Isabella Faraoni
email: faraoni@med.uniroma2.it

Francesca Romana Antonetti

John Cardone

Enzo Bonmassar

miR-155 gene: a typical multifunctional microRNA

Keywords: miRNAs, miR-155, hematopoiesis, inflammation, immunity, cancer, lymphoma, leukemia miR, microRNA gene, miRNA, microRNA, pri-miRNA, primary transcripts microRNA, DGCR8, Digeorge syndrome critical region gene 8, TRBP, TAR RNA binding protein, PACT, PKRactivating protein, RISC, RNA induced silencing complex, AGO, Argonaute, siRNAs, small interfering RNAs, CLL, Chronic Lymphocytic Leukemia, PD, Parkinson's disease, AD, Alzheimer's disease, FGF20, fibroblast growth factor 20, BACE 1, β-amyloid precursor protein-cleaving enzyme 1, Integration Cluster, HSC, hematopoietic stem cell, LPS, lipopolysaccharide, poly IC, polyriboinosinic-polyribocytidylic Lymphoma, DLBCL, Diffuse Large B cell Lymphoma AML, Acute Myeloid Leukemia, PDAC, Pancreatic Ductal Adenocarcinoma, Ang II, angiotensin

In the last years small RNA molecules, i.e. microRNA (miRNA) encoded by miR genes, have been found to play a crucial role in regulating gene expression of a considerable part of plant's and animal's genome. Here, we report the essential information on biogenesis of miRNAs and recent evidence on their important role in human diseases. Emphasis has been given to miR-155, since this molecule represents a typical multifunctional miRNA. Recent data indicate that miR-155 has distinct expression profiles and plays a crucial role in various physiological and pathological processes such as haematopoietic lineage differentiation, immunity, inflammation, cancer, and cardiovascular diseases. Moreover, miR-155 has been found to be implicated in viral infections, particularly in those caused by DNA viruses. The available experimental evidence indicating that miR-155 is overexpressed in a variety of malignant tumors allows us to include this miRNA in the list of genes of paramount importance in cancer diagnosis and prognosis. Exogenous molecular control in vivo of miR-155 expression could open up new ways to restrain malignant growth and viral infections, or to attenuate the progression of cardiovascular diseases.

Introduction

Micro-RNAs (miRNAs) are a class of evolutionarily conserved, single-stranded non-coding RNA molecules of 19-24 nucleotides that control gene expression at a post-transcriptional level. They derive from the metabolic processing of long RNA transcripts encoded by miR genes. They have been detected in plants, animal species and viruses, and are involved in numerous cellular processes including proliferation, differentiation, apoptosis and metabolism [START_REF] Bartel | MicroRNAs: genomics, biogenesis, mechanism, and function[END_REF]. In most cases, miRNAs are able to keep under tight control cell functions by means of their capacity of gene or gene cluster silencing.

To date they have been predicted to target and control the expression of at least 30% of the entire mammalian genome [START_REF] Filipowicz | Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight?[END_REF]. Of primary medical importance are the findings that deregulated miR gene expression is involved in different human diseases including cancer [START_REF] Singh | MicroRNAs--micro in size but macro in function[END_REF].

Biogenesis of miRNA

In the last few years, it has been found that miR genes may be present either as single units scattered in the genome or may be organized in gene clusters. A typical example of these gene clusters is provided by the miR-17-92 cluster, which comprises six miRs (miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1, miR-92a-1). Clustered miRs are likely transcribed together in polycistronic transcripts and usually control mRNAs with related functions. Therefore, genomic regions around known miR gene locations are particularly promising for finding new miRNA-coding DNA sequences [START_REF] Brameier | Ab initio identification of human microRNAs based on structure motifs[END_REF]. Some miRs can be grouped as "families". They have been identified studying sequence homology that is mainly present at the 5' end of mature miRNA [START_REF] Lagos-Quintana | New microRNAs from mouse and human[END_REF].

An interesting aspect of miR genes is related to their genome position. They are mainly located within introns and intergenic regions, largely composed by non-coding DNA sequences previously considered as "junk DNA". Biogenesis of miRNA is a complex multi-step process starting in the nucleus and ending in the cytoplasm throughout many post-transcriptional modifications. In the first step of this process the RNA polymerase II transcribes miR genes generating primary transcripts (pri-miRNA) with a 5'-cap and a 3' poly-A tail. Pri-miRNAs are large transcripts that can contain multiple miRNA sequences which at this stage are already folded into hairpin structures (Fig. 1). The nuclear microprocessor that contains the RNase III enzyme Drosha and a double-stranded-RNA-binding protein DGCR8 (Digeorge syndrome critical region gene 8) processes the pri-miRNA into ~70 nucleotide miRNA

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precursor known as pre-miRNA. The pre-miRNA stem-loop structures are then transported to the cytoplasm through the exportin 5 complex [START_REF] Denli | Processing of primary microRNAs by the Microprocessor complex[END_REF]. The cytoplasmic endonuclease Dicer, complexed with its co-factors TAR RNA binding protein (TRBP) and PKR-activating protein (PACT), starts to process the pre-miRNA [START_REF] Kok | Human TRBP and PACT directly interact with each other and associate with dicer to facilitate the production of small interfering RNA[END_REF][START_REF] Chendrimada | TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing[END_REF]. Dicer cleaves the pre-miRNA stem-loop forming a 21-24 bp duplex miRNA, which contains 2 nucleotides that overhang at the 3' of each strand. One strand is then selected on the basis of the stability of the 5' end (guide strand) whereas the other strand (passenger strand) is usually degraded. At this stage the miRNA is loaded into the RNA induced silencing complex (RISC)

containing Argonaute (AGO) proteins [START_REF] Diederichs | Dual role for argonautes in microRNA processing and posttranscriptional regulation of microRNA expression[END_REF][START_REF] Rana | Illuminating the silence: understanding the structure and function of small RNAs[END_REF]. After integration into the active RISC complex, miRNAs base pair with their complementary mRNA molecules. Degradation of the target mRNA occurs only when the miRNA and the target mRNA are exactly (perfect match) or nearly exactly complementary to each other. This process is the same of the RNA interference induced by artificial small interfering RNAs (siRNAs). On the other hand, if the complementarity between miRNA and target mRNA is only partial (imperfect match), translation repression will take place. In animals, miRNA molecules can bind several target sequences, mainly present at the 3' untranslated region (3'UTR) of mRNA with various degrees of complementarity [START_REF] Kertesz | The role of site accessibility in microRNA target recognition[END_REF]. It follows that each single miRNA is able to interact and regulate a large number of genes. It has been estimated that a single miRNA molecule is able to bind about 200 different transcripts [START_REF] Krek | Combinatorial microRNA target predictions[END_REF]. In turn, biogenesis and functions of miRNA are finely governed by transcription factors and dynamic cellular signals [START_REF] Smalheiser | Regulation of mammalian microRNA processing and function by cellular signaling and subcellular localization[END_REF].

Role of miRNA in diseases

In humans, more than 800 miR genes have been identified even if the total number is estimated to be not less than one thousand. Although research on miRNAs is only at the beginning, it is already clear that they are involved in a broad-spectrum of diseases, including cancer. The role of miRNAs in malignant transformation and tumour progression emerged with the finding that miR-15a and miR-16 are located on chromosome 13q14.3 within a 30-kb region that is frequently deleted in Chronic Lymphocytic Leukemia (CLL) determining down-regulation of both miRNAs in the majority of the analyzed CLL cell samples [START_REF] Calin | Frequent deletions and down-regulation of micro-RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia[END_REF]. Interestingly, more than 50% of miR genes were found to be located at fragile sites or at chromosomal regions showing amplification, deletion or translocation in cancer [START_REF] Calin | MicroRNA profiling reveals distinct signatures in B cell chronic lymphocytic leukemias[END_REF]. Deregulated miRNA expression has now been associated with several tumour types including colon, breast, and lung cancer [START_REF] Lu | MicroRNA expression profiles classify human cancers[END_REF][START_REF] Volinia | A microRNA expression signature of human solid tumors defines cancer gene targets[END_REF]. Moreover, altered miRNA expression has been found in

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haematological malignancies, including several types of lymphoma and leukemia [START_REF] Calin | MicroRNA profiling reveals distinct signatures in B cell chronic lymphocytic leukemias[END_REF][START_REF] Lawrie | MicroRNA expression distinguishes between germinal center B cell-like and activated B cell-like subtypes of diffuse large B cell lymphoma[END_REF][START_REF] Xu | MicroRNA gene expression in malignant lymphoproliferative disorders[END_REF] suggesting that miR genes play a key role as oncogenic or tumour suppressor genes.

A certain number of miRNAs has also been found to be implicated in neurological diseases, such as Parkinson's disease (PD) and Alzheimer's disease (AD). In PD, fibroblast growth factor 20 (FGF20)

was previously identified as a risk factor in several association studies. A risk allele stems from a single nucleotide polymorphism in the 3'UTR of FGF20, responsible of disruption of a binding site for microRNA-433. This is followed by increased translation of FGF20 leading to α-synuclein over expression, which has previously been shown to cause PD.

Little is known about the role of miRNAs in AD. A number of experimental and clinical data suggest that β-amyloid is central to the pathophysiology of AD and that the β-amyloid precursor proteincleaving enzyme 1 (BACE1) is essential for the generation of the neurotoxic β-amyloid peptide. It has been shown that miR-107 levels decrease significantly in the majority of patients including those at the early stage of the disease. In a parallel way, the BACE1 mRNA levels tend to increase as miR-107 levels decrease in the progression of AD. The 3'UTR of BACE1 mRNA can be targeted in multiple sites by miR-107 suggesting that miR-107 is one of the multifactorial causes involved in AD progression [START_REF] Wang | The expression of microRNA miR-107 decreases early in Alzheimer's disease and may accelerate disease progression through regulation of beta-site amyloid precursor protein-cleaving enzyme 1[END_REF].

Expression and function of miRNA-155

The present minireview focuses on the miR-155 (Fig. 2) since this molecule represents a typical multifunctional miRNA. To date, increased evidence points out that miR-155 is involved in numerous biological processes including haematopoiesis, inflammation and immunity. It is also implicated in the regulation of type 1 Angiotensin II receptor (AT1R) an important molecule that takes part in renal function and contraction of vascular smooth muscle.

Deregulation of miR-155 has been found to be associated with different kinds of cancer, cardiovascular diseases and viral infections. Since investigation on the functional activity of miR-155 started few years ago, it is reasonable to predict that many other functions will be uncovered in the near future.

MiR-155 maps within and is processed from an exon of a non-coding RNA transcribed from the B-cell Integration Cluster (BIC) located on chromosome 21 [START_REF] Lagos-Quintana | Identification of tissue-specific microRNAs from mouse[END_REF]. BIC shows strong sequence homology

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among human, mouse and chicken and is highly, although not exclusively, expressed in lymphoid organs implying an evolutionary conserved function.

Hematopoiesis

More than one miRNA is involved in regulation and differentiation of cells of haematopoietic origin.

MiR-155 is expressed in both lymphoid and myeloid cells but at different levels depending on the cell type. Masaki S. and colleagues have shown that during erythroid differentiation in vitro, purified human progenitors cells show a progressive decrease of miR-155 expression, that was found to be 200 fold lower in mature red cells. On the contrary, in the same cells during the last 12 days of differentiation miR-451 is up-regulated to 270-fold, thus identifying this two miRNAs as key molecules in erythroid maturation [START_REF] Masaki | Expression patterns of microRNAs 155 and 451 during normal human erythropoiesis[END_REF].

Georgantas RW III et al. found that a substantial amount of genes specifying hematopoietic cell differentiation are expressed by CD34+ hematopoietic stem cells (HSC) but are held in check by a number of miRNAs until differentiation occurs. More exhaustive information was obtained for miR-155. Chemically-induced erythroid and megakaryocytic differentiation of K562 cell line was found to be significantly reduced by target cell transduction with miR-155. The inhibitory function of this miR gene was confirmed in in vitro cultured normal HSCs in which transduction with miR-155 generated 5fold less myeloid and 3-fold less erythroid colonies. Furthermore, in this model, colonies were much smaller than controls [START_REF] Georgantas | CD34+ hematopoietic stem-progenitor cell microRNA expression and function: a circuit diagram of differentiation control[END_REF]. These results strongly suggest that miR-155 is an essential molecule in the control of human protein translation in both myelopoiesis and erythropoiesis.

Inflammation

The inflammatory response to infections involves the induction of several hundred genes, a process that must be finely regulated to achieve pathogen clearance and to prevent the pathological consequences of a deregulated gene expression.

The possibility that environmental factors, such as inflammatory ligands, may alter miR gene expression is just beginning to be explored. Recently, miR-155 has been identified and characterized as a component of the primary macrophage response to different types of inflammatory mediators.

For instance, it has been shown that miR-155 is induced by bacterial lipopolysaccharide (LPS) in a human monocytic cell line [START_REF] Taganov | NF-kappaB-dependent induction of microRNA miR-146, an inhibitor targeted to signaling proteins of innate immune responses[END_REF]. Inflammatory mediators including IFN-β, polyriboinosinic-

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polyribocytidylic acid (poly IC) or Tumour Necrosis Factor-α (TNF-α) can induce miR-155 in macrophages and monocytes. Furthermore, pharmacological inhibition of the c-Jun N-terminal kinase (JNK) blocks induction of miR-155, suggesting that miR-155-inducing signals use the JNK pathway [START_REF] O'connell | MicroRNA-155 is induced during the macrophage inflammatory response[END_REF]. Moreover, it has been hypothesized that the regulation of miR-155 levels could be controlled, at least in part, by the mitogen-activated protein kinase (MAPK) pathway [START_REF] O'connell | MicroRNA-155 is induced during the macrophage inflammatory response[END_REF].

Rheumatoid Arthritis (RA) is a chronic, systemic autoimmune disorder characterized by activation of the immune system at joint level, where it leads to inflammation and irreversible damage. Stanczyk et al. found expression of miR-155 and miR-146a in synovial fibroblasts and tissue of RA patients higher than that detectable in patients with osteoarthritis. They observed a further up-regulation of miR-155 after stimulation in vitro with TNF-α, IL-1β and toll-like receptor ligand (LPS, poly IC). Moreover, in RA synovial fibroblasts these authors found that over expression of miR-155 induced by LPS, IL-1β and TNF-α, down-regulates production of matrix metalloproteinase (MMPs) 1 and 3, that are important markers of the destructive and inflammatory effects of RA. Interestingly, the expression of miR-155 in CD14+ monocytes/macrophages present in the synovial fluid of RA patients was found 4.4 fold higher than that detectable in peripheral blood CD14+ cells. In summary, miR-155 might function as a protective miRNA that locally down-regulates the expression of certain MMPs, thus controlling tissue damage due to inflammation [START_REF] Stanczyk | Altered expression of MicroRNA in synovial fibroblasts and synovial tissue in rheumatoid arthritis[END_REF].

Immunity

BIC/miR-155 expression is greatly increased in activated B and T cells, as well as in activated macrophages and dendritic cells. On the other hand, miR-155 was found to be required for

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response and cytokine production. The authors stressed the finding that immune responses in miR-155-deficient mice is deranged toward a Th2 pattern, with substantial increase of IL-10, a cytokine endowed with immunodepressive activity against cell-mediated responses.

The intrinsic requirement for miR-155 was studied in detail in antigen B cell responses. In miR-155deficient mice, B cells generate reduced extrafollicular and germinal center responses and fail to produce high-affinity IgG1 antibodies [START_REF] Vigorito | microRNA-155 regulates the generation of immunoglobulin classswitched plasma cells[END_REF]. Microarray analysis of wild-type and miR-155-deficient B cells activated under conditions that promote class switching to IgG1, reveals that miR-155 regulates the expression of a substantial number of target genes, many of which can be predicted by in silico analysis. One of the genes highly expressed in miR-155-deficient B cells encodes for the transcription factor PU.1 which has a highly conserved miR-155 binding site in its 3′UTR. PU.1 is known to be important in many aspects of hematopoiesis, in particular for the early activation of B cells and regulation of B cell function. Moreover, PU.1 over expression in wild-type B cells results in reduced numbers of IgG1-switched cells. These results strongly suggest that miR-155 is required for inducing down-regulation of PU.1 in antigen driven maturation and IgG1 class-switched differentiation in B cells [START_REF] Vigorito | microRNA-155 regulates the generation of immunoglobulin classswitched plasma cells[END_REF].

Recently, two independent groups identified the enzyme activation-induced cytidine deaminase (AID)

as another important target of miR-155 in vivo. AID deaminates cytosine residues and introduces U:G mismatches in DNA. Therefore the enzyme promotes high frequency mutations that are physiologically involved in somatic hypermutation required for high-affinity IgG antibody repertoire in antigen-activated B cells. Solid experimental evidence indicates that miRNA-155 is a negative regulator of AID [START_REF] Teng | MicroRNA-155 is a negative regulator of activation-induced cytidine deaminase[END_REF][START_REF] Dorsett | MicroRNA-155 suppresses activation-induced cytidine deaminase-mediated Myc-Igh translocation[END_REF]. Nucleotide substitutions in the miR-155 binding site located in the 3'UTR of AID gene prolongs the half-life of its transcript and increases the amount of AID protein. Therefore, one would expect that B cells of miR-155 deficient mice would be excellent producers of high affinity IgG antibodies. However, as previously mentioned, in vivo mouse studies provided just opposite results [START_REF] Vigorito | microRNA-155 regulates the generation of immunoglobulin classswitched plasma cells[END_REF], pointing out that the high-affinity antibody response is finely regulated by different gene products presumably influenced by miR-155 in different and still not entirely understood ways.

Tili et al. investigated the relationship between miR-155 and innate immunity focusing their attention

on a microbial-associated activating molecule such as LPS and the pro-inflammatory cytokine TNF-α.

In LPS-stimulated macrophages TNF-α is one of the main cytokines involved in the innate immunity response. Studies on mouse macrophage/monocyte leukaemia line (i.e. Raw 264.7 cells) showed

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that enhancement of TNF-α by LPS is accompanied by down-regulation of miR-125b and upregulation of miR-155. A similar expression pattern has been also found in C57BL/6 mice subjected to i.p. inoculation with LPS [START_REF] Tili | Modulation of miR-155 and miR-125b levels following lipopolysaccharide/TNF-alpha stimulation and their possible roles in regulating the response to endotoxin shock[END_REF]. A number of studies suggest that miR-125b directly targets the 3'UTR of TNF-α transcripts. It follows that down-regulation of miR-125b induced by LPS removes, at least in part, the inhibitory effect of this miRNA on TNF-α translation. On the other hand, up-regulation of miR-155 provoked by exposure to LPS, enhances TNF-α production. In this case, however, the underlying mechanism has not been fully clarified. It has been hypothesized that miR-155 could increase directly TNF-α levels by augmenting transcript stability through binding to its 3'UTR.

Alternatively, miR-155 could target gene transcripts coding for proteins that are known to be repressor of TNF-α translation.

The correlation between miR-155 and responses induced by LPS are consistent with the finding that miR-155 targets transcripts coding for proteins involved in LPS/TNFα signaling including the Fasassociated death domain protein (FADD), the IkB kinase epsilon (IKKε) and the receptor (TNFR superfamily)-interacting serine-threonine kinase1 (RIPK1). Moreover, in vivo studies showed that transgenic mice over expressing miR-155 in B cell lineage (Eµ-miR-155) produce more TNF-α when challenged with LPS and are hypersensitive to LPS/D-galactosamine induced septic shock [START_REF] Tili | Modulation of miR-155 and miR-125b levels following lipopolysaccharide/TNF-alpha stimulation and their possible roles in regulating the response to endotoxin shock[END_REF] with respect to their normal counterparts. All together, these data suggest that the interplay between miR-155 and LPS/TNF-α may be implicated in the endotoxin shock.

miR-155 and neoplastic diseases

There are several lines of evidence that miR-155 is over expressed in a number of neoplastic diseases and that it plays a significant role in the process of carcinogenesis, acting predominantly as an oncomir [START_REF] Esquela-Kerscher | Oncomirs -microRNAs with a role in cancer[END_REF]. Several mechanisms have been suggested to explain this biological activity. For example, miR-155 was found to be one of the most potent miRNA suppressing apoptosis in human Tcell leukemia Jurkat cells and in MDA-MB-453 breast cancer cells. The mechanism underlying the effect of miR-155 could be ascribed to a blockade of caspase-3 activity [START_REF] Ovcharenko | Genome-scale microRNA and small interfering RNA screens identify small RNA modulators of TRAIL-induced apoptosis pathway[END_REF]. In fact, over expression of miR-155 is followed by a substantial decrease of tumor protein 53-induced nuclear protein 1 (TP53INP1), that is a nuclear protein able to induce cell cycle arrest and apoptosis through caspase-

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3 activation [START_REF] Gironella | Tumor protein 53-induced nuclear protein 1 expression is repressed by miR-155, and its restoration inhibits pancreatic tumor development[END_REF]. This role in apoptosis could be responsible for the oncogenicity of miR-155 in several types of cancer.

miR-155 and B-Cell Lymphoma

The BIC gene was originally described as a common site of viral DNA integration in virus-induced lymphomas in chicken. In humans, BIC RNA levels are low in normal lymphoid tissues but increase in various B-cell malignancies, including Hodgkin's Lymphoma and some subtypes of Non Hodgkin's Lymphoma [START_REF] Kluiver | BIC and miR-155 are highly expressed in Hodgkin, primary mediastinal and diffuse large B cell lymphomas[END_REF][START_REF] Eis | Accumulation of miR-155 and BIC RNA in human B cell lymphomas[END_REF].

Hodgkin's Lymphoma is clinically characterized by the orderly spread of disease from one lymph node group to another and by the development of systemic symptoms in an advanced stage of the disease. High expression of BIC/miR-155 was observed in Reed-Sternberg cells of Hodgkin's Lymphoma [START_REF] Van Den Berg | High expression of B-cell receptor inducible gene BIC in all subtypes of Hodgkin lymphoma[END_REF] and in the lymphocytic and histiocytic cells of the variant nodular lymphocytepredominant Hodgkin's Lymphoma [START_REF] Kluiver | BIC and miR-155 are highly expressed in Hodgkin, primary mediastinal and diffuse large B cell lymphomas[END_REF].

The majority of Non Hodgkin's Lymphomas does not express BIC/miR-155. However, BIC was observed in Primary Mediastinal B-cell Lymphoma (PMBL), in Diffuse Large B cell Lymphoma (DLBCL) [START_REF] Kluiver | BIC and miR-155 are highly expressed in Hodgkin, primary mediastinal and diffuse large B cell lymphomas[END_REF] and in children's Burkitt's Lymphoma [START_REF] Metzler | High expression of precursor microRNA-155/BIC RNA in children with Burkitt lymphoma[END_REF].

PMBL is a rare form of Non Hodgkin's Lymphoma that origins from a rare type of B-cell lymphocytes in the thymus gland. Presence of BIC/miR-155 was detected in frozen tissues and PMBL cell lines [START_REF] Kluiver | BIC and miR-155 are highly expressed in Hodgkin, primary mediastinal and diffuse large B cell lymphomas[END_REF].

DLBCL is the most common form of adult Non Hodgkin's lymphoma accounting for nearly 40% of all lymphoid tumors. DLBCL is characterized by marked clinical and pathological heterogeneity that is reflected at the molecular level. Gene expression and immuno-histochemical studies have revealed the presence of two distinct molecular subtypes of DLBCL related to the type of cell they arise from, i.e. germinal center B cell-like (GCB) and activated B cell-like (ABC). Prognosis is worse in the ABC than in GCB subtype. BIC/miR-155 is expressed at a higher level in ABC-type than in GCB-type cell lines, suggesting that the quantification of this miRNA may have a role in establishing the prognosis [START_REF] Lawrie | MicroRNA expression distinguishes between germinal center B cell-like and activated B cell-like subtypes of diffuse large B cell lymphoma[END_REF]. Recently 42 genes down-regulated in ABC subtypes were correlated with the high expression of miR-155. Although only nine of them are predicted targets of this miRNA, an indirect effect of miR-155 on the other genes cannot be excluded. Some of these genes regulated by miR-155 are implicated in immune system homeostasis and at least three of them have oncogenic effects [START_REF] Rai | Coordinated expression of microRNA-155 and predicted target genes in diffuse large B-cell lymphoma[END_REF].

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Burkitt's Lymphoma is a very fast growing form of Non Hodgkin's Lymphoma. In tumor cells of 11 children with Burkitt's Lymphoma, Metzler M. et al. [START_REF] Metzler | High expression of precursor microRNA-155/BIC RNA in children with Burkitt lymphoma[END_REF] found high expression of miR-155, that was more pronounced than that detectable in Acute Lymphoblastic Leukemia (ALL) of children or in mononuclear cells of healthy donors. However, these results were not confirmed by other authors [START_REF] Kluiver | Lack of BIC and microRNA miR-155 expression in primary cases of Burkitt lymphoma[END_REF]. To solve this controversy, different Burkitt's Lymphoma cell lines were induced to express BIC by BCR stimulation. It was shown that BIC up-regulation is not accompanied by increase of miR-155 expression suggesting two levels of regulation of mature miR-155 expression, either at transcriptional and at processing levels [START_REF] Kluiver | Regulation of pri-microRNA BIC transcription and processing in Burkitt lymphoma[END_REF]. However, a recent study failed to show the presence of an intrinsic blockade of BIC/miR-155 processing in cultured or primary Burkitt's Lymphoma cells [START_REF] Zhang | BIC is processed efficiently to microRNA-155 in Burkitt lymphoma cells[END_REF].

As previously mentioned, miR-155 targets efficiently the 3'UTR sequence of AID gene [START_REF] Teng | MicroRNA-155 is a negative regulator of activation-induced cytidine deaminase[END_REF][START_REF] Dorsett | MicroRNA-155 suppresses activation-induced cytidine deaminase-mediated Myc-Igh translocation[END_REF]. This biochemical event reveals a potentially different biological role of miR-155. Although AID activity is predominantly restricted to IgG genes, off-target lesions of DNA could be responsible of doublestrand breaks favouring oncogenic translocation, including Myc translocation [START_REF] Dorsett | MicroRNA-155 suppresses activation-induced cytidine deaminase-mediated Myc-Igh translocation[END_REF]. Since miR-155 is a negative modulator of AID levels, it is reasonable to hypothesize that this miRNA could reduce the chances of malignant transformation in selected tissues that can be targeted by AID-mediated mutagenesis [START_REF] Dorsett | MicroRNA-155 suppresses activation-induced cytidine deaminase-mediated Myc-Igh translocation[END_REF]. On the other hand, a direct evidence that miR-155 is involved in B cell malignancy has been obtained experimentally in vivo in transgenic mice carrying miR-155 that is specifically over expressed in B cell lineage [START_REF] Costinean | Pre-B cell proliferation and lymphoblastic leukemia/high-grade lymphoma in E(mu)-miR155 transgenic mice[END_REF]. In this experimental model miR-155 expression is under the control of a VH promoter and is active in the late stage of B cell development. Transgenic mice exhibit initially a preleukemic pre-B cell proliferation followed by the development of a high-grade B cell lymphoma approximately at the age of 6 months [START_REF] Costinean | Pre-B cell proliferation and lymphoblastic leukemia/high-grade lymphoma in E(mu)-miR155 transgenic mice[END_REF].

miRNA-155 and Leukemia

Quite recently, O'Connell and others, found increased expression of miR-155 in bone marrow blasts of leukaemic patients bearing M4 or M5 subtypes of Acute Myeloid Leukaemia (AML) [START_REF] O'connell | Sustained expression of microRNA-155 in hematopoietic stem cells causes a myeloproliferative disorder[END_REF]. Two experimental models were used in mouse to investigate the possible relationship between miR-155 and AML. Expanded proliferation of granulocytes/monocytes in the bone marrow was found in mice injected with HSCs transduced with miR-155. Moreover, transduction of miR-155 in cells of the mouse macrophage RAW264.7 line, induces deregulation of a large number of genes, including 89 genes that were predicted to be targets of miR-155 on the basis of in silico analysis. Ten of these

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target genes (e.g. PU.1 and C/EBPβ) turned out to be involved in haematopoietic and myeloproliferative disorders [START_REF] O'connell | Sustained expression of microRNA-155 in hematopoietic stem cells causes a myeloproliferative disorder[END_REF]. Modulation of PU.1 may have an oncogenic effect since it could play a role in differentiation or activation of macrophages and B-cells and in the development of myeloid leukaemia [START_REF] Metcalf | Inactivation of PU.1 in adult mice leads to the development of myeloid leukaemia[END_REF]. In addition, C/EBPβ is a transcription factor which plays an important role during heamatopoietic cell growth and differentiation and has been found to be a potential target of miR-155 in in vivo AML development [START_REF] O'connell | Sustained expression of microRNA-155 in hematopoietic stem cells causes a myeloproliferative disorder[END_REF].

Recent investigations showed that miR-155 is over expressed in CLL, [START_REF] Fulci | Quantitative technologies establish a novel microRNA profile of chronic lymphocytic leukaemia[END_REF][START_REF] Marton | Small RNAs analysis in CLL reveals a deregulation of miRNA expression and novel miRNA candidates of putative relevance in CLL pathogenesis[END_REF]. There are no molecular data on the role of miR-155 in CLL, although this miRNA appears to be dramatically over expressed, leading to an average up-regulation of 5.3 fold that can peak to 23 fold [START_REF] Fulci | Quantitative technologies establish a novel microRNA profile of chronic lymphocytic leukaemia[END_REF].

miR-155 and Solid tumors

miR-155 gene was found to be over expressed in several solid tumours, such as thyroid carcinoma [START_REF] Nikiforova | MicroRNA expression profiling of thyroid tumors: biological significance and diagnostic utility[END_REF], breast cancer [START_REF] Volinia | A microRNA expression signature of human solid tumors defines cancer gene targets[END_REF][START_REF] Iorio | MicroRNA gene expression deregulation in human breast cancer[END_REF][START_REF] Yan | MicroRNA miR-21 overexpression in human breast cancer is associated with advanced clinical stage, lymph node metastasis and patient poor prognosis[END_REF], colon cancer [START_REF] Volinia | A microRNA expression signature of human solid tumors defines cancer gene targets[END_REF], cervical cancer [START_REF] Wang | Aberrant expression of oncogenic and tumor-suppressive microRNAs in cervical cancer is required for cancer cell growth[END_REF], pancreatic ductal adenocarcinoma (PDAC) [START_REF] Lee | Expression profiling identifies microRNA signature in pancreatic cancer[END_REF][START_REF] Szafranska | MicroRNA expression alterations are linked to tumorigenesis and non-neoplastic processes in pancreatic ductal adenocarcinoma[END_REF], and lung cancer, where it is considered to be a marker of poor prognosis [START_REF] Volinia | A microRNA expression signature of human solid tumors defines cancer gene targets[END_REF][START_REF] Jay | MiRNA profiling for diagnosis and prognosis of human cancer[END_REF][START_REF] Yanaihara | Unique microRNA molecular profiles in lung cancer diagnosis and prognosis[END_REF]. Several studies have recently assessed the miRNA expression profiles of different types of thyroid cancer [START_REF] Nikiforova | MicroRNA expression profiling of thyroid tumors: biological significance and diagnostic utility[END_REF][START_REF] He | The role of microRNA genes in papillary thyroid carcinoma[END_REF][START_REF] Pallante | MicroRNA deregulation in human thyroid papillary carcinomas[END_REF][START_REF] Visone | Specific microRNAs are downregulated in human thyroid anaplastic carcinomas[END_REF][START_REF] Weber | A limited set of human MicroRNA is deregulated in follicular thyroid carcinoma[END_REF]. Thyroid cancer refers to any of the four kinds of malignant tumors of the thyroid gland: papillary, follicular, medullary or anaplastic. Nikiforava et al.

analyzed frozen tissues from surgically removed thyroid samples and found that miR-155 was 9.5, 5.5 and 13.2 fold over expressed in papillary, follicular and anaplastic carcinomas with respect to normal counterpart [START_REF] Nikiforova | MicroRNA expression profiling of thyroid tumors: biological significance and diagnostic utility[END_REF]. Carcinogenic mutations in thyroid cancer have been well studied. Papillary carcinomas positive for BRAF, RAS and RET/PTC mutations showed high expression levels of miR-155, revealing a significant relationship between the presence of this miRNA and those specific mutations. Moreover, the authors showed the diagnostic utility of evaluating the expression levels of some miRNAs in the preoperative assessment of thyroid nodules in samples obtained from 14 malignant thyroid tumors. A diagnostically valid assay is important to distinguish thyroid tumors from normal thyroid tissue and hyperplastic nodules. The authors analyzed the expression of miR-155 in fine-needle aspiration samples and found up regulation of this miRNA in papillary carcinoma samples compared to hyperplastic nodules [START_REF] Nikiforova | MicroRNA expression profiling of thyroid tumors: biological significance and diagnostic utility[END_REF]. Although further analyses are needed, these results suggest a diagnostically valid assay in order to distinguish thyroid tumors from normal thyroid tissue and hyperplastic nodules.

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Two independent studies detected a 10-14 fold up regulation of miR-155 in PDAC with respect to normal pancreatic tissue [START_REF] Lee | Expression profiling identifies microRNA signature in pancreatic cancer[END_REF][START_REF] Szafranska | MicroRNA expression alterations are linked to tumorigenesis and non-neoplastic processes in pancreatic ductal adenocarcinoma[END_REF]. In a recent work, a loss of TP53INP1 was demonstrated in early stages of pancreatic cancer. This is a protein known to interact with transcriptional activation of p53 in response to genotoxic stress. Induction of TP53INP1 over expression in tumour cells is followed by apoptosis and cell cycle arrest. Surprisingly, in PDAC the mRNA levels of TP53INP1 are similar to those detectable in peritumoral regions, whereas the protein is almost undetectable in cancer cells.

Therefore, these results point out that miR-155 operates an efficient post-transcriptional regulation of TP53INP1, as confirmed by transfection experiments. It follows that the loss of TP53INP1 in PDAC cells is probably due to miR-155 over expression [START_REF] Gironella | Tumor protein 53-induced nuclear protein 1 expression is repressed by miR-155, and its restoration inhibits pancreatic tumor development[END_REF].

miR-155 in Cardiovascular diseases

The renin-angiotensin system and its effector molecule Angiotensin II (Ang II) play an important role in the regulation of hemodynamic physiological responses such as arteriolar vasoconstriction, blood pressure, aldosterone production and renal function. Most of the physiological effects of Ang II are mediated by AT1R.

The expression of the AT1R is regulated predominantly by post-transcriptional mechanisms.

Functional studies have shown that transfection of miR-155 into human primary lung fibroblasts reduces the endogenous expression of the AT1R compared with non-transfected cells. Luciferase report assays showed that miR-155 could directly bind to the 3'UTR of AT1R mRNAs and translationally represses the protein [START_REF] Martin | MicroRNA-155 regulates human angiotensin II type 1 receptor expression in fibroblasts[END_REF]. Interestingly, a single nucleotide polymorphism (+1166 A/C) in the human AT1R was known to be associated with hypertension, cardiac hypertrophy and myocardial infarction but its role remained uncertain because it is located within an untranslated region. This polymorphism was then found to overlap with the miR-155 target site in the 3'UTR of AT1R gene (Fig. 2). Laboratory experiments showed that this polymorphism reduces the ability of miR-155 to interact with the cis regulatory site. Consequently, miR-155 cannot attenuate efficiently the translation of the receptor protein [START_REF] Martin | The human angiotensin II type 1 receptor +1166 A/C polymorphism attenuates microrna-155 binding[END_REF]. An additional hint suggesting that miR-155 could be involved in blood pressure regulation stems from the observation published by Sethupathy [START_REF] Sethupathy | Human microRNA-155 on chromosome 21 differentially interacts with its polymorphic target in the AGTR1 3' untranslated region: a mechanism for functional singlenucleotide polymorphisms related to phenotypes[END_REF] on homozygous twins discordant for trisomy 21. These authors found that the subject with the extra chromosome over expresses miR-155 and shows impairment of AT1R accompanied by low blood

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pressure. Altogether, these results seem to link miR-155 to arterial blood pressure pattern, suggesting that a deficit of miR-155 function could be implicated in hypertension and cardiovascular diseases.

miR-155 in Viral infection

Large experimental evidence is now available showing that miRNAs are also encoded by viral genomes. To date, known viral miRNAs derive exclusively from dsDNA viruses (herpesvirus, polyomavirus and adenovirus). Nevertheless, for other virus families, the possibility of encoding miRNAs is not excluded. Viral miRNAs have been shown to interfere with cellular mRNAs of infected cells regulating gene expression. Gottwein and colleagues showed that a miRNA encoded by Kaposi's-sarcoma-associated herpes virus (KSHV) known as miR-K12-11, functions as an orthologue of cellular miR-155 providing a replicative advantage to the KHSV through the down regulation of the expression of genes with known roles in cell growth and apoptosis [START_REF] Gottwein | A viral microRNA functions as an orthologue of cellular miR-155[END_REF][START_REF] Skalsky | Kaposi's sarcoma-associated herpesvirus encodes an ortholog of miR-155[END_REF].

BIC/miR-155 expression was analyzed in a panel of Epstein-Barr virus (EBV) positive B lymphocytes expressing type III and type I latency genes and in EBV-negative cell lines. The results showed that the type III EBV-positive cells strongly express miR-155 and that the induction of miR-155 by EBV contributes to alter gene expression by targeting a number of transcriptional regulatory genes [START_REF] Yin | MicroRNA-155 is an Epstein-Barr virus-induced gene that modulates Epstein-Barr virus-regulated gene expression pathways[END_REF].

The direct targeting of the transcription factor BACH1 by miR-155 and by its viral orthologue miR-K12-11 suggests that suppression of BACH1 as well as that of other miR-155 targets could be a conserved activity of the members of herpesvirus family.

Conclusions

The discovery of RNA interference is one of the major scientific breakthroughs in recent years and has revolutionized the way we look at gene regulation, so that the Nobel Prize CC Mello has given his lecture with the provocative title "Return to the RNA World: rethinking gene expression and evolution" [START_REF] Mello | Return to the RNAi world: rethinking gene expression and evolution[END_REF]. It was only in October of 2001 that three independent groups proposed the term microRNA for a distinct class of small RNAs implicated in a post-transcriptional regulatory mechanisms, recognizing that miRNA function could be more general and important than previously

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thought [START_REF] Lagos-Quintana | Identification of novel genes coding for small expressed RNAs[END_REF][START_REF] Lau | An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans[END_REF][START_REF] Lee | An extensive class of small RNAs in Caenorhabditis elegans[END_REF]. After few years the number of studies on miRNAs has been exponentially increased but the scale of the phenomenon suggests that we are still at the beginning.

In this review we report the most important results referred to miR-155 function. It was shown that miR-155 has a distinctive expression pattern during the different stages of HSC maturation.

Moreover, it has a key role in modulating humoral and cell-mediated immune response. Mice deficient for miR-155 are apparently normal during the first months after birth. However, later, in the course of their life, the animals develop lung and intestinal lesions compatible with a status of immunological dysfunction that was confirmed by lack of adequate immune response following intravenous sensitization with salmonella [START_REF] Rodriguez | Requirement of bic/microRNA-155 for normal immune function[END_REF][START_REF] Thai | Regulation of the germinal center response by microRNA-155[END_REF][START_REF] Vigorito | microRNA-155 regulates the generation of immunoglobulin classswitched plasma cells[END_REF]. Worth of note is the finding that miR-155 is greatly expressed in activated B, T cells, macrophages and dendritic cells. In addition the miR-155 down-regulation in erythroid differentiation suggests a role of this gene in erythropoiesis.

MiR-155 has been also found to be involved in cardiovascular diseases. Since over expression of this miRNA is accompanied by impairment of AT1R activity and low blood pressure, it has been suggested that a deficit of miR-155 function could be implicated in hypertension. Moreover, miR-155 appears to be involved in biochemical mechanisms associated with viral infections. For example, induction of miR-155 by EBV contributes to alter gene expression in infected cells, in part targeting a number of transcriptional regulatory genes.

To date the world literature has revealed that among the presently known miRNAs, miR-155 is one of the miRNAs most consistently involved in neoplastic diseases. Indeed, the frequently detected upregulation of miR-155 in malignant cells allows to consider this gene predominantly as an oncogene playing a role in the pathogenesis of many human cancers, such as malignancies of the haematopoietic system (i.e. Hodgkin's Lymphoma, some types of Non Hodgkin's Lymphoma, AML, and CLL) and solid tumors (i.e. breast, colon, cervical, pancreatic, lung and thyroid cancer). This hypothesis is reinforced experimentally by the finding that over expression of miR-155 in B cells of transgenic mice induces polyclonal preleukemic B cell proliferation followed by overt B cell lymphoma [START_REF] Costinean | Pre-B cell proliferation and lymphoblastic leukemia/high-grade lymphoma in E(mu)-miR155 transgenic mice[END_REF]. However, as reported by Dorsett et al [START_REF] Dorsett | MicroRNA-155 suppresses activation-induced cytidine deaminase-mediated Myc-Igh translocation[END_REF], the restrain operated by miR-155 on AID expression, could also suggest a possible tumor suppression function of this microRNA in a selected cellular setting. In any case, the findings that miR-155 is involved in inflammatory processes including RA [START_REF] Stanczyk | Altered expression of MicroRNA in synovial fibroblasts and synovial tissue in rheumatoid arthritis[END_REF], appear to highlight a potential link between inflammation and cancer, where the up-regulation of miR-155 could play an important role. As a consequence, rather attractive is the hypothesis that early

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resolution of chronic inflammation could be followed by significant reduction of carcinogenesis through, at least in part, a modification of miR-155 expression pattern.

Up to now, not entirely elucidated are the mechanisms underlying the oncogenic potential or the participation to pathogenesis of non-neoplastic diseases of miR-155. However, a number of genes implicated in differentiation, inflammation, apoptosis and transcriptional regulation, could be controlled in their expression by miR-155.

The total number of predicted targets of miR-155 that can be obtained from the miRBase Sequence database [http://microrna.sanger.ac.uk] is 991. Moreover, other methods of target prediction, that are not considered in this data base, can be also used, thus increasing the number of potential targets.

Not all in silico predicted targets have been found to be responsive to the miRNA upon experimental validation [START_REF] Voorhoeve | Classifying microRNAs in cancer: the good, the bad and the ugly[END_REF]. Presently, only a limited number of these genes has been experimentally validated by

Luciferase report assay after transfection with the relevant 3'UTR portion of mRNA, as summarized in Table 1. Several transcripts of genes coding for transcriptional regulatory proteins, protein receptors, kinases, nuclear and DNA binding proteins are the direct targets of miR-155. It follows that this gene could be now considered an important pleiotropic regulator of cell homeostasis.

Perspectives

Even though the mechanism that regulates miRNA function and expression has not been completely clarified, the information presently available (i.e. over expression in a wide type of tumors) allows us to recognize miR-155 as a gene of paramount clinical importance either in cancer diagnosis and therapy [START_REF] Jay | MiRNA profiling for diagnosis and prognosis of human cancer[END_REF][START_REF] Krützfeldt | Silencing of microRNAs in vivo with 'antagomirs[END_REF]. It is reasonable to predict that evaluation of mRNA-155 in tissues or biological fluids could be utilized as a biochemical parameter for tumor detection and prognosis [START_REF] Lawrie | MicroRNA expression distinguishes between germinal center B cell-like and activated B cell-like subtypes of diffuse large B cell lymphoma[END_REF][START_REF] Jay | MiRNA profiling for diagnosis and prognosis of human cancer[END_REF][START_REF] Yanaihara | Unique microRNA molecular profiles in lung cancer diagnosis and prognosis[END_REF].

Moreover, a number of groups have shown that antisense oligonucleotides complementary to the guide strand of miRNAs named anti-miRNA oligonucleotides (AMO or antimirs), or antagomirs (when they are conjugated with cholesterol), can target specific miRNAs abolishing their function in in vitro cultured cells, or in vivo in mice and in monkeys [START_REF] Krützfeldt | Silencing of microRNAs in vivo with 'antagomirs[END_REF][START_REF] Meister | Mechanisms of gene silencing by double-stranded RNA[END_REF][START_REF] Elmén | LNAmediated microRNA silencing in non-human primates[END_REF][START_REF] Tong | Modulation of miRNA activity in human cancer: a new paradigm for cancer gene therapy?[END_REF]. Therefore, it could be suggested that an efficacious device able to down-regulate miR-155 expression in a clinical setting would provide a novel, and possibly specific way to control the growth of a wide range of solid and haematopoietic malignancies in conjunction with classical cytotoxic therapy. In addition, it was recently demonstrated

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that a number of drugs of clinical relevance can modulate in vitro the miRNA expression in the treated cells thus suggesting that miRNAs can be suitable targets for therapeutic effects of anticancer agents [START_REF] Rossi | Modification of miR gene expression pattern in human colon cancer cells following exposure to 5-fluorouracil in vitro[END_REF][START_REF] Blower | MicroRNA expression profiles for the NCI-60 cancer cell panel[END_REF]. We are just at the beginning of a long and still unexplored avenue of highly promising field of molecular investigation. For example, if one considers the possible role of miR-155 in human pathology not involving malignant growth, no data are presently available to suggest whether drugs used in various areas of therapeutic intervention (e.g. immune-modulating cytokines, haematopoietic factors, anti-inflammatory and immune-active agents, antiviral agents, cardiovascular drugs and other agents acting directly or indirectly on the functional activity of vital organs) are able to influence the expression of this miR gene. Considering the multi-target influence afforded by a single miRNA, it is reasonable to hypothesize that studies directed at establishing the effect of drugs on miR gene expression (i.e. a novel "pharmacomirnomics" area of investigation) could disclose possible and upto now unrevealed mode of action of drugs. This would appear to be particularly attractive in those cases in which the pharmacological agents are known to possess pleiotropic effects that could be explained through a miRNA-mediated mechanism. Biogenesis of mature miRNA.

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Transcription of a miR gene by RNA polymerase II generates pri-miRNA (with a 5' 7mG cap and a 3' poly-A tail) that is processed in the nucleus into pre-miRNA by a complex containing Drosha and DGCR8 proteins. Pre-miRNA is exported to the cytoplasm and is processed into a duplex miRNA by another complex in which Dicer, TRBP, PACT proteins play the main role.

After cleavage by the Dicer complex the resulting miRNA duplex is unwound and the passenger strand is degraded The mature miRNA is loaded into the RISC to bind the mRNA target. If the complementarity between the 3'UTR mRNA and the miRNA is extensive the target mRNA is degraded, whereas, if the complementarity is partial, the translation of the target mRNA is repressed.

  lymphocyte development and generation of B and T cell responses in vivo after BCR or TCR activation. In fact, BIC/miR155 deficient mice immunized with the tetanus toxin fragment C protein are characterized by a reduced production of IgM and switched antigen-specific antibodies. Moreover, splenocytes of immunized mice failed to produce significant levels of IL-2 and IFN-γ, thus indicating an important role for miR-155 during both T-cell independent and dependent responses. In addition to defective B and T cell immunity, miR-155 deficient mice have shown an impaired antigen-presenting function of dendritic cells [28]. Comparable results were obtained by Thai et al [29] using a similar experimental mouse model. They showed that miR-155 deficient mice had reduced germinal center function, T-cell dependent antibody A C C E P T E D M A N U S C R I P T

Figure 1 .

 1 Figure 1.

Figure 2 .

 2 Figure 2. Maturation and interaction of miR-155 with a validated mRNA target. A. Pre-miR-155 stem-loop structure. Nucleotide sequence of miR-155 RNA duplex is indicated in red. B. Mature single strand miR-155. C. Example of interaction of miR-155 with target 3'UTR of mRNA transcript of AT1R gene. In this case, a 50% mismatch has been found between miR-155 sequence and the major AT1R allele. The arrow indicates the base that can be substituted by C in the case of single nucleotide polymorphism that is accompanied by failure of translation control by miR-155 [64].

Acknowledgment

We thank Dr Carla Mastrilli of the Department of Neuroscience of our University for her technical and administrative support. This work has been supported in part by a grant from the Istituto Superiore di Sanità, Italy-USA Oncology Program, contract 527/B-3B/6 and in part by the Italian Ministry of Employment, Health and Social Policies.

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Lucía Gutiérrez

Carmen Quintana

Cristina Patiño

Javier Bueno

Hélène Coppin

Marie P Roth

Francisco J Lázaro

Iron speciation study in Hfe knockout mice tissues: Magnetic and ultrastructural characterisation

Keywords: Ferritin, Iron overload diseases, Hfe knockout, Magnetic susceptibility, Biomineralisation

Liver, spleen and heart tissues of DBA/2 Hfe knockout mice have been characterised by low temperature AC magnetic susceptibility measurements together with Transmission Electron Microscopy (TEM) and Selected Area Electron Diffraction in order to investigate the chemical iron speciation in a murine model of iron overload diseases. With emphasis on ferritin-like species, the temperature dependent in-phase and out-of-phase susceptibility profiles agree with the elemental analysis in that, in this model, iron accumulation takes place in the hepatic tissue while in the spleen and heart tissues no differences have been observed between knockout and wild type animals. The comparison of the magnetic properties between perfused and non-perfused liver tissues has made it possible to estimate the magnetic contribution of usually present blood remains. The TEM observations reveal that, besides the isolated ferritins and ferritincontaining lysosomes-siderosomes present in the hepatocytes, other iron deposits, of heterogeneous size, morphology and crystalline structure (haematite and/or goethite), are present in the cytoplasm, near the membrane, and in extracellular spaces.

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Introduction

Genetic haemochromatosis is an autosomal recessive disorder characterized by excessive iron absorption from the duodenum and iron release from the macrophages. In humans, gradual deposition of iron occurs in the liver and in a number of other tissues including the pancreas, joints, skin, heart and the gonadotrophin-secreting cells of the anterior pituitary [START_REF] Andrews | Disorders of Iron Metabolism[END_REF][START_REF] Pietrangelo | Hereditary Hemochromatosis--A New Look at an Old Disease[END_REF]. Disease manifestations include hepatic fibrosis, diabetes mellitus, arthropathy, pigmentation, cardiomyopathy and hypogonadotrophic hypogonadism. Liver fibrosis may progress towards cirrhosis, a complication associated with a 200-fold increased risk of hepatocellular carcinoma, the most common cause of death in this condition. Most patients with genetic haemochromatosis are homozygotes for a single point mutation, C282Y, in the HFE gene [START_REF] Feder | A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis[END_REF]. Population studies have shown that the prevalence of genetic haemochromatosis is one in 300 homozygotes for the C282Y mutation in populations of Northern European extraction.

In the last decades an intense effort has been made in the investigation of the chemical speciation of the tissue iron deposits in cases of iron overload by using

Mössbauer spectroscopy [START_REF] Andrews | Studies on haemosiderin and ferritin from iron-loaded rat liver[END_REF][START_REF] St | A Mössbauer spectroscopic study of the form of iron in iron overload[END_REF][START_REF] St | Core structures of haemosiderins deposited in various organs in beta-thalassemia hemoglobin-E disease[END_REF][START_REF] St | The form of iron oxide deposits in thalassemic tissues varies between different groups of patients: a comparison between Thai beta-thalassemia/hemoglobin E patients and Australian beta-thalassemia patients[END_REF][START_REF] St | Iron overload diseases: the chemical speciation of non-heme iron deposits in iron loaded mammalian tissues[END_REF][START_REF] Ward | Further characterization of forms of hemosiderin in iron-overloaded tissues[END_REF][START_REF] Webb | Biologically significant iron(III) oxyhydroxy polymers: Mössbauer spectroscopic study of ferritin and hemosiderin in pancreas tissue of betathalassemia hemoglobin E disease[END_REF], Transmission Electron Microscopy (TEM) [START_REF] Richter | The cellular transformation of injected colloidal iron complexes into ferritin and hemosiderin in experimental animals -a study with the aid of electron microscopy[END_REF][START_REF] Richter | Studies of iron overload. Rat liver siderosome ferritin[END_REF][START_REF] Iancu | Ferritin and hemosiderin in pathological tissues[END_REF][START_REF] Iancu | Ultrastructural sequences during liver iron overload in genetic hemochromatosis[END_REF],

infrared spectroscopy [START_REF] Chua-Anusorn | Chemical speciation of iron deposits in thalassemic heart tissue[END_REF] and magnetic measurements [START_REF] Allen | Lowfrequency low-field magnetic susceptibility of ferritin and hemosiderin[END_REF][START_REF] Lázaro | Magnetic characterisation of rat muscle tissues after subcutaneous iron dextran injection[END_REF][START_REF] Gutiérrez | Bioinorganic transformations of liver iron deposits observed by tissue magnetic characterisation in a rat model[END_REF][START_REF] Lázaro | Biological tissue magnetism in the frame of iron overload diseases[END_REF][START_REF] Hackett | The magnetic susceptibilities of iron deposits in thalassaemic spleen tissue[END_REF], as the biomineralisation of iron in the tissues may be related to its toxicity. In particular, the study of the magnetic properties of these tissues is of special interest to improve noninvasive, magnetism-based, techniques for diagnosis and follow-up treatment of iron overload diseases [START_REF] Brittenham | Noninvasive measurement of iron: report of an NIDDK workshop[END_REF][START_REF] Brittenham | Magnetic susceptibility measurement of human iron stores[END_REF][START_REF] Nielsen | Non-invasive liver iron quantification by SQUID-biosusceptometry and serum ferritin iron as new diagnostic parameters in hereditary hemochromatosis[END_REF][START_REF] Jensen | Evaluation of iron overload[END_REF][START_REF] Gandon | Non-invasive assessment of hepatic iron stores by MRI[END_REF][START_REF] St | Noninvasive measurement and imaging of liver iron concentrations using proton magnetic resonance[END_REF].

Murine models of iron overload, like Hfe knockout mice (Hfe -/-), provide a useful alternative to humans for a better understanding of the physiologic pathways involved in the disease process [START_REF] Zhou | HFE gene knockout produces mouse model of hereditary hemochromatosis[END_REF][START_REF] Bahram | Experimental hemochromatosis due to MHC class I HFE deficiency: Immune status and iron metabolism[END_REF]. It has been previously reported that, compared with other mouse strains, DBA/2 mice are particularly susceptible to iron loading in response to Hfe disruption [START_REF] Dupic | Inactivation of the hemochromatosis gene differentially regulates duodenal expression of iron-related mrnas between mouse strains[END_REF]. As DBA/2 Hfe -/-mice constitutively become iron overloaded, the model constructed with these animals is expected to be a closer

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representation of the human disease, at least in terms of genetic origin, than other models like the iron overload induced by parenteral injection of iron dextran.

In this paper, the low temperature AC magnetic susceptibility data of liver, spleen and heart tissues of DBA/2 Hfe knockout (KO) and wild type (WT) mice will be presented with the aim of investigating the tissue iron speciation in iron overload diseases. The magnetic results together with elemental analyses and ultrastructural information of the iron deposits, obtained from Transmission Electron Microscopy and Selected Area Electron Diffraction (SAED), will be analysed.

Materials and methods

Animals

A total of 12 DBA/2 mice, 8 female and 4 male, have been used in this study.

Half of the animals were Hfe knockout and the rest were wild type, in a way that each KO mice had a WT counterpart of the same age and gender (see Table 1 for details).

All the experiments were performed in accordance with institutional and governmental guidelines and the experimental protocols were approved by the Midi-Pyrénées Animal Ethics Committee. WT and KO mice were housed in the IFR30 animal facility (4-6 animals/cage, 12:12 h light-dark cycle; 22 ± 1 °C, 60 ± 5% humidity). The animals had free access to R03 diet (UAR, Epinay-sur-Orge, France) containing 280 mg Fe/kg and distilled water.

Elemental analysis

The animal tissues were analysed by Inductively Coupled Plasma Atomic Emission Spectrometry (ICP-AES) after microwave acid digestion (solution 4:1 (v/v) of HNO 3 (65%) and H 2 O 2 (30%)). The magnetically characterised freeze-dried samples (see description below) were subsequently analysed for elemental content in order to

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allow a quantitative interpretation of the magnetic data. The elemental analysis was focused on the determination of Fe, but the presence of other magnetogenic elements as Co, Ni, Mn and Cu, which may contribute to the magnetic results, was also included in the analysis.

Transmission Electron Microscopy

Two different protocols were used for the fixation of the tissues. Half of the animals were sacrificed under pentobarbitone anaesthesia and portions of the livers, spleens and hearts were prepared for TEM observations immediately after tissue dissection. Fragments of the different tissues were fixed for 2 h at room temperature and overnight at 4 ºC in a solution of 2% paraformaldehyde (PAF) and 2% glutaraldehyde (Gluta) in 0.1 M phosphate buffer (PB). The rest of the animals were also sacrificed under pentobarbitone anaesthesia although the fixation of the tissues was performed by perfusion. The same fixative (2% PAF, 2% Gluta, 0.1 M PB) was injected directly in the heart and pumped during 8 min at 20 ml/min. Then a different solution containing 0.1 M phosphate buffer was pumped at the same rate during 2 min. A part of the liver was then dissected and left in the fixative at 4 ºC overnight.

All the tissues were then postfixed with 1% osmium tetroxide for 45 min at 4 ºC.

After four washes with buffer, samples were treated with 2% uranyl acetate, dehydrated in graded series of acetone (50, 70, 90, 100%) for 15 min each at 4 ºC and embedded in Epon resin at room temperature for 24 h. The polymerization was done at 60 ºC for 48 h.

Ultra-thin sections (40-60 nm) were collected on formvar-coated grids and some of them were also lightly stained with 2% uranyl acetate for 30-60 s or stained with saturated uranyl acetate and lead citrate by standard procedures.

The TEM characterization was carried out in a Jeol 1010 microscope operated at 100 kV and in a Jeol 1200FX. X-ray microanalysis was also performed with these equipments in order to determine the presence of iron in the tissues.

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AC susceptibility

Samples of the tissues for the magnetic characterisation were prepared as follows. In the case of the non-perfused animals, the spleen, heart and liver tissues were immediately frozen at -80 ºC after dissection. These tissues were freeze-dried during 48 h in a Heto PowerDry PL3000 equipment and then ground to powder in order to have a homogeneous sample. The fixed liver samples of the perfused animals were stored at 4 ºC, freeze-dried during 24 h in the same equipment and then ground to powder. The powder of each liver sample was placed in individual gelatine capsules for the magnetic measurements.

Due to the small available amount of spleen and heart tissues, pools of samples were prepared for the magnetic characterisation. The spleen tissues of the KO mice (two female and one male) were joined into a single gelatine capsule; doing the same thing with the WT counterparts. The heart tissue samples were prepared following identical procedure.

The magnetic characterisation has been carried out in a Quantum Design MPMS-5S SQUID magnetometer with an AC susceptibility option. The measurements were performed with an AC amplitude of 0.45 mT and at a frequency of 1 Hz in the temperature range 1.8-300 K. The presence of adsorbed oxygen in the samples, which would contribute to the magnetic susceptibility [START_REF] Kanoh | Magnetic spin states of O 2 confined in a graphitic slitshaped nanospace at low temperature[END_REF], has been checked and avoided by repeated runs above and below its boiling temperature in all the cases.

Results

Elemental analysis

The elemental iron content of each tissue is shown in Table 1. For livers, it varies in a range between 1.729 and 3.903 mg Fe/g dry tissue for KO mice and between

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0.371 and 0.636 mg Fe/g dry tissue for the WT ones. For spleen and heart tissues, there are no such big differences in the iron content between the KO and WT tissue pools.

The content of other magnetogenic elements (Cu, Co, Mn and Ni) is always less than 0.003 wt% of the freeze-dried tissues.

Ultrastructural observations

TEM observations of the KO liver tissues show hepatocytes with a dense cytoplasm where the nucleus and all the organelles are well preserved (Fig. 1). In these tissues, in lightly stained sections, several types of electron dense deposits, in which the presence of iron has been confirmed by X-ray microanalysis, are observed in the cytoplasm of hepatocytes and Kupffer cells and also in intercellular spaces. It is important to note that, in spite of the iron excess in the KO livers, the physiological tissue structure seems not to be altered, as the reported in other animal models of iron overload [START_REF] Gutiérrez | Bioinorganic transformations of liver iron deposits observed by tissue magnetic characterisation in a rat model[END_REF].

Observations at higher magnification (Fig. 2) allow to classify the iron containing deposits into three types: i) cores of ferritin molecules (1) dispersed in the cytoplasm of cells, sometimes localised near the endoplasmic reticulum (ER), ii) cores of ferritin-haemosiderin molecules (2) in lysosomes-sideromes of a size that ranges between 0.3 and 0.8 µm and iii) deposits of heterogeneous shape and size (3), observed mainly in the cytoplasm near the membrane and in the extracellular space.

Selected Area Electron Diffraction (SAED) performed on deposits type 1 and 2

show a mineralisation similar to ferrihydrite (Table 2), the most common mineral found in physiological ferritin cores [START_REF] Towe | Mineralogical constitution of colloidal 'hydrous ferric oxides[END_REF][START_REF] Cowley | The structure of ferritin cores determined by electron nanodiffraction[END_REF]. A set of rings, different from those of ferrihydrite and consistent with haematite and/or goethite phases, appear in the diffraction pattern of type 3 deposits (Table 2), although a deeper study on their nature is still needed.

AC magnetic susceptibility

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The temperature dependence of the AC susceptibility per mass of sample of mice liver tissues is shown in Figs. 3 and4. For the sake of clarity only the results from six animals are shown in the graphs, although data from all the characterised tissues have been listed in Table 1.

A low temperature paramagnetic tail can be observed in the in-phase susceptibility χ'(T) of all the liver samples. The in-phase component of some of the samples also shows a maximum, or a small shoulder, around 11 K that, together with the non-zero out-of-phase susceptibility χ''(T) at the same temperature range, provide evidence for a magnetic relaxation phenomenon. The out-of-phase susceptibility of all the liver tissues shows a single maximum around 8 K and stays negligible, within the accuracy of the experiments, above approximately 25 K reminding previous results obtained for rat liver ferritin [START_REF] Gutiérrez | Bioinorganic transformations of liver iron deposits observed by tissue magnetic characterisation in a rat model[END_REF]. The value of the susceptibility per mass of sample of the out-of-phase maxima of all the samples is shown in Table 1.

The spleen and heart pools of the KO and WT animals present a low temperature paramagnetic tail in the in-phase susceptibility in all the cases (Fig. 5). The out-of-phase susceptibility of the corresponding four samples is plotted in Fig 6 . It can be observed that the heart samples show a nearly negligible out-of-phase susceptibility in all the temperature range, while a small and rather noisy maximum at low temperatures can be observed for the spleen samples. For both tissues, the susceptibility results do not show significant differences between the KO and the WT animals.

Discussion

From the data in Table 1, it can be observed that the KO animals have, as expected, a greater concentration of iron in the liver tissue than the WT ones. No significant differences are observed between 9 and 14 week old animals, in agreement with unpublished observations indicating that the hepatic iron concentration in Hfe -/- mice increases sharply after weaning but stabilizes after few weeks. Besides, the iron

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contents of the KO mice tissues obtained from the elemental analysis are also similar to those previously determined by biochemical methods in the same animal model of iron overload [START_REF] Dupic | Inactivation of the hemochromatosis gene differentially regulates duodenal expression of iron-related mrnas between mouse strains[END_REF].

Although their protein shell is not visible in these TEM micrographs, a large amount of ferritin cores can be observed in the cytoplasm. The actual presence of the ferritin molecules is evidenced from the shape (approximately spherical) and the size (not larger than about 8 nm) of their electron-dense cores that, besides, in lightly stained sections, appear mutually separated by the non electron-dense protein shells [START_REF] Iancu | Ferritin and hemosiderin in pathological tissues[END_REF]. The presence of ferritin molecules near the ER may indicate a profuse synthesis of this protein in the liver tissues.

The size and shape of the iron containing particles inside the lysosomessiderosomes is similar to those of the ferritin cores. However, the apparent mutual contact between them may indicate that the protein shell has undergone a certain degradation, analogously to what has been observed in the so-called secondary lysosomes [START_REF] Harrison | Ferritins: Molecular properties, iron storage function and cellular regulation[END_REF][START_REF] Iancu | Ferritin and hemosiderin in pathological tissues[END_REF]. This type of deposit has been mentioned in the degradation sequence of ferritin, in the lysosomes, to form haemosiderin [START_REF] Richter | Studies of iron overload. Rat liver siderosome ferritin[END_REF][START_REF] Iancu | Ultrastructural sequences during liver iron overload in genetic hemochromatosis[END_REF]. In fact some authors have called them "prehaemosiderin", indicating that the mineral cores would not be altered yet [START_REF] St | A Mössbauer spectroscopic study of the form of iron in iron overload[END_REF]. In addition, it has to be taken into account that when observing closed packed ferritin particles in structures such as these lysosomes, mineral particles may appear to be in contact even if the protein shell is intact. However, independently of the term used to describe these clusters or the state of the protein shell, it is clear that they contain a large part of the total iron accumulated in the liver.

The intracellular and extracellular type 3 iron deposits observed in the micrographs show neither morphological nor crystalline similarities with the ferritin cores. Their location near the membranes and in the extracellular space may indicate a process of iron removal from the cell, possibly explaining why the tissues are relatively well preserved in spite of the iron excess. In fact, the beneficial role of biomineralisation processes in preventing iron toxicity has been pointed out in relation to the formation of haemosiderin [START_REF] Ward | Does the haemosiderin iron core determine its potential for chelation and the development of iron-induced tissue damage?[END_REF]. Goethite deposits in tissues have previously been

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observed in conditions of iron overload [START_REF] St | Core structures of haemosiderins deposited in various organs in beta-thalassemia hemoglobin-E disease[END_REF][START_REF] St | The form of iron oxide deposits in thalassemic tissues varies between different groups of patients: a comparison between Thai beta-thalassemia/hemoglobin E patients and Australian beta-thalassemia patients[END_REF][START_REF] Ward | Does the haemosiderin iron core determine its potential for chelation and the development of iron-induced tissue damage?[END_REF], while haematite, as far as we know, has only been mentioned as a minor phase in physiological ferritin cores [START_REF] Cowley | The structure of ferritin cores determined by electron nanodiffraction[END_REF][START_REF] Quintana | Electron nanodiffraction and highresolution electron microscopy studies of the structure and composition of physiological and pathological ferritin[END_REF]. It is therefore in this work, in our opinion, the first time that the presence of haematite-like deposits are detected in iron overload situations.

As previous studies have reported the transformation from ferrihydrite either to haematite or to goethite, we can hypothesize that our type 3 deposits may be originated from the ferritin ferrihydrite cores. However, while it is known that haematite can be formed from the dehydration of ferrihydrite, the formation of goethite from ferrihydrite would imply its dissolution and further nucleation [START_REF] Cornell | The Iron oxides: Structure, Properties, Reactions, occurrences and uses[END_REF].

As other authors have reported the transformation from ferrihydrite to other iron oxide structures by the effect of the electron beam [START_REF] Pan | Electron beam damage studies of synthetic 6-line ferrihydrite and ferritin molecule cores within a human liver biopsy[END_REF], we have done dedicated experiments to assure that the crystalline structure of type 3 deposits is not due to such effect. In our case, the SAED patterns of ferrihydrite deposits have not been altered after several minutes of irradiation.

From previous works on the study of homologous tissues in rats [START_REF] Gutiérrez | Bioinorganic transformations of liver iron deposits observed by tissue magnetic characterisation in a rat model[END_REF] and from available AC magnetic susceptibility data of ferritin standards [START_REF] Allen | Lowfrequency low-field magnetic susceptibility of ferritin and hemosiderin[END_REF][START_REF] Gutiérrez | Bioinorganic transformations of liver iron deposits observed by tissue magnetic characterisation in a rat model[END_REF][START_REF] Luis | Resonant spin tunneling in small antiferromagnetic particles[END_REF], the χ''(T) maximum observed in these mice tissues can be assigned to the presence of ferritin or, alternatively, to the cores of partially degraded ferritin whose crystalline structure and size are still similar to those of ordinary ferritin. However, the χ''(T) maxima of the mice tissues are located at slightly lower temperatures (7 -9 K) than those previously observed for rat liver ferritin (8 -12 K) [START_REF] Gutiérrez | Bioinorganic transformations of liver iron deposits observed by tissue magnetic characterisation in a rat model[END_REF] measured in the same conditions; a phenomenon that may result from small differences in the size of the iron-containing crystallites inside the ferritin cages or from a qualitative different biomineralisation between the two animal genera.

With respect to the contribution of type 3 deposits to the total magnetic susceptibility of the tissues, we expect it to be under the detection limits of the magnetic characterisation. This assumption is based (i) on the fact that these aggregates are quite sparse in the volume in comparison with the ferritin cores and (ii) on the relatively large

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size of these deposits that, given the antiferromagnetic character of haematite and goethite, result in smaller magnetic moments per iron ion.

When plotting the out-of-phase susceptibility per mass of sample, the height of the χ''(T) maximum (see data in Fig. 4 only for selected samples and in Table 1 for all of them) informs about the amount of iron in the form of ferritin cores. In Fig. 4, it can be observed that, independently on whether the animals were perfused or not, the χ''(T) maxima for the KO mice are always higher than for their WT counterparts. This result indicates that the genetically provoked iron overload consists of, not only a greater amount of total iron, but a greater amount of iron in the form of ferritin.

In order to study the differences in shape, width and location of the χ''(T) maxima it is usually adequate to scale the curves to their maximum height. In Fig. 7 it can be observed that there is a rather good coincidence of the χ''(T) profile for all the liver samples (perfused and non-perfused, WT and KO) suggesting that in all these cases a very similar biomineralisation, in terms of the formed iron (oxyhydr)oxide, may exist. However, when looking in more detail, it can be observed that, while the high temperature flank of the χ''(T) profile very well coincides in all the cases, likely due to the size limitation of the ferritin cavity, there are significant differences in the low temperature flank between perfused and non-perfused animals. This result would indicate that, assuming a similar biomineralisation in all the cases, the size distribution of the iron-containing crystallites in the case of the non-perfused animals is more extended to small sizes than in the case of the perfused ones.

Although, as mentioned, no big differences between the χ''(T) data for perfused and non-perfused animals, of the same genotype and gender, are observed, they do differ in the in-phase component. The low temperature tail observed in χ'(T) originates at paramagnetic iron-containing species, that is, molecules or microscopical entities in which the iron atoms may be either bounded to organic ligands (e.g. deoxyhaemoglobin, transferrin) or not, but never in the form of iron (oxyhydr)oxide nanoparticles. This paramagnetic contribution may correspond either to intracellular iron-containing species or to other substances remaining in the blood vessels. The observed χ'(T) data for the

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non-perfused animals, independently of gender and genotype, always present a much greater paramagnetic contribution than for the perfused ones. This result appears logical because in the perfusion process the fixative is introduced through the blood vesselsthe iron-containing species from the blood are substantially removedreducing their paramagnetic contribution.

Within a deeper analysis of the iron speciation in the liver tissues, the χ''(T) data, expressed per mass of iron, are shown in Fig. 8. In this type of representation, if all of the iron atoms in a given tissue were present in the form of a single iron-containing species (e.g. ferritin in this case), the corresponding χ''(T) data, per mass of iron, would coincide with that of isolated ferritin. However, the variable presence of other species not contributing to χ'', like paramagnetic iron, diamagnetic iron, or other weakly magnetic forms of iron as the invoked haematite and/or goethite deposits, will result in that the greater the ratio Fe ferritin /Fe total , the higher the χ''(T) ferritin profile when represented per mass of iron. In particular, in Fig. 8, the χ''(T) data points for perfused animals significantly appear higher than for the non-perfused ones, meaning that the first ones contain a bigger proportion of ferritin iron. The previously discussed observation of a smaller paramagnetic component in perfused animal tissues (see Fig. 3) independently of their gender or genotype, as it clearly implies removal of iron ions is also consistent with the same idea. It is also worth noting that the height of the χ''(T) maxima per mass of iron for the perfused mice livers is very similar in all the cases.

This coincidence means that the Fe ferritin /Fe total ratio is similar for all those samples in spite of their different iron contents. Within the group of the non-perfused liver tissues, it can be observed that the KO females present a higher iron-specific out-of-phase susceptibility maximum in comparison with their WT counterparts explained by a higher Fe ferritin /Fe total ratio, due to the iron loading. However, the biological explanation of the fact that the iron-specific out-of-phase susceptibility maximum of the KO nonperfused male has the same height as its WT counterparts is not clear for us yet.

The χ'' maximum for spleen tissues (Fig. 6), although more noisy, is located at similar temperatures as liver tissues, most likely corresponding also to ferritin iron. For

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13 heart tissues, χ''(T) is nearly negligible in all the temperature range meaning that the detection of ferritin iron in these samples is beyond the limits of the technique. For both spleen and heart tissues, the susceptibility of the pool samples in the case of KO animals does not differ significantly from the WT ones indicating that there is no particular iron accumulation in these tissues, in agreement with the determined elemental iron contents (Table 1).

The magnetic data in this mouse model of iron overload can be compared with the rat model constructed by means of a single injection of iron dextran [START_REF] Lázaro | Magnetic characterisation of rat muscle tissues after subcutaneous iron dextran injection[END_REF][START_REF] Gutiérrez | Bioinorganic transformations of liver iron deposits observed by tissue magnetic characterisation in a rat model[END_REF][START_REF] Lázaro | Biological tissue magnetism in the frame of iron overload diseases[END_REF]. In that model iron deposits were observed in liver, spleen, muscle, heart and kidney tissues while in the present case of DBA/2 Hfe KO mice iron overload it has only been observed in the liver.

For all the studied tissues, in the high temperature range, χ'(T) show Curie-type behaviour as it is typical for a paramagnet, a superparamagnet, or a mixture of both. As it has been previously explained [START_REF] Lázaro | Magnetic characterisation of rat muscle tissues after subcutaneous iron dextran injection[END_REF], the high temperature paramagnetic and superparamagnetic contributions are mixed up on a single Curie-law susceptibility whose information is condensed into the value of the effective moment  eff per iron ion (for a deeper explanation of the calculation of the  eff value of tissue samples see Ref. [START_REF] Lázaro | Magnetic characterisation of rat muscle tissues after subcutaneous iron dextran injection[END_REF]). As each iron-containing species usually has a characteristic effective moment per iron ion, namely, 5.46  B for deoxyhaemoglobin, 0  B for oxyhaemoglobin and 3.4  B

for ferritin [START_REF] Pauling | The magnetic properties and structure of hemoglobin, oxyhemoglobin and carbonmonoxyhemoglobin[END_REF][START_REF] Alpert | Magnetic susceptibility measurements of deoxygenated hemoglobins and isolated chains[END_REF][START_REF] Pauling | Magnetic properties and structure of oxyhemoglobin[END_REF][START_REF] Gutiérrez | Bioinorganic transformations of liver iron deposits observed by tissue magnetic characterisation in a rat model[END_REF], the effective moment per iron ion determined for the whole tissue sample results from the average of the  eff values, weighted with the relative abundances of each species.

The effective moments per iron ion of all the characterised samples are shown in Table 1. It can be seen that  eff = 2.93 ± 0.36 (mean + s.d.) in the case of non-perfused animals and 3.23 ± 0.17 for the perfused ones. This significant result may indicate that the iron removed in the perfusion process, although being in part of paramagnetic nature, may include an important content of low magnetic moment iron, which may be of diamagnetic and/or antiferromagnetic (large particle size) nature.

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Conclusions

In this work, for the first time, AC magnetic susceptibility measurements together with TEM analysis have been used to study the iron deposits formed in the tissues of DBA/2 Hfe KO mice.

No differences between KO and WT mice have been observed in the iron content of spleen and heart tissues. However, an increased iron accumulation in liver tissues has been found for the KO mice.

The differences in shape and location of the χ''(T) maxima, corresponding to the ferritin cores contribution, observed between different animal genera (data of these mice and previous data for rats [START_REF] Gutiérrez | Bioinorganic transformations of liver iron deposits observed by tissue magnetic characterisation in a rat model[END_REF]) may indicate a slightly different qualitative biomineralisation or different size distribution of the iron-containing crystallites.

The perfusion procedure has revealed that a considerable part of the paramagnetic contribution observed in the non-perfused tissues comes from ironcontaining species in the remaining blood.

Iron deposits whose morphology, size and crystalline structure (haematite and/or goethite) appear different from those of ferritin and those of lysosomes-siderosomes containing ferritin-haemosiderin have been observed in the liver tissues by TEM and SAED analysis. This finding deserves a more detailed study, as it may be related to the apparently healthy aspect of the tissues, and opens the way to future studies on biomineralisation processes in cases of iron overload and related to potential treatments of the disease. The formation of these iron-containing deposits may be a way to avoid free iron and the oxidative stress related to it.

The combination of magnetic characterisation and TEM analysis has been proven to be of great usefulness to study the nature and the evolution of tissue iron deposits in conditions of iron overload.

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Table 1.

Height values of the out-of-phase susceptibility maximum ('' (T) max ) for the different tissue samples, genotype (WT = wild type; KO = knockout), gender (M = male; F = female), age of the animals, elemental iron content and effective moment per iron ion ( eff ). The '' (T) maximum and the iron content are expressed per mass of dry tissue. Temperature dependence of the out-of-phase susceptibility '' (T), at 1 Hz, per mass of freeze-dried tissues of the spleen and heart tissue pools corresponding to mice of different genotype.

Comparative plot of the '' (T) profiles, scaled to their respective maxima, for the magnetically characterised liver tissues.

Fig. 1 .Fig. 2 TEM

 12 Fig. 1. Liver tissue TEM micrograph of a KO female mouse 9 weeks old. Electrondense iron deposits (marked with arrows) can be observed in the cytoplasm of the hepatocytes.

Fig. 3 Temperature

 3 Fig.3Temperature dependence of the in-phase susceptibility '(T), at 1 Hz, per mass of freeze-dried liver tissue corresponding to mice of different gender and genotype, some of them subjected to perfusion. Filled symbols correspond to non-perfused animals and empty symbols to the perfused ones.

Figure 4 Temperature

 4 Figure 4Temperature dependence of the out-of-phase susceptibility '' (T), at 1 Hz, per mass of freeze-dried liver tissue corresponding to mice of different gender and genotype, some of them subjected to perfusion. Filled symbols correspond to non-perfused animals and open symbols to the perfused ones.

Fig. 5 Temperature

 5 Fig. 5 Temperature dependence of the in-phase susceptibility ' (T), at 1 Hz, per mass of freeze-dried tissues of the spleen and heart tissue pools corresponding to mice of different genotype.

Fig. 8 Temperature 7 A

 87 Fig.8Temperature dependence of the out-of-phase susceptibility '' (T), at 1 Hz, per mass of elemental iron for the liver tissues. Filled symbols correspond to non-perfused animals and open symbols to the perfused ones.

Table 2 .

 2 Calculated d spacings (nm) from the SAED ring data for the different deposits observed in the liver sections. Deposits type 1 and 2 correspond to isolated spherical particles and lysosomes-siderosomes containing spherical electron-dense particles respectively, while type 3 correspond to the very electron-dense deposits shown in Fig.

	Animal Organ Genotype Gender	Age (weeks)	Perfusion	'' (T) max (m 3 /kg)	[Fe] (mg/g)	 eff ( B )
	1	Liver	WT	M	9	No	1.910 -10	0.436 2.43
	2	Liver	KO	M	9	No	1.110 -9	2.579 2.69
	3	Liver	WT	F	9	No	3.110 -10	0.614 2.95
	4	Liver	WT	F	9	No	3.110 -10	0.636 2.92
	5	Liver	KO	F	9	No	3.110 -9	3.903 3.15
	6	Liver	KO	F	9	No	1.910 -9	2.537 3.46
	7	Liver	WT	M	14	Yes	2.410 -10	0.439 3.72
	8	Liver	KO	M	14	Yes	2.010 -9	1.729 3.23
	9	Liver	WT	F	14	Yes	4.010 -10	-	-
	10	Liver	KO	F	14	Yes	2.810 -9	2.496 3.28
	11	Liver	WT	F	14	Yes	4.910 -10	0.371 3.61
	12	Liver	KO	F	14	Yes	3.010 -9	2.764 3.37
	1+3+4 Spleen	WT	2F+1M	9	No	8.810 -10	1.996 2.18
	2+5+6 Spleen	KO	2F+1M	9	No	7.510 -10	1.835 2.51
	1+3+4	Heart	WT	2F+1M	9	No	-	0.450 3.17
	2+5+6	Heart	KO	2F+1M	9	No	-	0.375 3.44

Acknowledgements

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M A N U S C R I P T We thank Ana Rosa Abadía, Laure Verret and Claire Rampon for technical support. Financial support from Instituto de Salud Carlos III through project PI060549 is acknowledged. Lucía Gutiérrez thanks Gobierno de Aragón and Fondo Social Europeo for Grant B091/2005.

We are also very grateful to the anonymous referees for having generously given their time in helping us to improve this manuscript.

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