Hematology-2013-Camaschella-1-8

Iron and hepcidin: a story of recycling and balance
Clara Camaschella1
1Vita-Salute University and San Raffaele Scientific Institute, Milan, Italy
To avoid iron deficiency and overload, iron availability is tightly regulated at both the cellular and systemic levels. The
liver peptide hepcidin controls iron flux to plasma from enterocytes and macrophages through degradation of the
cellular iron exporter ferroportin. The hepcidin-ferroportin axis is essential to maintaining iron homeostasis. Genetic
inactivation of proteins of the hepcidin-activating pathway causes iron overload of varying severity in human and mice.
Hepcidin insufficiency and increased iron absorption are also characteristic of anemia due to ineffective erythropoiesis
in which, despite high total body iron, hepcidin is suppressed by the high erythropoietic activity, worsening both iron
overload and anemia in a vicious cycle. Hepcidin excess resulting from genetic inactivation of a hepcidin inhibitor, the
transmembrane protease serine 6 (TMPRSS6) leads to a form of iron deficiency refractory to oral iron. Increased
hepcidin explains the iron sequestration and iron-restricted erythropoiesis of anemia associated with chronic
inflammatory diseases. In mice, deletion of TMPRSS6 in vivo has profound effects on the iron phenotype of
hemochromatosis and beta-thalassemia. Hepcidin manipulation to restrict iron is a successful strategy to improve
erythropoiesis in thalassemia, as shown clearly in preclinical studies targeting TMPRSS6; attempts to control anemia of
chronic diseases by antagonizing the hepcidin effect are ongoing. Finally, the metabolic pathways identified from iron
disorders are now being explored in other human pathologic conditions, including cancer.




Introduction
Iron is essential for multiple cell functions, but is also potentially
deleterious because of its ability to generate free oxygen radicals.
Due to the absence of an active excretory mechanism, iron balance
in mammals is maintained by limiting its intestinal uptake and by
continuously recycling and reusing cellular iron. Multiple safety
mechanisms, such as binding to chaperone proteins, storage in
ferritin, and export through ferroportin (FPN), protect cells from
free iron toxicity. The mechanisms of cellular iron handling are
summarized in Figure 1. Iron is used in mitochondria for heme
synthesis and iron sulfur cluster biogenesis. There is increasing
interest in the latter pathway because iron sulfur clusters are
prosthetic groups for key enzymes of DNA duplication, repair, and
epigenetics. Iron-regulatory proteins (IRPs) and hepcidin exert iron
homeostatic control at the cell and systemic levels, respectively.1
Disruption of iron control mechanisms leads to genetic iron
disorders and may also contribute to the pathophysiology of
common pathologic conditions including inflammation, neurodegeneration,
metabolic disorders, and cancer.
At the cellular level, IRP1 and IRP2 orchestrate the coordinated
expression of iron importers (transferrin receptor 1 [TFR1] and
divalent metal transporter 1 [DMT1]) and of storage (ferritin
light and heavy chains) and export (FPN) proteins. IRPs regulate
their targets posttranscriptionally by binding to special stem loop
elements in the untranslated regions of mRNA-encoding proteins
involved in iron metabolism; binding activity is high in iron
deficiency and hypoxia and is suppressed by iron and oxygen (for
review, see Hentze et al1). Recently, differential target specificity
of the 2 IRPs has been identified, with IRP1 specifically
controlling the hypoxia mediator HIF2-alpha2 and IRP2 controlling
ferritin.3 Control of HIF2-alpha by IRP is one of the multiple
links between iron and hypoxia. Undoubtedly, conditional deletion
of either IRP in animal models will clarify other tissue- and
IRP-specific roles.
At the systemic level, the liver peptide hepcidin regulates iron
homeostasis by binding and degrading the sole cellular iron exporter
FPN, which is highly expressed at the basolateral surface of
duodenal enterocytes and on the cell membrane of macrophages. In
this way, hepcidin restricts the amount of iron delivered to its
plasma carrier transferrin.4 The concentration of both circulating
and tissue iron provides distinct signals that modulate hepcidin. The
result is low hepcidin and active iron delivery to plasma in iron
deficiency and high hepcidin with reduced iron flux to plasma in
iron overload (Figure 2).
How IRP-based and systemic regulatory pathways interconnect and
work together in general iron homeostasis is only partially understood
and is a subject of intensive investigation.
Hepcidin up-regulation
Hepcidin transcription in hepatocytes is dependent on the bone
morphogenic protein (BMP)-SMAD signaling cascade (Figure
3A).1 BMP6 is the iron-related BMP receptor (BMPR) ligand in
vivo, as shown by Bmp6 / mice, which have severe iron overload
and very low hepcidin.5 In the liver, BMP6 is mainly expressed in
nonparenchymal cells such as sinusoidal endothelial and Kupffer
cells.6 Binding of the ligand to BMPR complex on the hepatocyte
surface triggers phosphorylation of SMAD proteins, which translocate
to the nucleus to activate target genes including hepcidin
(Figure 3A). In mice, liver-specific disruption of the BMPR ALK2
and ALK-3 or of SMAD4 molecule results in iron overload with
low hepcidin.7 Hemojuvelin (HJV), a protein mutated in juvenile
hemochromatosis type A (Table 1), is the essential BMP
coreceptor in this pathway. In humans, its inactivation causes
severe, early onset iron overload indistinguishable from hemochromatosis
caused by inactivation of the hepcidin gene itself.8
Hemochromatosis type 1, 2 and 3 (Table 1) and their corresponding
murine models show defective BPM signaling that results in
hepcidin insufficiency. Whereas the function of membrane-HJV

in hepcidin activation is clearly defined, that of other hemochromatosis
proteins (TFR2 and HFE) remains uncertain. To add
further complexity, in mice, hepcidin, Hfe, and Hjv are modulated
by microRNA miR-122.9
In inflammation, hepcidin is activated by IL-6, IL-1-beta, and other
cytokines as well as by lipopolysaccharide through the JAK2/STAT3
signaling pathway (Figure 3B). The integrity of the BMP pathway is
essential for a full hepcidin response in inflammation1 and the
cross-talk between the 2 pathways is the subject of intensive
investigation.
Hepcidin down-regulation
Erythropoiesis consumes most of the recycled and absorbed iron
( 25 mg/daily). Therefore, it is not surprising that hepcidin
expression is suppressed in all conditions characterized by increased
erythroid demand for iron and elevated circulating erythropoietin
levels, such as iron deficiency, hypoxia, and erythropoietic expansion.
A key hepcidin inhibitor was discovered by positional cloning
of the gene responsible for the phenotype of the Mask mice, which
have microcytic anemia, high hepcidin, and are unable to absorb
oral iron.10 Matriptase-2, encoded by TMPRSS6, is a liver-expressed
type II transmembrane serine protease that is inactivated in Mask
mice due to truncation of the protein and loss of the catalytic
domain. TMPRSS6 interacts with and cleaves the BMP coreceptor
HJV, switching off BMP signaling and thereby decreasing hepcidin
transcription (Figure 3A).11 It is unknown whether, in vivo,
TMPRSS6 cleaves other substrates. In iron deficiency, the function
of TMPRSS6 is essential to suppress hepcidin and to allow iron
absorption. In vitro, the expression of TMPRSS6 is up-regulated by
hypoxia and iron deficiency and its proteolytic activity is inhibited
by hepatocyte growth factor activator inhibitor type-2 (HAI-2), an
inhibitor of the homologous protease matriptase-1.12 The regulation
of TMPRSS6 in vivo is largely unknown.
Several other candidate hepcidin inhibitors have been proposed.
Any direct effect of the hypoxia mediator HIF-1-alpha on the
hepcidin promoter remains uncertain. Growth differentiation factor
15 (GDF15) and twisted gastrulation protein homolog 1 (TWGS1)
are released by erythroblasts and have been proposed as hepcidin
suppressors, especially in thalassemia, but their physiologic or
pathologic role remains uncertain.1 The longstanding known soluble
(s)TFR1 and the recently identified sTFR2 (C.C., unpublished data,
April 2013) could in theory contribute to hepcidin suppression, but
experimental proof is lacking. In vitro, soluble HJV produced by
furin cleavage13 competes as a decoy molecule with membrane-HJV
for BMP6 binding, but its in vivo function is unclear. HJV is highly
expressed in muscle, where it might have an iron-unrelated function,
because muscle-specific Hjv-knockout mice have normal iron
homeostasis.14 It is conceivable that multiple inhibitors triggered by
different signals from iron-deficient, expanded, or abnormal erythropoiesis
all converge on the same final pathway.
Lessons from human iron disorders
Genetic disruption of the finely tuned regulation of the hepcidin-
FPN axis causes either iron overload or iron deficiency (Table 1).
Hemochromatosis, a heterogeneous genetic disorder leading to iron
overload and ultimately to organ failure, provided important clues to
understanding hepcidin up-regulation, and autosomal recessive
iron-refractory iron-deficiency anemia (IRIDA) provided clues to
the mechanisms of hepcidin suppression.


Hemochromatosis patients are natural human mutants of iron
homeostasis. In the recessive disease, mutations affect genes
involved in hepcidin activation (HFE, HJV, HAMP, TFR2); in the
dominant type, they affect the hepcidin receptor FPN (Table 1; for
review, see Pietrangelo15). In hemochromatosis, the severity of iron
overload correlates with the degree of hepcidin deficiency, suggesting
a hierarchy of the corresponding proteins in the regulatory
pathway. HAMP (hepcidin) and HJV, the genes of the most severe
juvenile form of hemochromatosis, have a central role in hepcidin
regulation, whereas HFE and TFR2 have ancillary roles. In the
current model, HFE and TFR2 function as a complex to activate
hepcidin1 (Figure 3A); however, distinct disorders result from their
individual inactivation. HFE disease has adult onset, low penetrance,
and male predominant expression, suggesting a modest
biological effect of the protein. Although the number of cases
reported is limited, TFR2 hemochromatosis affects both genders
and has early onset, but its clinical course is not as severe as the
juvenile form. These clinical observations suggest 2 distinct,
perhaps age-dependent, mechanisms of hepcidin regulation. In
addition, the hepcidin response after an oral iron challenge that
increases only plasma and not tissue iron is blunted in HFE but
absent in TFR2 patients,16 and the same response is observed in the
corresponding animal models.17 The evidence that normal HFE and
TFR2 do not compensate for deficient HJV suggests that all of these
hepcidin-regulatory proteins converge on the same pathway. Indeed,
HFE, TFR2, and HJV in vitro interact to form a cell surface
complex.18 The simplest explanation is that TFR2, which binds
iron-loaded transferrin, controls hepcidin according to plasma iron
levels to avoid iron overload when iron demands are high, as in
young individuals. HFE might control hepcidin according to tissue
iron concentration to avoid iron accumulation in adults. An additive
effect of HFE and TFR2 is evident in a patient who presented with
mutations in both genes and had severe disease.15
On the opposite side, the only known genetic disorder with high
hepcidin is the recessive IRIDA19 due to TMPRSS6 inactivation,
pointing to the unique role of this protease in hepcidin suppression.
Inhibition of hepcidin expression in iron deficiency serves to
increase the iron supply to plasma. IRIDA patients have moderate
anemia, severe microcytosis and hypochromia, very low transferrin
saturation, and inappropriately normal/high hepcidin levels.19 Anemia
is more severe in children than in adults,20 supporting the
concept that TMPRSS6 mediates the physiologic response to
increased iron demand.
All of these clinical observations point to a single iron-responsive
hepcidin regulatory pathway with hepcidin production reflecting the
balance between positive (BMP6) and negative (TMPRSS6) hepcidin
regulators (Figure 4). Interestingly, hepcidin up-regulation by
increased plasma iron does not require BMP6 to increase. One
possible mechanism causing a rapid increase in hepcidin when
BMP6 is low is blocking TMPRSS6 activity. Hemochromatosis
resulting from mutations of FPN is dominant (Table 1) and the
homozygous state has not been reported, likely because homozygous
inactivation of FPN would be incompatible with life; this again
points to the critical importance of cellular iron export and
macrophage iron recycling. Disorders of FPN are heterogeneous:
mutations that decrease its surface expression or the ability to export
iron result in relatively benign iron accumulation in macrophages.15
In contrast, mutations at the hepcidin-binding site of FPN cause true


hemochromatosis with parenchymal iron overload because FPN is
not degraded by hepcidin (hepcidin resistance). The similarity of the
phenotype caused by hepcidin deficiency and by FPN mutations that
cause resistance to hepcidin attests to the essential role of the
hepcidin-FPN interaction in iron homeostasis.15
Hepcidin insufficiency causes the development of secondary iron
overload also in “iron-loading anemias,” which include the nontransfusion-
dependent thalassemia syndromes such as thalassemia
intermedia21 and congenital dyserythropoietic and inherited nonsyndromic
sideroblastic anemias (Table 1). Beta-thalassemia intermedia,
which has a clinical course of severity intermediate between
transfusion-dependent patients and asymptomatic carriers, is the
prototype of conditions characterized by ineffective erythropoiesis
and high iron stores. Despite often severe iron overload, hepcidin is
suppressed by the expanded erythropoiesis. The observation of
increased iron absorption irrespective of high iron stores in ironloading
anemias antedated the discovery of hepcidin.
We learned from patients with inflammatory disorders that hepcidin
production is up-regulated by cytokines. Hepcidin is an acute phase
protein and an essential mediator of the complex anemia of
inflammation or anemia of chronic diseases, a multifactorial form
of anemia present in numerous disorders22 in which the blockage of
iron absorption and recycling plays a major role. In addition to the
systemic effects of increased hepcidin production by the liver,
inflammatory macrophages also express hepcidin and may induce
iron retention by an autocrine mechanism. The blockage of macrophage
iron recycling and the resulting hypoferremia is considered a
protective mechanism against extracellular pathogens, likely reflecting
the true “antimicrobial” function of hepcidin. Recent studies
indicate that serum ferritin is predominantly secreted by macrophages;
if so, hepcidin-induced iron sequestration in macrophages
and the resulting stimulation of ferritin synthesis would explain the
high serum ferritin observed in inflammation and also the high
correlation between serum hepcidin and ferritin levels reported not
only in healthy subjects23 but also in inflammation.
Lessons from animal models of TMPRSS6
inactivation
Tmprss6 / mice show the same phenotype of iron deficiency with
high hepcidin described in Mask mice. Crossing Tmprss6 / mice
with iron-loaded mice provided important insights into the hierarchy
of the hepcidin pathway proteins (Figure 5). Tmprss6-Bmp6
double knockout mice are as severely iron loaded as Bmp6 / mice,
implying that the function of Tmprss6 requires an active Bmp-Smad
pathway.24 Tmprss6-Hjv double mutant mice are as iron loaded as
Hjv / mice, a finding consistent with Hjv being downstream of
Tmprss6 and likely its substrate.25 On the contrary, the genetic loss
of Tmprss6 in Hfe / mice reverts the body iron status to an
IRIDA-like phenotype,26 indicating that Hfe acts genetically upstream
of Tmprss6. Hfe was hypothesized to reduce the activity of
Tmprss6, promoting Bmp/Smad signaling and increasing hepcidin.
26 In addition, Tmprss6-TFR2–null mice have iron deficiency,
with increased RBC and reticulocyte count and even more severe
microcytosis than Tmprss6 / mice27 (C.C., unpublished data, April
2013). Hepcidin is increased, although it does not attain the levels
found in Tmprss6 / mice, likely because the expanded erythropoiesis
in the double mutants drives partial hepcidin suppression.
Interestingly, TFR2 is a component of the erythropoietin receptor
complex and is necessary for its efficient transport to the erythroblast
surface.28 The development of a chimeric mouse with TFr2-
deficient BM is in progress in our laboratory and hopefully will help
to clarify the function of TFR2 in erythropoiesis.Unexpected results were obtained when crossing Tmprss6 / with
Hbbth3/ mice, which recapitulate features of thalassemia intermedia
patients, such as microcytic anemia, ineffective erythropoiesis,
splenomegaly, low hepcidin levels, and liver iron overload. As
expected, the ablation of Tmprss6 increased hepcidin expression
and reduced liver iron concentration, but, surprisingly, it also
decreased ineffective erythropoiesis and spleen size, improving


Table 1. Classification of hepcidin disorders
Gene Phenotype Mechanism Current therapy Potential treatment
Genetic disorders
I. Low hepcidin: iron overload
Hemochromatosis type 1 HFE Classic type Reduced hepcidin activation Phlebotomy Hepcidin agonists
Hemochromatosis type 2A HJV Juvenile type Ablation of BMP co-receptor Phlebotomy Hepcidin agonists
Hemochromatosis type 2B HAMP Juvenile type Ablation of hepcidin Phlebotomy Hepcidin agonists
Hemochromatosis type 3 TFR2 Early onset Reduced hepcidin activation Phlebotomy Hepcidin agonists
Hemochromatosis type 4A FPN Macrophage type Decreased iron export Phlebotomy Unknown
Hemochromatosis type 4B FPN Classic type Hepcidin-resistance Phlebotomy Unknown
II. High hepcidin: iron deficiency
IRIDA TMPRSS6 Iron deficiency, iron
refractoriness
Lack of hepcidin inhibition Parenteral iron Hepcidin antagonists
Other disorders
I. Low hepcidin: iron overload
Beta-thalassemia intermedia Anemia, iron overload Hepcidin suppression Iron chelation Hepcidin agonists
Congenital dyserythropoietic anemias Anemia, iron overload Hepcidin suppression Iron chelation Hepcidin agonists
Sideroblastic anemia Anemia, iron overload Hepcidin suppression Iron chelation Hepcidin agonists
II. High hepcidin: iron deficiency
Inflammation/cancer ACD Cytokine effects No specific treatment Hepcidin antagonists
Hepcidin overexpression EPO (selected
cases)
Chronic renal failure Severe anemia EPO insufficiency EPO iron Hepcidin antagonists
ACD Reduced hepcidin
clearance
Hepcidin-producing adenoma Iron deficiency Autonomous production Adenoma surgical
removal
Unknown
ACD indicates anemia of chronic diseases; and EPO, erythropoietin.


anemia and erythrocyte survival.29 These results provided the proof
of principle for novel thalassemia therapeutic strategies based upon
Tmprss6 manipulation. In addition, the characterization of this
double mutant mouse showed that full hepcidin suppression by
ineffective erythropoiesis requires a functional Tmprss6, in agreement
with the observation that Tmprss6 / mice are resistant to the
effect of exogenous erythropoietin.29,27 Only hypoxia seems to fully
suppress hepcidin in Tmprss6 / animals,27 suggesting an effect
downstream of Tmprss6 and consistent with the concept of multiple
inhibitors of the pathway.
Lessons from genome-wide association studies for
iron and erythrocyte parameters
Several genome-wide association studies have shown that common
TMPRSS6 genetic variants are associated with erythrocyte traits and
iron parameters, highlighting a role for TMPRSS6 in the control of
erythropoiesis in healthy individuals.30 A common associated
single nucleotide polymorphism (rs855791) causes a nonsynonymous
alanine to valine change in the catalytic domain of the protease (A736V), leading to the hypothesis that the hematological
effects are mediated by variations of hepcidin levels secondary
to the different inhibitory activity of TMPRSS6 polymorphic
alleles. The rs855791 allele (736V), associated with low MCV
and MCH, induced the hepcidin promoter more efficiently in an
in vitro assay and was associated with higher serum hepcidin and
lower iron levels than the 736A allele in a large series of healthy
individuals.31 Variants of iron genes, such as TMPRSS6 and
HFE, are associated with serum hepcidin, iron parameters and
erythrocyte traits, underlining the influence of hepcidin and iron
levels on normal erythropoiesis.31 These results further reinforce
the concept that TMPRSS6 modulates the BMP-SMAD pathway
in physiological conditions and that hepcidin production results
from the balance between opposing forces (Figure 4). This also
implies that the role of TMPRSS6 is especially relevant when
BMP6 is low. As a corollary, in the latter condition, the only
possibility for rapidly increasing hepcidin expression is to
suppress TMPRSS6 activity. From these data, TMPRSS6 appears
to be an important target for the manipulation of hepcidin
levels in different pathological conditions.
TMPRSS6 as a therapeutic target in disorders of low
hepcidin
Because the hepcidin pathway is deranged in several disorders, its
manipulation is an attractive novel therapeutic strategy. Hepcidin
agonists might replace hepcidin when insufficient, restoring the
correct balance and controlling iron overload in disorders with low
hepcidin (Table 1). Small synthetic peptides (minihepcidins) may
decrease serum iron in healthy mice and prevent iron overload and
promote at least partial iron redistribution in hepcidin-deficient mice
(Table 2).32 The pharmacologic inhibition of TMPRSS6 is emerging
as an alternative strategy to increase hepcidin levels. This strategy
has been exploited in preclinical studies using Tmprss6 small
interfering RNA formulated in lipid nanoparticles with high liver
affinity33 or by Tmprss6 allele-specific oligonucleotides34 (Table 2).
Tmprss6 silencing successfully increased hepcidin levels and reduced
serum and liver iron concentration in both hemochromatosis
Hfe / and thalassemia Hbbth3/ murine models. In thalassemic
mice, the effect overlaps that observed in the Tmprss6 / Hbbth3/
double mutant,29 with improvement of RBC maturation and survival
and partial correction of anemia. The positive effect was shown to
be thalassemia specific because Hfe / mice showed mild anemia as
a side effect of Tmpss6 inhibition.
On the opposite side, excessive hepcidin production can be antagonized
at different levels. Antagonists are under development with
the aim of reducing excessive hepcidin in anemia of chronic
diseases and are being tested in animal models of inflammation
(Table 2). However, it remains to be proven that reduction of
hepcidin alone can improve anemia in inflammatory disorders
because of the multifactorial nature of anemia of inflammation.
Iron as a modifier of erythropoiesis
Why does enhancing hepcidin production improve anemia in
beta-thalassemia? The analysis of the cross-talk between iron
homeostasis and erythropoiesis in Hbbth3/ mice led to the conclusion
that limiting iron may be beneficial in transfusion-independent
beta-thalassemia. Beta-thalassemia erythroblasts are unable to produce
adequate amounts of beta globin chains but synthesize normal
amounts of alpha chains and heme. Excess unpaired alpha chains
causes severe oxidative damage through hemichrome formation that
induces apoptosis and death of the erythroid precursors, leading to
“a high proliferation low differentiation state.”21 Limiting iron
might reduce the proliferation of immature cells and increase RBC
maturation. Positive effects were reported first in Hbbth1/th1 mice, a
model of mild thalassemia intermedia, treated by infusions of
exogenous transferrin35; then in Hbbth3/ mice engineered to overexpress
moderate amounts of hepcidin36; and finally by Tmprss6
pharmacological ablation.33,34 In all cases, the RBC count was
increased, although MCV and MCH were further reduced. This
suggests that in all of these situations, less iron was delivered to a
higher number of erythroid precursors than in control thalassemia
animals. Because heme is a strong regulator of protein translation in
erythroblasts, it is highly likely (but not yet proven) that decreased
heme synthesis due to low iron impairs alpha globin translation,
reducing globin chain imbalance, oxidative damage, and ineffective
erythropoiesis.37
Anemia may also improve after iron removal by chelators in
sideroblastic anemia, which is characterized by mitochondrial iron
accumulation in ringed sideroblasts and excessive reactive oxygen
species formation, and in some myelodysplastic syndromes.38
Limiting iron excess improves erythropoiesis in a proportion of
these patients, reinforcing the concept that iron overload has a
negative effect on erythropoiesis and that iron is a modifier of
erythroid maturation. Potential role of hepcidin in common nonhematologic
disorders
Derangement of iron metabolism at the systemic and/or cellular
level may accompany common pathological processes such as
infections, inflammatory and metabolic disorders, neurodegeneration,
and cancer. Hepcidin production by inflammatory macrophages
to control local iron availability is a phenomenon that might
be relevant in disorders characterized by low-grade inflammation,
such as obesity, diabetes, and metabolic syndrome.
The hepcidin-FPN axis has been proposed to mediate the acute and
chronic changes in iron distribution that contribute to host defense
in major infections.39 The relevance of iron for cell proliferation and
its propensity to generate reactive oxygen species explain the
increasing interest for the study of iron metabolism in cancer,
considering that hypoxia and inflammation create a microenvironment
that favors iron supply to neoplastic cells. The hepcidin-FPN
axis plays an important role in breast cancer, in which cell retention
of iron due to low FPN expression or the high hepcidin/high FPN
combination is a prognostic signature of cancer itself,40 an issue
worth investigating in hematologic malignancies as well.
The lessons learned from rare genetic iron disorders are beginning
to shed light on important pathogenic or protective mechanisms in
major human diseases.
Acknowledgments
This work was partially supported by Telethon Fondazione Onlus
Rome (Grants GGP12025 and MIUR PRIN 2010-2011) and the
Italian Ministry of Health (Grant RF-2010-2312048).
Disclosures
Conflict-of-interest disclosure: The author declares no competing
financial interests. Off-label drug use: None disclosed.
Correspondence
Clara Camaschella, MD, Vita-Salute University and San Raffaele
Scientific Institute, Via Olgettina, 60, 20132 Milano, Italy;
Phone: 39-02-2643-7782; Fax: 39-02-2641-2640; e-mail:
camaschella.clara@hsr.it.
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