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Cinzia GARUTI

Personale tecnico amministrativo
Dipartimento di Scienze Mediche e Chirurgiche Materno-Infantili e dell'Adulto

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Pubblicazioni

2023 - CREB-H is a stress-regulator of hepcidin gene expression during early postnatal development [Articolo su rivista]
Vecchi, C.; Montosi, G.; Garuti, C.; Canali, S.; Sabelli, M.; Bergamini, E.; Ricci, A.; Buzzetti, E.; Corradini, E.; Pietrangelo, A.
abstract

Hepcidin, the hepatic iron hormone, is the central regulator of iron homeostasis. Cyclic AMP-Responsive Element-Binding protein 3-like 3 (CREB3L3/CREB-H) is a liver homeostatic regulator of essential nutrients (i.e. glucose and lipids) and has been previously involved in hepcidin response to pathologic stress signals. Here, we asked whether CREB-H has also a physiologic role in iron homeostasis through hepcidin. To this end, we analyzed hepcidin gene expression and regulation in the liver of wild type and Creb3l3 knockout mice during early postnatal development, as a model of "physiologic" stressful condition. The effect of iron challenge in vivo and BMP6 stimulation in vitro have been also addressed. In addition, we investigated the BMP signaling pathway and hepcidin promoter activity following CREB3L3 silencing and hepcidin promoter mutation in HepG2 cells. Creb3l3 knockout suckling and young-adult mice showed a prominent serum and hepatic iron accumulation, respectively, due to impaired hepcidin mRNA expression which progressively returned to normal level in adult mice. Interestingly, upon iron challenge, while the upstream BMP/SMAD signaling pathway controlling hepcidin was equally responsive in both strains, hepcidin gene expression was impaired in knockout mice and more iron accumulated in the liver. Accordingly, hepcidin gene response to BMP6 was blunted in primary CREB-H knockout hepatocytes and in HepG2 cells transfected with CREB-H siRNA or carrying a hepcidin promoter mutated in the CREB-H binding site. In conclusion, CREB-H has a role in maintaining the homeostatic balance of iron traffic through hepcidin during the critical postnatal period and in response to iron challenge.Key messagesCREB-H KO mice develop liver iron overload shortly after weaning that normalizes in adulthood.CHEB-H is involved in hepcidin gene response to oral iron in vivo.CREB-H loss hampers hepcidin promoter response to BMP6.CREB-H is a key stress-sensor controlling hepcidin gene transcription in physiologic and pathophysiologic states.

2017 - Human macrophage ferroportin biology and the basis for the ferroportin disease [Articolo su rivista]
Sabelli, Manuela; Montosi, Giuliana; Garuti, Cinzia; Caleffi, Angela; Oliveto, Stefania; Biffo, Stefano; Pietrangelo, Antonello
abstract

Ferroportin (FPN1) is the sole iron exporter in mammals, but its cell-specific function and regulation are still elusive. This study examined FPN1 expression in human macrophages, the cells that are primarily responsible on a daily basis for plasma iron turnover and are central in the pathogenesis of ferroportin disease (FD), the disease attributed to lack-of-function FPN1 mutations. We characterized FPN1 protein expression and traffic by confocal microscopy, western blotting, gel filtration, and immunoprecipitation studies in macrophages from control blood donors (donor) and patients with either FPN1 p.A77D, p.G80S, and p.Val162del lack-of-function or p.A69T gain-of-function mutations. We found that in normal macrophages, FPN1 cycles in the early endocytic compartment does not multimerize and is promptly degraded by hepcidin (Hepc), its physiological inhibitor, within 3-6 hours. In FD macrophages, endogenous FPN1 showed a similar localization, except for greater accumulation in lysosomes. However, in contrast with previous studies using overexpressed mutant protein in cell lines, FPN1 could still reach the cell surface and be normally internalized and degraded upon exposure to Hepc. However, when FD macrophages were exposed to large amounts of heme iron, in contrast to donor and p.A69T macrophages, FPN1 could no longer reach the cell surface, leading to intracellular iron retention. Conclusion: FPN1 cycles as a monomer within the endocytic/plasma membrane compartment and responds to its physiological inhibitor, Hepc, in both control and FD cells. However, in FD, FPN1 fails to reach the cell surface when cells undergo high iron turnover. Our findings provide a basis for the FD characterized by a preserved iron transfer in the enterocytes (i.e., cells with low iron turnover) and iron retention in cells exposed to high iron flux, such as liver and spleen macrophages. (Hepatology 2017;65:1512-1525).

2016 - The SMAD pathway is required for hepcidin response during endoplasmic reticulum stress [Articolo su rivista]
Canali, Susanna; Vecchi, Chiara; Garuti, Cinzia; Montosi, Giuliana; Babitt, Jodie L; Pietrangelo, Antonello
abstract

Hepcidin, the iron hormone, is regulated by a number of stimulatory and inhibitory signals. The cAMP responsive element binding protein 3-like 3, CREB3L3, mediates hepcidin response to endoplasmic reticulum (ER) stress. In this study we asked whether hepcidin response to ER stress also requires the SMAD1/5/8 pathway that has a major role in hepcidin regulation in response to iron and other stimuli. We analyzed hepcidin mRNA expression and promoter activity in response to ER stressors in HepG2 cells in the presence of the BMP type I receptor inhibitor LDN-193189, mutated hepcidin promoter or siRNA against different SMAD proteins. We then used a similar approach in vivo in wild-type, Smad1/5 or Creb3l3 -/- animals undergoing ER stress. In vitro, LDN-193189 prevented hepcidin mRNA induction by different ER stressors. Seemingly, mutation of a BMP-responsive element in the hepcidin promoter prevented ER stress-mediated upregulation. Moreover, in vitro silencing of SMAD proteins by siRNA, in particular SMAD5, blunted hepcidin response to ER stress. On the contrary, hepcidin induction by ER stress was maintained when using antibodies against canonical BMP receptor ligands. In vivo, hepcidin was induced by ER stress and prevented by LDN-193189. In addition, in Smad1/5 knock-out mice, ER stress was unable to induce hepcidin expression. Finally, in Creb3l3 knock-out mice, in response to ER stress, SMAD1/5 were correctly phosphorylated and hepcidin induction was still appreciable, although to a lesser extent as compared to control mice. In conclusion, our study indicates that hepcidin induction by ER stress involves the central regulatory SMAD1/5 pathway.

2014 - GLUCONEOGENIC SIGNALS DIRECTLY CONTROL IRON HOMEOSTASIS THROUGH HEPCIDIN [Abstract in Atti di Convegno]
Vecchi, Chiara; Montosi, Giuliana; Garuti, Cinzia; Corradini, Elena; Sabelli, Manuela; Qian, J; Liu, C; Canali, S; Pietrangelo, Antonello
abstract

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2014 - Gluconeogenic Signals Regulate Iron Homeostasis via Hepcidin in Mice. [Articolo su rivista]
Vecchi, Chiara; Montosi, Giuliana; Garuti, Cinzia; Corradini, Elena; Sabelli, Manuela; Canali, Susanna; Pietrangelo, Antonello
abstract

Hepatic gluconeogenesis provides fuel during starvation, and is abnormally induced in obese individuals or those with diabetes. Common metabolic disorders associated with active gluconeogenesis and insulin resistance (obesity, metabolic syndrome, diabetes, and nonalcoholic fatty liver disease) have been associated with alterations in iron homeostasis that disrupt insulin sensitivity and promote disease progression. We investigated whether gluconeogenic signals directly control Hepcidin, an important regulator of iron homeostasis, in starving mice (a model of persistently activated gluconeogenesis and insulin resistance).|We investigated hepatic regulation of Hepcidin expression in C57BL/6Crl, 129S2/SvPas, BALB/c, and wild-type and Creb3l3-/- null mice. Mice were fed a standard, iron-balanced chow diet or an iron-deficient diet for 9 days before death, or for 7 days before a 24- to 48-hour starvation period; liver and spleen tissues then were collected and analyzed by quantitative reverse-transcription polymerase chain reaction and immunoblot analyses. Serum levels of iron, hemoglobin, Hepcidin, and glucose also were measured. We analyzed human hepatoma (HepG2) cells and mouse primary hepatocytes to study transcriptional control of Hamp (the gene that encodes Hepcidin) in response to gluconeogenic stimuli using small interfering RNA, luciferase promoter, and chromatin immunoprecipitation analyses.|Starvation led to increased transcription of encodes phosphoenolpyruvate carboxykinase 1 (a protein involved in gluconeogenesis) in livers of mice, increased levels of Hepcidin, and degradation of Ferroportin, compared with nonstarved mice. These changes resulted in hypoferremia and iron retention in liver tissue. Livers of starved mice also had increased levels of Ppargc1a messenger RNA and Creb3l3 messenger RNA, which encode a transcriptional co-activator involved in energy metabolism and a liverspecific transcription factor, respectively. Glucagon and a cyclic adenosine monophosphate analog increased promoter activity and transcription of Hamp in cultured liver cells; levels of Hamp were reduced after administration of small interfering RNAs against Ppargc1a and Creb3l3. PPARGC1A and CREB3L3 bound the Hamp promoter to activate its transcription in response to a cyclic adenosine monophosphate analog. Creb3l3-/- mice did not up-regulate Hamp or become hypoferremic during starvation.|We identified a link between glucose and iron homeostasis, showing that Hepcidin is a gluconeogenic sensor in mice during starvation. This response is involved in hepatic metabolic adaptation to increased energy demands; it preserves tissue iron for vital activities during food withdrawal, but can cause excessive iron retention and hypoferremia in disorders with persistently activated gluconeogenesis and insulin resistance.

2014 - SEX HORMONES DIFFERENTLY REGULATE HEPATIC HEPCIDIN EXPRESSION AND SYSTEMIC IRON HOMEOSTASIS IN VIVO [Abstract in Rivista]
Garuti, Cinzia; Montosi, Giuliana; Barelli, S; Pietrangelo, Antonello; Corradini, Elena
abstract

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2013 - SEX HORMONES DIFFERENTLY REGULATE HEPCIDIN EXPRESSION AND IRON HOMEOSTASIS IN VIVO. [Abstract in Rivista]
Garuti, Cinzia; Montosi, Giuliana; S., Barelli; Pietrangelo, Antonello; Corradini, Elena
abstract

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2013 - THE BMP'S PATHWAY IN HEPCIDIN REGULATION DURING ENDOPLASMIC RETICULUM STRESS RESPONSE. [Abstract in Rivista]
Canali, Susanna; Vecchi, Chiara; Garuti, Cinzia; Pietrangelo, Antonello
abstract

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2011 - THE MOLECULAR BASIS FOR THE HEPATIC REGULATION OF HEPCIDIN, THE IRON HORMONE, BY BONE MORPHOGENETIC PROTEINS. [Abstract in Rivista]
Corradini, Elena; Meynard, D; Montosi, Giuliana; Garuti, Cinzia; Wu, Q; Ventura, Paolo; Babitt, Jl; Lin, Hy; Pietrangelo, Antonello
abstract

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2010 - Altered hepatic BMP signaling pathway in human HFE hemochromatosis. [Articolo su rivista]
Bolondi, G; Garuti, Cinzia; Corradini, Elena; Zoller, H; Vogel, W; Finkenstedt, A; Babitt, Jl; Lin, Hy; Pietrangelo, Antonello
abstract

Human hemochromatosis (HC) has been associated with the common C282Y polymorphism of HFE or rare pathogenic mutations of TfR2, HJV, FPN and HAMP. All forms of human HC seem to be caused by low or inadequate levels of hepcidin, the iron hormone. We and others have recently shown that Hfe(-/-) mice exhibit an impairment in the bone morphogenetic protein (BMP) signaling pathway controlling hepcidin. However, all data indicating the central role of BMPs in hepcidin regulation and an impaired BMP/SMAD signaling in HC have been collected in mice. In this study we investigated whether also in humans the expression of BMP signaling targets, SMAD7 and Id1, are associated with liver iron concentration (LIC) and whether such regulation is disrupted in HFE-HC. We correlated the mRNA expression, assessed by RT-PCR, of HAMP, SMAD7 and Id1 with LIC in liver biopsies from patients with normal iron status. HFE-HC or non-HC hepatic iron overload. We found that in human liver, not only HAMP, but also SMAD7 and Id1 mRNA significantly correlate with the extent of hepatic iron burden. However, this correlation is lost in patients with HFE-HC, but maintained in subjects with non-hemochromatotic iron overload. These data indicate that in human HFE-HC a disrupted BMP/SMAD signaling in the liver is key in the pathogenesis of the disease.

2010 - BMP6 treatment compensates for the molecular defect and ameliorates hemochromatosis in Hfe knockout mice. [Articolo su rivista]
Corradini, Elena; Schmidt, Pj; Meynard, D; Garuti, Cinzia; Montosi, Giuliana; Chen, S; Vukicevic, S; Pietrangelo, Antonello; Lin, Hy; Babitt, J. L.
abstract

BACKGROUND AND AIMS: Abnormal hepcidin regulation is central to the pathogenesis of HFE hemochromatosis. Hepatic bone morphogenetic protein 6 (BMP6)-SMAD signaling is a main regulatory mechanism controlling hepcidin expression, and this pathway was recently demonstrated to be impaired in Hfe knockout (Hfe(-/-)) mice. To more definitively determine whether HFE regulates hepcidin expression through an interaction with the BMP6-SMAD signaling pathway, we investigated whether hepatic Hfe overexpression activates the BMP6-SMAD pathway to induce hepcidin expression. We then investigated whether excess exogenous BMP6 administration overcomes the BMP6-SMAD signaling impairment and ameliorates hemochromatosis in Hfe(-/-) mice.METHODS: The BMP6-SMAD pathway and the effects of neutralizing BMP6 antibody were examined in Hfe transgenic mice (Hfe Tg) compared with wildtype (WT) mice. Hfe(-/-) and WT mice were treated with exogenous BMP6 and analyzed for hepcidin expression and iron parameters.RESULTS: Hfe Tg mice exhibited hepcidin excess and iron deficiency anemia. Hfe Tg mice also exhibited increased hepatic BMP6-SMAD target gene expression compared with WT mice, while anti-BMP6 antibody administration to Hfe Tg mice improved the hepcidin excess and iron deficiency. In Hfe(-/-) mice, supraphysiologic doses of exogenous BMP6 improved hepcidin deficiency, reduced serum iron, and redistributed tissue iron to appropriate storage sites.CONCLUSIONS: HFE interacts with the BMP6-SMAD signaling pathway to regulate hepcidin expression, but HFE is not necessary for hepcidin induction by BMP6. Exogenous BMP6 treatment in mice compensates for the molecular defect underlying Hfe hemochromatosis, and BMP6-like agonists may have a role as an alternative therapeutic strategy for this disease.

2010 - Hepcidin expression does not rescue the iron-poor phenotype of Kupffer cells in Hfe-null mice after liver transplantation. [Articolo su rivista]
Garuti, Cinzia; Tian, Y; Montosi, Giuliana; Sabelli, Manuela; Corradini, Elena; Graf, R; Ventura, Paolo; Vegetti, Alberto; Clavien, Pa; Pietrangelo, Antonello
abstract

BACKGROUND & AIMS: Hemochromatosis is a common hereditary disease caused by mutations in HFE and characterized by increased absorption of iron in the intestine. However, the intestine does not appear to be the site of mutant HFE activity in the disease; we investigated the role of the liver-the source of the iron regulatory hormone hepcidin-in pathogenesis in mice. METHODS: We exchanged livers between Hfe wild-type (+/+) and Hfe null (-/-) mice by orthotopic liver transplantation (OLT) and assessed histopathology, serum and tissue iron parameters, and hepatic hepcidin messenger RNA expression. RESULTS: At 6-8 months after OLT, Hfe(-/-) mice that received Hfe(-/-) livers maintained the hemochromatosis phenotype: iron accumulation in hepatocytes but not Kupffer cells (KC), increased transferrin levels, and low levels of iron in the spleen. Hfe(+/+) mice that received Hfe(-/-) livers had increased levels of iron in serum and liver and low levels of iron in spleen. However, they did not develop the iron-poor KCs that characterize hemochromatosis: KCs appeared iron rich, although hepatic hepcidin expression was low. Transplantation of Hfe(+/+) livers into Hfe(-/-) mice prevented hepatic iron accumulation but did not return spleen and plasma levels of iron to normal; KCs still appeared to be iron poor, despite normal hepcidin expression. CONCLUSIONS: In Hfe(-/-) mice, transplantation of livers from Hfe(+/+) mice reversed the iron-loading phenotype associated with hemochromatosis (regardless of Hfe expression in intestine). However, KCs still had low levels of iron that were not affected by hepatic hepcidin expression. These findings indicate an independent, iron-modifying effect of HFE in KCs.

2009 - Bone Morphogenetic Protein Signaling Is Impaired in an Hfe Knockout Mouse Model of Hemochromatosis. [Articolo su rivista]
Corradini, Elena; Garuti, Cinzia; Montosi, Giuliana; Ventura, Paolo; Andriopoulos, B; Lin, Hy; Pietrangelo, Antonello; Babitt, Jl
abstract

Mutations in HFE are the most common cause of the iron-overload disorder hereditary hemochromatosis. Levels of the main iron regulatory hormone, hepcidin, are inappropriately low in hereditary hemochromatosis mouse models and patients with HFE mutations, indicating that HFE regulates hepcidin. The bone morphogenetic protein 6 (BMP6)-SMAD signaling pathway is an important endogenous regulator of hepcidin expression. We investigated whether HFE is involved in BMP6-SMAD regulation of hepcidin expression. METHODS: The BMP6-SMAD pathway was examined in Hfe knockout (KO) mice and in wild-type (WT) mice as controls. Mice were placed on diets of varying iron content. Hepcidin induction by BMP6 was examined in primary hepatocytes from Hfe KO mice; data were compared with those of WT mice. RESULTS: Liver levels of Bmp6 messenger RNA (mRNA) were higher in Hfe KO mice; these were appropriate for the increased hepatic levels of iron in these mice, compared with WT mice. However, levels of hepatic phosphorylated Smad 1/5/8 protein (an intracellular mediator of Bmp6 signaling) and Id1 mRNA (a target gene of Bmp6) were inappropriately low for the body iron burden and Bmp6 mRNA levels in Hfe KO, compared with WT mice. BMP6 induction of hepcidin expression was reduced in Hfe KO hepatocytes compared with WT hepatocytes. CONCLUSIONS: HFE is not involved in regulation of BMP6 by iron, but does regulate the downstream signals of BMP6 that are triggered by iron.

2007 - Effect of anti-osteoarthritic drugs on interleukin 1-dependent activation of NF-kappa B in human chondrocytes [Abstract in Atti di Convegno]
S., Barelli; Garuti, Cinzia; Montosi, Giuliana; Pietrangelo, Antonello
abstract

No abstract available

2007 - STAT3 is required for IL-6-gp130-dependent activation of hepcidin in vivo [Articolo su rivista]
Pietrangelo, Antonello; U., Dierssen; L., Valli; Garuti, Cinzia; A., Rump; Corradini, Elena; M., Ernst; C., Klein; C., Trautwein
abstract

BBackground & Aims: Hepcidin is a peptide hormone that is central to the regulation of iron homeostasis. In response to interleukin 6 (IL-6), hepatocytes produce hepcidin that decreases iron release/transfer from enterocytes and macrophages and causes hypoferremia. To clarify the molecular pathways involved in hepcidin activation by IL-6, we used different mice strains in which the main IL-6/gp130 signaling pathways have been genetically disrupted. Methods: We generated mice with hepatocyte-specific deletion of the IL-6 signaltransducing gp130 receptor (alfgp130 (LoxP/LoxP)), with a gp130 receptor lacking the essential region for STAT1 and -3 activation (alrpCre gp130(Delta STAT/LoxP)) or mice expressing a gp130 allele lacking the essential tyrosine for RAS-MAPK activation (alfpCregp130(Y757F/LoxP)). We studied gp130-dependent pathways and hepcidin mRNA expression by Western blot, reverse-transcription polymerase chain reaction, and Northern blot in vivo and ex vivo. Results: IL-6 stimulated phospho STAT3, serum amyloid A (SAA), and suppressor of cytokine signaling 3 (SOCS3) expression in livers of mild-type and alfpCregp130(Y757F/LoxP) mice, whereas this response was blocked in alfpCre gp130(LoxP/LoxP) and alfpCre gp130(Delta STAT/LoxP) mice. In wild-type and alfpCregP130(Y757F/LoxP) animals, significantly higher hepcidin mRNA expression was found 3 to 6 hours after IL-6 stimulation. In contrast, no IL-6-dependent regulation of hepcidin mRNA expression was found in alfpgp130(Delta STAT/LoxP) and AlfpCre gp130 (LoxP.LoxP) animals. In primary hepatocytes, higher hepcidin mRNA expression after IL-6 stimulation was only observed when gp130-STAT3-dependent signaling was intact. Conclusions: We have demonstrated that both in vivo and in vitro STAT3 is the key transcription factor responsible

2005 - Haptoglobin modifies the hemochromatosis phenotype in mice [Articolo su rivista]
E., Tolosano; S., Fagoonee; Garuti, Cinzia; L., Valli; N. C., Andrews; F., Altruda; Pietrangelo, Antonello
abstract

Classic hereditary hemochromatosis (HH) is a common genetic disorder of iron metabolism caused by a mutation in the HFE gene. Whereas the prevalence of the mutation is very high, the clinical penetrance of the disease is low, suggesting that the HFE mutation is a necessary but not sufficient cause of clinical HH. Several candidate modifier genes have been proposed in mice and humans, including haptoglobin. Haptoglobin is the plasma protein with the highest binding affinity for hemoglobin. It delivers free plasma hemoglobin to the reticuloendothelial system, thus reducing loss of hemoglobin through the glomerull and allowing heme-iron recycling. To gain insight into the role of haptoglobin as a modifier gene in HH, we used Hfe and haptoglobin double-null mice. Here, we show that Hfe and haptoglobin compound mutant mice accumulate significantly less hepatic iron than Hfe-null mice, thus demonstrating that haptoglobin-mediated heme-iron recovery may contribute significantly to iron loading in HH.

2005 - Juvenile hemochromatosis associated with pathogenic mutations of adult hemochromatosis genes [Articolo su rivista]
Pietrangelo, Antonello; Caleffi, Angela; Henrion, J.; Ferrara, F.; Corradini, Elena; Kulaksiz, H.; Stremmel, W.; Andreone, P.; Garuti, Cinzia
abstract

Background & Aims: Juvenile hemochromatosis is a severe form of hereditary iron overload that has thus far been linked to pathogenic mutations of the gene coding for hemojuvelin (HJV), on chromosome 1, or, more rarely, that coding for hepcidin (HAMP), on chromosome 19. A milder adult-onset form is due to pathogenic mutations of HFE or, rarely, serum transferrin receptor 2. Methods: We studied a pedigree with siblings affected by both juvenile and adult-onset hereditary hemochromatosis. Affected subjects underwent full clinical evaluation, as well as microsatellite and gene sequencing analysis. Results: Two siblings (male and female, aged 24 and 25 years, respectively) were hospitalized for severe endocrinopathy and cardiomyopathy. At age 18 and 17 years, they had presented with impotence and amenorrhea, respectively, and increased serum iron levels. Hypogonadotropic hypogonadism was confirmed in both, and liver biopsy showed marked hepatic iron accumulation and micronodular cirrhosis. Iron levels were normalized after 24 months (female) and 36 months (male) of weekly phlebotomies. Microsatellite analysis showed no linkage with chromosome I and 19, and gene sequencing showed no hemojuvelin or hepcidin gene mutations. Instead, combined mutations of HFE (C282Y/H63D compound heterozygosity) and serum transferrin receptor 2 (Q317X homozygosity) were found. A 21-year-old brother with a milder phenotype resembling classic adult-onset hereditary hemochromatosis carried only the Q317X serum transferrin receptor 2 homozygote mutation. Conclusions: Juvenile hereditary hemochromatosis is not a distinct monogenic disorder invariably due to hemojuvelin or hepcidin mutations: it may be genetically linked to the adult-onset form of hereditary hemochromatosis.

2005 - Kupffer cells and macrophages are not required for hepatic hepcidin activation during iron overload [Articolo su rivista]
Montosi, Giuliana; Corradini, Elena; Garuti, Cinzia; S., Barelli; S., Recalcati; G., Cairo; L., Valli; Pignatti, Elisa; Vecchi, Chiara; F., Ferrara; Pietrangelo, Antonello
abstract

Hepcidin, the iron hormone, is produced by the liver in response to iron and inflammation. Its synthesis during inflammation is triggered by cytokines, but the details of iron activation are obscure. We tested the role of Kupffer cells and macrophages by studying iron-loaded or inflamed mice with selective inactivation of Kupffer cells or the in vitro effect of conditioned human macrophages on hepcidin expression. Hepcidin messenger RNA (mRNA) expression was studied by Northern blot and reverse transcriptase polymerase chain reaction analysis in mice that were treated with 40 mg/kg gadolinium (III) chloride (GdCl3) as a Kupffer cell inactivating agent and subjected to inflammatory challenges with either lipopolysaccharide (LPS) and turpentine or iron overload by iron-dextran administration. Similar analyses were performed in human hepatoma cells (HepG2) cultured with medium from LPS- or iron-conditioned macrophages from blood donors or patients with HFE-linked hereditary hemochromatosis (HH). In vivo, LPS and particularly turpentine stimulated hepcidin mRNA expression, and this effect was prevented by the inactivation of Kupffer cells. Also, iron overload markedly upregulated hepatic hepcidin mRNA, but this activity persisted in spite of Kupffer cell blockade. In vitro, the medium of LPS-treated normal or hemocromatotic macrophages turned on hepcidin expression. On the contrary, medium of iron-manipulated macrophages, regardless of their HFE status, did not affect hepcidin mRNA steady-state levels. In conclusion, Kupffer cells are required for the activation of hepcidin synthesis during inflammation, and HH inflamed macrophages are capable of mounting a normal response, eventually leading to hepcidin stimulation. However, both Kupffer cells and human macrophages are dispensable for the regulatory activity exerted by iron on hepatic hepcidin.

2005 - Lack of enterocyte iron accumulation in the ferroportin disease [Articolo su rivista]
Corradini, Elena; Montosi, Giuliana; F., Ferrara; A., Caleffi; Pignatti, Elisa; S., Barelli; Garuti, Cinzia; Pietrangelo, Antonello
abstract

Ferroportin-associated iron overload (also known as the ferroportin disease) is a common cause of hereditary hyperferritinemia. It was originally proposed that loss-of-protein function mutations account for iron overload in the FD. This hypothesis is consistent with the 14 phenotype reported in most patients with FD of early iron accumulation in tissues, particularly in macrophages, in spite of relatively normal-low circulatory iron. It was still unclear, however, how FPN mutations would affect iron retention in enterocytes. We studied histologically the intestine of six patients with different FPN mutations as compared to other subjects with various iron disorders. We found that regardless of the underlying FPN mutation, no iron accumulation was found in absorbing enterocytes while, intestinal villi showed marked signs of iron accumulation in the cells of lamina propria. Not surprisingly, in the liver, iron excess was found mainly in Kupffer cells. These results indicate that FPN haploinsufficiency is not limiting for iron export from enterocytes.

2004 - Erratum: Iron overload in Africans and African-Americans and a common mutation in the SCL40A1 (ferroportin 1) gene (Blood Cells, Molecules, and Diseases (2003) 31 (299-304) DOI: 10.1016/S1079-9796(03)00164-5) [Articolo su rivista]
Gordeuk, V. R.; Caleffi, A.; Corradini, E.; Ferrara, F.; Jones, R. A.; Castro, O.; Onyekwere, O.; Kittles, R.; Pignatti, E.; Montosi, G.; Garuti, C.; Gangaidzo, I. T.; Gomo, Z. A. R.; Moyo, V. M.; Rouault, T. A.; Macphail, P.; Pietrangelo, A.
abstract

2003 - Iron overload in Africans and African-Americans and a common mutation in the SCL40A1 (ferroportin 1) gene [Articolo su rivista]
Gordeuk, Vr; Caleffi, Angela; Corradini, Elena; Ferrara, Francesca; Jones, Ra; Castro, O; Onyekwere, O; Kittles, R; Pignatti, Elisa; Montosi, Giuliana; Garuti, Cinzia; Gangaidzo, It; Gomo, Zar; Moyo, Vm; Rouault, Ta; Macphail, P; Pietrangelo, Antonello
abstract

The product of the SLC40A1 gene, ferroportin 1, is a main iron export protein. Pathogenic mutations in ferroportin 1 lead to an autosomal dominant hereditary iron overload syndrome characterized by high serum ferritin concentration, normal transferrin saturation, iron accumulation predominantly in macrophages, and marginal anemia. Iron overload occurs in both the African and the African-American populations, but a possible genetic basis has not been established. We analyzed the ferroportin 1 gene in 19 unrelated patients from southern Africa (N = 15) and the United States (N = 4) presenting with primary iron overload. We found a new c. 744 C→T (Q248H) mutation in the SLC40A1 gene in 4 of these patients (3 Africans and 1 African-American). Among 22 first degree family members, 10 of whom were Q248H heterozygotes, the mutation was associated with a trend to higher serum ferritin to amino aspartate transferase ratios (means of 14.8 versus 4.3 μg/U; P = 0.1) and lower hemoglobin concentrations (means of 11.8 versus 13.2 g/dL; P = 0.1). The ratio corrects serum ferritin concentration for alcohol-induced hepatocellular damage. We also found heterozygosity for the Q248H mutation in 7 of 51 (14%) southern African community control participants selected because they had a serum ferritin concentration below 400 μg/L and in 5 of 100 (5%) anonymous African-Americans, but we did not find the change in 300 Caucasians with normal iron status and 25 Caucasians with non-HFE iron overload. The hemoglobin concentration was significantly lower in the African community controls with the Q248H mutation than in those without it. We conclude that the Q248H mutation is a common polymorphism in the ferroportin 1 gene in African populations that may be associated with mild anemia and a tendency to iron loading.

2003 - The role of the iron responsive element in the control of ferroportin1/IREG1/MTP1 gene expression [Articolo su rivista]
A., Lymboussaki; Pignatti, Elisa; Montosi, Giuliana; Garuti, Cinzia; Dj, Haile; Pietrangelo, Antonello
abstract

Background/Aims: MTP1/Ferroportin1/IREG1, the product of the SLC40A1 gene, is a main iron export protein in mammals. However, the way this gene is regulated by iron is still unclear. The aim of this study was to investigate the functional role of genomic SLC40A1 elements in response to iron. Methods: Vectors containing either similar to 2.6 kb 5' flanking region or deletion constructs, including one devoid of an iron responsive element (SLC40A1-DeltaIRE-Luc), were analyzed by luciferase reporter gene in transfected HepG2, CaCO2 and U937 cells. Expression of iron genes and activity of the iron regulatory protein were also studied. Results: Iron increased and desferrioxamine decreased luciferase activity in all the cell types using both the full-length construct and the promoter deletion constructs, in the absence of changes in SLC40A1 or luciferase mRNA levels. To test the role of the SLC40A1 5' untranslated region, we first demonstrated that wild type and not SLC40A1-DeltaIRE-Luc could bind iron regulatory protein. Then, in cells transfected with SLC40A1-DIRE-Luc, we found that, in spite of iron regulatory protein activation, the response to iron manipulation was lost. Conclusions: We demonstrate that the iron responsive element in the SLC40A1 gene is functional and that it controls gene expression through the cytoplasmic iron regulatory protein system. (C) 2003 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

2002 - Iron-induced oxidant stress in nonparenchymal liver cells: Mitochondrial derangement and fibrosis in acutely iron-dosed gerbils and its prevention by silybin [Articolo su rivista]
Pietrangelo, Antonello; Montosi, Giuliana; Garuti, Cinzia; Contri, Miranda; F., Giovannini; D., Ceccarelli; A., Masini
abstract

Hepatic fibrosis due to iron overload is mediated by oxidant stress. The basic mechanisms underlying this process in vivo are still little understood. Acutely iron-dosed gerbils were assayed for lobular accumulation of hepatic lipid peroxidation by-products, oxidant-stress gene response, mitochondrial energy-dependent functions, and fibrogenesis. Iron overload in nonparenchymal cells caused an activation of hepatic stellate cells and fibrogenesis. Oxidant-stress gene response and accumulation of malondialdehyde-protein adducts were restricted to iron-filled nonparenchymal cells, sparing nearby hepatocytes. Concomitantly, a significant rise in the mitochondrial desferrioxamine-chelatable iron pool associated with the impairment of mitochondrial oxidative metabolism and the hepatic ATP decrease, was detected. Ultrastructural mitochondrial alterations were observed only in nonparenchymal cells. All biochemical and functional derangements were hindered by in vivo silybin administration which blocked completely fibrogenesis. Iron-induced oxidant stress in nonparenchymal cells appeared to bring about irreversible mitochondrial derangement associated with the onset of hepatic fibrosis.

2001 - Autosomal-dominant hemochromatosis is associated with a mutation in the ferroportin (SLC11A3) gene [Articolo su rivista]
Montosi, Giuliana; Donovan, A.; Totaro, Antonella; Garuti, Cinzia; Pignatti, Elisa; Cassanelli, S.; Trenor, C. C.; Gasparini, P.; Andrews, N. C.; Pietrangelo, Antonello
abstract

Hemochromatosis is a progressive iron overload disorder that is prevalent among individuals of European descent. It is usually inherited in an autosomal-recessive pattern and associated with missense mutations in HFE, an atypical major histocompatibility class I gene. Recently, we described a large family with autosomal-dominant hemochromatosis not linked to HFE and distinguished by early iron accumulation in reticuloendothelial cells. Through analysis of a large pedigree, we have determined that this disease maps to 2q32. The gene encoding ferroportin (SLC11A3), a transmembrane iron export protein, lies within a candidate interval defined by highly significant lod scores. We show that the iron-loading phenotype in autosomal-dominant hemochromatosis is associated with a nonconservative missense mutation in the ferroportin gene. This missense mutation, converting alanine to aspartic acid at residue 77 (A77D), was not seen in samples from 100 unaffected control individuals. We propose that partial loss of ferroportin function leads to an imbalance in iron distribution and a consequent increase in tissue iron accumulation.

2001 - Frequency and biochemical expression of C282Y/H63D hemochromatosis (HFE) gene mutations in the healthy adult population in Italy [Articolo su rivista]
Cassanelli, S; Pignatti, Elisa; Montosi, Giuliana; Garuti, Cinzia; Mariano, M; Campioli, D; Carbonieri, A; Baldini, Enrica; Pietrangelo, Antonello
abstract

Background/Aims: The actual prevalence of the main hemochromatosis (HFE) mutations in the Italian adult population and their phenotypic expression have not yet been established. This information is key to advocate a mass-screening program. Methods: Two thousand one hundred adults were tested for the C282Y/H63D HFE gene mutations by an automated genotyping assay as well as transferrin saturation (TS) and serum ferritin levels. Results: No homozygotes for the C282Y mutation were found. Heterozygosity for the C282Y mutation was 3.1%, while heterozygosity and homozygosity for the H63D mutation were 21.5% and 2.5%, respectively. TS was significantly higher in C282Y heterozygotes and H63D homozygotes as compared to wild-type individuals (P < 0.01). Interestingly, of the HFE wild-type subjects 5.9% had a TS value above the 45% threshold. Conclusions: This study shows that (i) the predicted prevalence for C282Y homozygosity in Italy is 1:3900; (ii) the C282Y/H63D wild-type population has an increased baseline of iron parameters possibly due to genetic factors not linked to the C282Y/H63D mutations; (iii) since in the latter population the actual tissue iron burden cannot be assessed, phenotypic (TS) screening in Italy is not recommended until the true prevalence of all mutations in the HFE gene and in other hemochromatosis genes will be established. (C) 2001 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved.

2001 - The low-affinity neurotrophin receptor (p75) mediates apoptosis in human keratinocytes [Abstract in Rivista]
Marconi, Alessandra; C., Fila; S., Bertellini; M., Pignatti; Garuti, Cinzia; Giannetti, Alberto; Pincelli, Carlo
abstract

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2000 - Iron-induced oxidant stress leads to irreversible mitochondrial dysfunctions and fibrosis in the liver of chronic iron-dosed gerbils. The effect of silybin [Articolo su rivista]
A., Masini; D., Ceccarelli; F., Giovannini; Montosi, Giuliana; Garuti, Cinzia; Pietrangelo, Antonello
abstract

Hepatic iron toxicity because of iron overload seems to be mediated by lipid peroxidation of biological membranes and the associated organelle dysfunctions. However, the basic mechanisms underlying this process in vivo are still little understood. Gerbils were dosed with weekly injections of iron-dextran alone or in combination with sylibin, a well-known antioxidant, by gavage for 8 weeks. A strict correlation was found between lipid peroxidation and the level of desferrioxamine chelatable iron pool. A consequent derangement in the mitochondrial energy-transducin capability, resulting from a reduction in the respiratory chain enzyme activities, occurred. These irreversible oxidative anomalies brought about a dramatic drop in tissue ATP level, The mitochondrial oxidative derangement was associated with the development of fibrosis in the hepatic tissue. Silybin administration significantly reduced both functional anomalies and the fibrotic process by chelating desferrioxamine chelatable iron.

2000 - Wild-type HFE protein normalizes transferrin iron accumulation in macrophages from subjects with hereditary hemochromatosis [Articolo su rivista]
Montosi, Giuliana; P., Paglia; Garuti, Cinzia; Ca, Guzman; Jm, Bastin; Mp, Colombo; Pietrangelo, Antonello
abstract

Hereditary hemochromatosis (HC) is one of the most common single-gene hereditary diseases. A phenotypic hallmark of HC is low iron in reticuloendothelial cells in spite of body iron overload. Most patients with HC have the same mutation, a change of cysteine at position 282 to tyrosine (C282Y) in the HFE protein. The role of HFE in iron metabolism and the basis for the phenotypic abnormalities of HC are not understood. To clarify the role of HFE in the phenotypic expression of HC, we stud led monocytes-macrophages from subjects carrying the C282Y mutation in the HFE protein and clinically expressing HC and transfected them with wild-type HFE by using an attenuated Salmonella typhimurium strain as a gene carrier. The Salmonella system allowed us to deliver genes of interest specifically to monocytes-macrophages with high transduction efficiency. The accumulation of Fe-55 delivered by Fe-55-Tf was significantly lower in macrophages from patients with HC than from controls expressing wild-type HFE. Transfection of HC macrophages with the HFE gene resulted in a high level of expression of HFE protein at the cell surface, The accumulation of Fe-55 delivered by 55Fe-Tf was raised by 40% to 60%, and this was reflected by an increase in the Fe-55-ferritin pool within the HFE-transfected cells. These results suggest that the Iron-deficient phenotype of HC macrophages is a direct effect of the HFE mutation, and they demonstrate a role for HFE in the accumulation of iron in these cells.

1999 - Hereditary hemochromatosis in adults without pathogenic mutations in the hemochromatosis gene [Articolo su rivista]
Pietrangelo, Antonello; Montosi, Giuliana; A., Totaro; Garuti, Cinzia; D., Conte; Cassanelli, Stefano; M., Fraquelli; C., Sardini; F., Vasta; P., Gasparini
abstract

Background and Methods Hereditary hemochromatosis in adults is usually characterized by mutations in the hemochromatosis (HFE) gene on the short arm of chromosome 6. Most patients have a substitution of tyrosine for cysteine at position 282 (C282Y). We studied a large family from Italy that includes persons who have a hereditary iron-overload condition indistinguishable from hemochromatosis but without apparent pathogenic mutations in the HFE gene. We performed biochemical, histologic, and genetic studies of 53 living members of the family, including microsatellite analysis of chromosome 6 and direct sequencing of the HFE gene, Results Of the 53 family members, 15 had abnormal serum ferritin levels, values for transferrin saturation that were higher than 50 percent, or both. Thirteen of the 15 had elevated body iron levels, diagnosed on the basis of the clinical evaluation and liver biopsy, and underwent iron-removal therapy. The other two, both children, did not undergo liver biopsy or iron-removal therapy. None of the 15 members had the C282Y mutation of the HFE gene; 5 of the 15 (as well as 5 healthy relatives) had another mutation of this gene, a substitution of aspartate for histidine at position 63, but none were homozygous for it. No other mutations were found after sequencing of the entire HFE gene for all family members. Microsatellite analysis showed no linkage of the hemochromatosis phenotype with the short arm of chromosome 6, the site of the HFE gene. Conclusions Hereditary hemochromatosis can occur in adults who do not have pathogenic mutations in the hemochromatosis gene. (N Engl J Med 1999;341:725-32,) (C)1999, Massachusetts Medical Society.

1998 - Diacerhein blocks iron regulatory protein activation in inflamed human monocytes [Articolo su rivista]
Pietrangelo, Antonello; Montosi, Giuliana; Recalcati, S; Garuti, Cinzia; Cairo, G.
abstract

Iron Regulatory Proteins (IRPs), by modulating expression of ferritin, which stores excess iron in a non toxic form, and transferrin receptor, which controls iron uptake, are the main controller of cellular iron metabolism. During inflammation, modification of IRP activity may affect iron availability, free radical generation and cytokine gene response in inflammatory cells. In the present study we tested the effect of inflammatory stimuli on IRP function in a human monocytic-macrophagic cell line and the possibility of interfering with these pathways by using an antiinflammatory compound, diacerhein (DAR). IRP activity was enhanced by interferon gamma/lipopolysaccarhide (IFN/LPS), and this effect was consistently counteracted by increasing concentrations of DAR. No direct effect of DAR on IRP activity was found in vitro. However, in vivo, similar IRP activation was achieved by exposing cells to nitric oxide (NO) donors and the LPS/IFN-induced activation of IRP was reversed by NO inhibitors. Interestingly, NO-induced IRP activation was efficiently blocked by DAR. These data show for the first time that a clinically useful antiinflammatory compound, DAR, interferes with IRP activation by NO in inflammed human cells.

1998 - Hepatic stellate cells are not subjected to oxidant stress during iron-induced fibrogenesis in rodents [Articolo su rivista]
Montosi, Giuliana; Garuti, Cinzia; S., Martinelli; Pietrangelo, Antonello
abstract

Oxidant stress plays a key role in hepatic fibrogenesis. This study was undertaken to assess whether, during iron overload-associated liver fibrosis in vivo, oxidant stress occurs in hepatic stellate cells (HSC) during active fibrogenesis. Gerbils were treated with iron-dextran, and, after hepatic fibrosis developed, livers were subjected to various combination of in situ hybridization and immunocytochemistry analyses. In iron-treated animals, no specific accumulation of ferritin protein was found in collagen mRNA-expressing cells. Moreover, the activity of the iron regulatory protein, the main sensor of cellular iron status, was unchanged in HSC from iron-treated animals. Although a significant amount of malondialdehyde-protein adducts was detected in gerbil liver during fibrogenesis, accumulation of these lipid peroxidation by-products was restricted to iron-laden cells adjacent to activated HSC. In cultured gerbil HSC, iron, aldehydes, and other pro-oxidants were able to enhance the expression of an oxidant stress-responsive gene, heme oxygenase (HO), with no change in collagen mRNA accumulation. In keeping with these findings, we found that, in vivo, activation of HO gene was present in iron-filled nonparenchymal cell aggregates, but absent in HSC. In conclusion, the data indicate that during iron overload-associated fibrogenesis, HSC are not directly subjected to oxidant stress, but are likely to be activated by paracrine signals arising in neighboring cells.

1998 - No HFE, no HLA, no 6p-linked adult hemochromatosis: A new genetic iron overload condition [Abstract in Atti di Convegno]
Pietrangelo, Antonello; Montosi, Giuliana; Garuti, Cinzia; Conte, D; Fraquelli, M; Gasparini, P.
abstract

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1998 - Oxidant stress: cell signalling and gene response in the liver: the iron-oxygen connection. [Relazione in Atti di Convegno]
Pietrangelo, Antonello; Montosi, Giuliana; Garuti, Cinzia; S., Martinelli
abstract

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1998 - Spatial and temporal dynamics of hepatic stellate cell activation during oxidant-stress-induced fibrogenesis [Articolo su rivista]
Montosi, Giuliana; Garuti, Cinzia; Iannone, Anna; Pietrangelo, Antonello
abstract

In vitro and in vivo studies indicate that oxidant stress is implicated in Liver fibrogenesis, However, it is still unknown whether, in vivo, oxidant stress directly affects the hepatic cells responsible for fibrogenesis, ie, the hepatic stellate cells (HSCs), This study was aimed at answering this question by assessing the temporal and spatial relationships between oxidant stress and activation of HSCs in an in vivo model of oxidant-stress-associated fibrogenesis, To this purpose, rats were treated with carbon tetrachloride (CCl4) and livers subjected to in situ perfusion with nitroblue tetrazolium, which, in the presence of superoxide ions, is reduced to an insoluble blue-colored formazan derivative and is readily detectable in the tissue by Light microscopy, Moreover, various combinations of in situ hybridization and immunocytochemical analyses were performed. An acute dose of CCl4 caused a transient production of superoxide radicals at 24 hours into pericentral necrotic areas, whereas HSC appearance and expression of collagen mRNA were detectable only at 48 and 72 hours. After chronic CCl4 intoxication, higher levels of oxygen radical production in necrotic areas were detectable along with dramatic and sustained activation of HSCs, However, maximal HSC activation was still delayed as compared with superoxide production. Expression of heme oxygenase, a gene responsive to a variety of oxidant stress mediators, was strongly enhanced by chronic CCl4 administration but remained unchanged in HSCs, both in situ and after isolation of pure HSC fractions from control and CCl4-treated animals. In conclusion, during postnecrotic fibrogenesis, oxidant stress anticipates HSC activation. HSCs do not directly face an oxidant stress while engaged in active fibrogenesis.

1998 - Ursodeoxycholic acid complexation with 2-hydroxypropyl-beta-cyclodextrin increases ursodeoxycholic acid biliary excretion after single oral administration in rats [Abstract in Rivista]
Ventura, Paolo; Panini, Rossana; Montosi, Giuliana; Garuti, Cinzia; Vandelli, Maria Angela; G., Brunetti; Pietrangelo, Antonello; Salvioli, Gianfranco
abstract

Complexation of ursodeoxycholic acid (UDCA) with 2-hydroxypropyl-beta-cyclodextrin (HPbCD) (1:1 molar ratio) improves the water solubility and dissolution rate of UDCA and hence its bioavailability. We compared UDCA bile recovery (percentage of administered UDCA dose excreted in bile) after single oral (gavage) administration of UDCA in three different pharmaceutical forms in a bile fistula rat model. Male Sprague-Dawley rats were randomly assigned to 11 groups (6 rats per groups); the bile duct was cannulated after gavage to collect 4-h bile samples. Groups 1, 2 and 3 (fed 5, 10 and 50 mg/kg body weight UDCA/HPbCD complex, respectively) showed (mean values ± SD) 39.2 ± 5.9%, 25.7 ± 4.1% and 9.4 ± 3.1% UDCA bile recovery, respectively; in groups 4, 5 and 6 (fed UDCA suspension formulation containing 5, 10 and 50 mg/kg body weight, respectively) UDCA bile recovery was 33.2 ± 3.6%, 16.9 ± 4.7% and 6.3 ± 0.8%, respectively; groups 7, 8 and 9 (fed UDCA capsule formulation containing 5, 10 and 50 mg/kg body weight, respectively) showed 30.6 ± 9.0%, 21.7 ± 6.4% and 4.7 ± 1.8% UDCA recovery. Groups 10 and 11 (controls) were fed with HPbCD and saline, respectively. Results indicate a significantly (p<0.05) higher bile UDCA recovery in rats fed UDCA-HPbCD complex than in those fed UDCA suspension or UDCA capsule formulations at the same dose. In this model, oral UDCA/HPbCD complex increased UDCA biliary excretion making for greater UDCA enrichment of the bile acid pool than identical doses of common pharmaceutical formulations.

1996 - Defective control or iron metabolism in pre-neoplastic liver nodules during experimental carcinogenesis [Abstract in Atti di Convegno]
Pietrangelo, Antonello; Montosi, Giuliana; Garuti, Cinzia; Stal, P; Eriksson, L.
abstract

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1995 - MOLECULAR AND CELLULAR ASPECTS OF LIVER-DISEASE IN GENETIC HEMOCHROMATOSIS [Abstract in Atti di Convegno]
Pietrangelo, Antonello; Montosi, Giuliana; Garuti, Cinzia; Stal, P; Hultcrantz, R.
abstract

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