania major, Leishmania infantum and

Leishmania braziliensis (Huynh and Andrews, 2008). There are reports suggesting that

expression of DMT1 and Nramp1 in the endosomal pathway (as discussed before) may result

in small amounts of the metal available to the parasite (Huynh et al., 2006; Huynh and

Andrews, 2008). This could constitute a challenge to Leishmania parasites, although this is

still an area without consensus. It may occur that Nramp1 may affect the parasite by

mechanisms unrelated to nutritional reasons (Gomez et al., 2007). It is possible that depletion

of ionic iron is partly overcome by the uptake of other sources of iron, like heme and Hb,

which may fulfill some of the parasite requirements (Carvalho et al., 2009). Recently, Das et

al. (2009) demonstrated that intracellular Leishmania donovani is able to deplete the

macrophage labile iron pool as a strategy to acquire iron and that this iron pool might be

successively renovated by induction of the expression of TfR1, with impaired ferritin

synthesis; the ability to increase TfR1 expression was corroborated in Leishmania major.

To obtain a complete picture of the iron-acquisition strategies (and their importance)

present in Leishmania more research is necessary, although, what is known, helps in the

23

comprehension of virulence mechanisms of these parasites and points towards possible

therapeutic targets such as the transporter LIT-1.

Malaria

Malaria is an important factor of morbidity and mortality, especially in endemic areas, that

are mainly located in the tropical regions. It is caused by protozoa of the genus Plasmodium,

being Plasmodium falciparum the etiologic agent responsible for the most severe cases.

During infection, parasites invade erythrocytes and, in some cases, reticulocytes. Hb, as a

source of aa, is very important for the parasite. It undergoes endocytosis and is degraded in

the acidic environment of the food vacuole that contains Asp and Cys proteases; part of the

resulting heme originates hemozoin, the “malaria pigment”, while a small amount may exit

the food vacuole to be degraded in the parasite cytosol via glutathione (Wilson and Britigan,

1998; Cabantchik et al., 1999). Hemozoin was considered an inert material, but its biological

activity is becoming apparent, for example, in what concerns to its role in catalyzing the

formation of free radicals and even its contribution in the pathogenesis of malarial anemia

(Gosh and Gosh, 2007).

Iron is important for the growth of malaria parasites but its main source to the parasite

remains unclear (Prentice, 2008). It is debatable whether a Tf-dependent process to acquire

this metal does exist in these parasites, with some authors defending that parasites insert Tf-

like receptors on the host erythrocyte membrane (Wilson and Britigan, 1998; Gosh and Gosh,

2007). An iron-regulated IRP-like protein was found in Plasmodium falciparum but it is

unclear whether it plays any role in parasite iron homeostasis (Loyevski et al., 2001).

24

Anemia is a common finding in malaria and may be the result of several mechanisms still

not completely understood such as, for example, hemolysis and inflammatory mediated

pathways (Gosh and Gosh, 2007; Ndyomugyenyi et al., 2008; Northrop-Clewes, 2008).

Indeed, the levels in sera of mediators related to inflammation are found to be raised in

malaria. This is the case of IL-6 and, as discussed before, this may constitute a defense

strategy against the parasite (Gosh and Gosh, 2007; Howard et al., 2007). In this context,

some hypothesis can be made and shall be tested, like the one presented by Oliveras-Vergés

and Espel-Masferrer (2007), which suggests a protective role of hepcidin against malaria

sporozoites in the liver through decreasing the availability of Ca2+

. In a study with Ghana

patients with malaria due to Plasmodium falciparum, an association between the

concentration of urinary hepcidin and parasitemia was found, with high levels of urinary

hepcidin corresponding to patients with high levels of parasitemia (Howard et al., 2007).

Iron metabolism in the host and treatments that affect the iron load of the body may

influence the course of malarial infection, as many studies about this subject report.

Iron deficiency may be beneficial against malaria and there are some studies supporting

this principle. For example, a study with mice, reported that iron-deficiency improves the

course of malaria, one reason being because it enhances clearance of infected erythrocytes

(Koka et al, 2007). A protective effect of iron deficiency to placental malaria in women and to

clinical malaria in children was also reported (Kabyemela et al., 2008; Friedman et al., 2009).

These findings might be explained by a lower iron availability to the parasite or by

strengthening of immune effector pathays such as generation of NO by macrophages

(Kabyemela et al., 2008; Friedman et al., 2009) (as already referred).

Although, in theory, one would think that iron supplementation / iron-rich diet has

detrimental effects on malaria outcomes and would increase susceptibility to this disease, not

25

all studies supported this hypothesis (Cabantchik et al., 1999; Mebrahtu et al., 2004; Richard

et al., 2006; Kabyemela et al., 2008; Prentice, 2008; Weinberg, 2009; Friedman et al., 2009).

These contradictory findings may have several explanations and variables such as health care

facilities, body iron status or acquired immunity may affect the results of the studies.

However, this field needs to be better clarified in order to devise strategies that can improve

health of populations living in endemic areas where malarial anemia and iron deficiency

anemia coexist and are prevalent, particularly in childhood and pregnancy.

In a study with Zambian children infected with Plasmodium falciparum that suffered from

its most feared complication, cerebral malaria, it was observed an improved parasite clearance

when the iron chelator desferrioxamine was administered additionally to the standard

treatment. In particular, deep coma recovery enhanced with the chelator. A subsequent

retrospective analysis of Tf saturations in these children supported the hypothesized role of

iron-generated free radicals in the pathogenesis of this kind of coma because recovery, using

the iron chelator, seemed to be specifically improved in children with high Tf saturations

(Cabantchik et al., 1999). Although this iron chelation treatment may have beneficial effects

on morbidity, the same does not seem to be true in what concerns to mortality which can even

be higher (Cabantchik et al., 1999). Other clinical studies with desferrioxamine were

preformed and showed beneficial effects on Plasmodium parasitemia, at least in a short period

of time (Cabantchik et al., 1999). Nevertheless, more research in this field is necessary to

support a clinical use of iron chelators as anti-malarials. Namely, it is important the

production and discovery of new substances characterized by higher efficacy and well

tolerability, with minimal adverse effects on the patient.

26

Trichomonas vaginalis

Trichomonas vaginalis, the etiologic agent of the worldwide sexual transmitted disease

trichomoniasis, usually resides in the urogenital cavities, particularly in the vagina (Jesus et

al., 2006).

The adaptability of Trichomonas vaginalis to its habitat may be explained by its capacity to

use several sources of iron. HoloLf is one of the vaginal iron sources for this parasite, that

possesses a specific receptor for it which expression and affinity are increased in iron-

depleted medium (Wilson and Britigan, 1998; León-Sicairos et al., 2005). Also, the iron

requirements of Trichomonas vaginalis can be satisfied by phagocytosis of erythrocytes

(particularly during menstruation) and it seems that the adhesion proteins AP51 and AP65

have a role in iron uptake, because they may have heme and Hb-binding properties (Ardalan

et al., 2009).

There are a number of studies about the influence of iron in Trichomonas vaginalis and

growing evidence suggests it affects the virulence of this parasite. In fact, the expression of

several proteins of this parasite was found to be modulated by iron although the underlying

mechanisms are only starting to be understood (Jesus et al., 2007). An iron-responsive

promoter was reported to be present in the ap65-1 gene and an identified IRP/IRE-like system

could regulate expression of the cysteine proteinase TVCP4 (Tsai et al., 2002; Solano-

González et al., 2007). Some proteins were found to be enhanced and others decreased by

similar iron conditions, what is possibly related to the necessity of the parasite to adapt to

changes in the environment as occurs with the menstrual cycle. Some adhesin molecules and

TV44 (an IgA-reactive surface protein) are examples of proteins which expression is

decreased (or even suppressed) in low-iron media (Moreno-Brito et al., 2005; Mundodi et al.,

2006). Also, ecto-phosphatase and ecto-ATPase activities are reduced under iron-depleted

27

conditions (Jesus et al., 2006). On the contrary, for instance, the activity of cysteine proteases

CP30 and CP65 (proteins that are related to cytotoxicity) is induced (Alvarez-Sánchez et al.,

2007; Kummer et al., 2008). The activity of hydrogenosomes (an organelle related with

energy metabolism and drug susceptibility) appear to be also regulated by iron (Kim et al.,

2006). Interestingly, when the iron-content of the medium is reduced morphological changes

can occur in the parasite which adopts a pseudocyst form (Jesus et al., 2007). To conclude,

further research is required to clarify all these findings and their importance for the in vivo

infection and also to characterize other possible mechanisms and proteins which function in

iron metabolism of this parasite.

Trypanosoma

Parasites from the genus Trypanosoma are an important cause of morbidity and mortality,

namely Trypanosoma cruzi, the etiologic agent of Chagas disease, and Trypanosoma brucei

ghambiense and Trypanosoma brucei rhodesiense, the etiologic agents of sleeping sickness,

being the first one endemic in America and the others in Africa (Fauci et al., 2008). Also for

these parasites, iron is an essential element, as anti-trypanosomal effects of iron chelators

suggest (Merschjohann and Steverding, 2006). Nevertheless, there is less information

available related to iron for Trypanosoma cruzi than for Trypanosoma brucei subspecies.

Trypanosoma brucei lives in the blood and acquires most of its iron from Tf. It presents

receptors for Tf, which enable acquisition of this iron source, because their expression is

increased in iron starvation conditions (Mussmann et al., 2004; Steverding, 2006).

During the course of African trypanosomiasis, the inflammatory environment can lead to

anemia. A performed study with an experimental Trypanosoma brucei infection in a murine

28

model reinforced the notion that iron sequestration in activated macrophages is enhanced

during the chronic phase of trypanosomiasis (Stijlemans et al., 2008). The same report

suggests that, in the acute phase of infection, an increase in iron recycling may become

favorable for iron acquisition by the parasite.

Brief notes regarding other Protozoa

Intraerythrocytic protozoa from the genus Babesia have animals as natural reservoirs but

have been responsible for some cases of infection in humans (reported worldwide), being

transmitted by ectoparasites from the genus Ixodes. The disease can vary from a mild flulike

syndrome to a malaria-like one, with the possibility to be life threatening. The severity of the

process depends also on host factors such as age or immunity for example. Hemolytic anemia

can occur, with the subsequent formation of haptoglobin-Hb complexes in the circulation and

iron may be lost through hemoglobinuria. (Fauci et al., 2008)

The relationship of Blastocystis hominis with iron metabolism is poorly studied and several

questions persist in what concern to its pathogenic mechanisms. It seems that when this

protozoan parasites intestinal tract it may contribute to the generation of iron deficiency

anemia (Yavasoglu et al., 2008).

Giardia lamblia, a flagellate intestinal protozoan, is the etiological agent of widespread

giardiasis. Giardiasis, particularly symptomatic one, usually course with malabsorption of

duodenum and proximal jejunum, due to diffuse reduction in the microvillus surface area by

the parasite, a process that can result in decreased ferremia and evolve to iron deficiency

anemia (Ertan et al., 2002; Monajemzadeh and Monajemzadeh, 2008; Kasirga et al., 2009). Lf

29

may play a role in the resistance to Giardia lamblia because, in vitro, it was found to be

cytotoxic for trophozoites (the active form of the parasite) (Wilson and Britigan, 1998).

Toxoplasma gondii, an obligate intracellular parasite, is the etiologic agent of

toxoplasmosis and may be responsible for several clinical manifestations, particularly in

congenital infection and in immunocompromised patients. In this infection, iron losses can be

increased, for example via hemoptysis which can coexist with pneumonia. (Fauci et al., 2008)

In studies with Toxoplasma gondii-infected human fibroblasts an increased activity of

IRP1 was reported, with subsequent up-regulation of TfR1. The mechanism responsible for

this finding appeared to be related with secreted factor(s) that need further research. (Gail et

al., 2004)

Conclusion

Iron is essential and plays important rules in the human host and also in protozoan parasite

which usually leads to a competition for this element during an infectious process. In our

organism, iron homeostasis is achieved mainly with regulation at the level of intestinal

absorption, because there are no active pathways for excretion of iron, and at the level of its

releasing from the storage compartment, where macrophage plays a central rule. The

discovery of hepcidin and its role on systemic iron regulation answered many questions about

the regulation of iron metabolism in our organism. A virulence factor of protozoans often

consists on the development of strategies to acquire iron, evading iron withholding strategies

of the host, such as the existence of receptors for iron containing compounds that usually exist

in the host environment, for example.

30

In spite of the existence of some protozoans where this subject is poorly studied, like the

case of Trypanosoma cruzi, therapeutical strategies such those aimed at preventing iron access

to the parasites may be promising. However, as happens with many other therapeutic

strategies in several pathologic situations, the risk of adverse effects exist, at least because

iron is also essential to our organism and because the possibility of drugs against parasite

molecules interact with host molecules is not disposable.

Further research is necessary to illuminate unanswered questions related to these subjects

and ideally many of those should be investigated in vivo (including, in some circumstances, in

humans), respecting, of course, ethic imperatives.

Acknowledgments

This work was supervised by Professor Ana Tomás (University of Porto) and by Professor

Anabela Mota Pinto (University of Coimbra) to whom the Author sincerelly thanks.

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