Cognitive Training Improves Disconnected Limbs' Mental Representation and Peripersonal Space after Spinal Cord Injury.

Paraplegia following spinal cord injury (SCI) affects the mental representation and peripersonal space of the paralysed body parts (i.e., lower limbs). Physical rehabilitation programs can improve these aspects, but the benefits are mostly partial and short-lasting. These limits could be due to the absence of trainings focused on SCI-induced cognitive deficits combined with traditional physical rehabilitation. To test this hypothesis, we assessed in 15 SCI-individuals the effects of adding cognitive recovery protocols (motor imagery-MI) to standard physical rehabilitation programs (Motor + MI training) on mental body representations and space representations, with respect to physical rehabilitation alone (control training). Each training comprised at least eight sessions administered over two weeks. The status of participants' mental body representation and peripersonal space was assessed at three time points: before the training (T0), after the training (T1), and in a follow-up assessment one month later (T2). The Motor + MI training induced short-term recovery of peripersonal space that however did not persist at T2. Body representation showed a slower neuroplastic recovery at T2, without differences between Motor and the Motor + MI. These results show that body and space representations are plastic after lesions, and open new rehabilitation perspectives.

Complement activation in the plasma and placentas of women with different subsets of antiphospholipid syndrome.

PROBLEM: As antiphospholipid antibody-positive women with adverse pregnancy outcomes have higher plasma complement activation product levels, and the placentas of women with antiphospholipid syndrome (APS) exhibit C4d complement component deposition, complement activation involvement has been hypothesized in APS pregnancy complications. METHOD OF STUDY: Plasma levels of C5a and C5b-9 complement components of 43 APS non-pregnant patients and 17 pregnant APS women were measured using enzyme-linked immunosorbent assay. The results were compared with those of 16 healthy non-pregnant women and eight healthy pregnant women, respectively. Placenta samples of five APS patients at high risk of pregnancy complications and of five healthy controls were subjected to immunoblotting analysis with specific antibodies to C5b-9 and CD46, CD55, CD59 complement regulators. RESULTS: The mean plasma C5a and C5b-9 levels were significantly higher in the non-pregnant APS patients with previous thrombosis ± pregnancy morbidity (P = .0001 and P = .0034, respectively) and in the pregnant APS women with adverse outcomes (P = .0093 for both). Similarly, C5b-9 amounts were significantly higher in the adverse pregnancy outcome placenta (P = .0115) than in those associated to a favorable outcome. The mean CD46, CD55 and CD59 amounts were, instead, lower, although not always significantly, in the placentas of all the high-risk APS women with respect to the control placentas. CONCLUSION: Data analysis demonstrated that there was significant complement activation in the more severe subset of APS patients and in only the adverse pregnancy outcome APS women. Further studies will clarify whether the lower CD46, CD55, and CD59 expressions in the APS placentas are limited to only high-risk APS patients.

Sucrosomial® Iron Supplementation in Mice: Effects on Blood Parameters, Hepcidin, and Inflammation.

Sucrosomial® Iron is a recently developed formulation to treat iron deficiency based on ferric pyrophosphate covered by a matrix of phospholipids plus sucrose esters of fatty acids. Previous data indicated that Sucrosomial® Iron is efficiently absorbed by iron-deficient subjects, even at low dosage, and without side effects. Its structural properties may suggest that it is absorbed by an intestinal pathway which is different to the one used by ionic iron. Although, studies in vitro showed that Sucrosomial® Iron is readily absorbed, no animal models have been established to study this important aspect. To this aim, we induced iron deficient anemia in mice by feeding them with a low-iron diet, and then we treated them with either Sucrosomial® Iron or sulfate iron by gavage for up to two weeks. Both iron formulations corrected anemia and restored iron stores in a two-week period, but with different kinetics. Ferrous Sulfate was more efficient during the first week and Sucrosomial® Iron in the second week. Of note, when given at the same concentrations, Ferrous Sulfate induced the expression of hepcidin and four different inflammatory markers (Socs3, Saa1, IL6 and CRP), while Sucrosomial® Iron did not. We conclude that anemic mice are interesting models to study the absorption of oral iron, and that Sucrosomial® Iron is to be preferred over Ferrous Sulfate because of similar absorption but without inducing an inflammatory response.

Hepcidin and DNA promoter methylation in hepatocellular carcinoma.

BACKGROUND: The liver hormone hepcidin regulates iron homoeostasis that is often altered in hepatocellular carcinoma (HCC). Epigenetic phenomena control gene expression through a dynamic fashion; therefore, considering the plasticity of both iron homoeostasis and epigenetic mechanisms and their role in liver carcinogenesis, we investigated whether hepcidin gene (HAMP) expression is modulated by DNA methylation, thus affecting iron status in human HCC. MATERIALS AND METHODS: Thirty-two patients affected by nonviral HCC were enrolled, and their main clinical and biochemical characteristics were obtained. Neoplastic and homologous non-neoplastic liver tissues were analysed for HAMP promoter DNA methylation, for HAMP gene expression and for iron content. An in vitro demethylation assay with a human hepatocarcinoma cell line was performed to evaluate the role of DNA methylation on HAMP transcriptional repression. RESULTS: Gene expression and DNA methylation analyses on tissues showed that HAMP was transcriptionally repressed in HCC tissues consensually with a promoter hypermethylation. Furthermore, patients with HCC had low serum hepcidin concentrations, and HCC tissues had relative iron depletion as compared to non-neoplastic liver tissues. The cell culture model showed the functional role of DNA hypermethylation by downregulating HAMP gene expression. Through a quantitative methylation analysis on HCC tissues, we then proved that methylation at definite CpG sites within consensus sequences for specific transcription factors is possibly the mechanism underlying HAMP repression. CONCLUSIONS: This study highlights a novel role for HAMP downregulation through DNA promoter hypermethylation and emphasises the significance of epigenetics in the regulation of iron metabolism in HCC.

Glycol-split nonanticoagulant heparins are inhibitors of hepcidin expression in vitro and in vivo.

Hepcidin controls systemic iron availability, and its excess contributes to the anemia of chronic diseases, the most prevalent anemia in hospitalized patients. We previously reported that heparins are efficient hepcidin inhibitors both in vitro and in vivo, but their anticoagulant activity limits therapeutic use. We studied nonanticoagulant heparins produced by N-acetylation and oxidation/reduction (glycol-split) that lost antithrombin-binding affinity. Four nonanticoagulant heparins inhibited hepcidin expression in hepatic HepG2 cells and primary hepatocytes. The 2 most potent ones used in mice suppressed liver hepcidin expression and serum hepcidin in 6 hours, with a significant decrease of spleen iron. This occurred also in lipopolysaccharide (LPS)-treated animals that mimic inflammation, as well as after chronic 1-week treatments, without evident adverse effects on coagulation. Heparin injections increased iron mobilization and facilitated the recovery from the anemia induced by heat-killed Brucella abortus, a model of inflammatory anemia. The heparins were used also in Bmp6(-/-) mice. A single dose of heparin reduced the already low level of hepcidin of these mice and prevented its induction by LPS. These nonanticoagulant compounds impair bone morphogenetic protein /sons of mothers against decapentaplegic signaling with no evident adverse effect in vivo, even when administered chronically. They may offer a strategy for the treatment of diseases with high hepcidin levels.

Serum hepcidin in inflammatory bowel diseases: biological and clinical significance.

BACKGROUND: Hepcidin, a peptide produced by hepatocytes, regulates body iron homeostasis. Inflammation increases serum hepcidin, and its determination can be useful in the differential diagnosis of anemias during inflammatory diseases. METHODS: We measured serum hepcidin-25 and hepcidin-20 isoforms in 54 patients with inflammatory bowel diseases (IBD) and 54 reference subjects (36 healthy controls and 18 anemic patients without inflammation or renal failure). Disease activity, blood counts, iron status, and erythropoiesis-related parameters were obtained for all study subjects. RESULTS: In IBD hepcidin-25, the peptide bioactive isoform correlated positively with C-reactive protein and serum ferritin; an inverse correlation was observed with transferrin, the soluble transferrin receptor, and the soluble transferrin receptor to Log(ferritin) ratio. Similar correlations were found in reference subjects. Patients with anemia of inflammation had higher hepcidin-25 levels than those with iron deficiency anemia or a combination of iron deficiency anemia and inflammation (P = 0.0061). In patients with inflammation and serum ferritin concentration 100 to 200 ng/mL, hepcidin-25 was low, suggesting that these patients had iron deficiency. A serum hepcidin-25 concentration below 2.0 nM differentiated 85% of patients with iron deficiency anemia (with or without inflammation) from patients with anemia of inflammation. In IBD, hepcidin-20 correlated with both hepcidin-25 and C-reactive protein. CONCLUSIONS: In IBD, iron stores, inflammation, and iron requirement for erythropoiesis influence serum hepcidin-25. Hepcidin-25 determination can be useful in the differential diagnosis of IBD-associated anemias. Serum hepcidin-20 is linked to hepcidin-25, but inflammation has an independent regulatory role on its concentration, indicating that hepcidin-20 may have a biological function.

Correction: Increased Serum Hepcidin Levels in Subjects with the Metabolic Syndrome: A Population Study.

[This corrects the article on p. e48250 in vol. 7.].

Serum hepcidin levels and muscle iron proteins in humans injected with low- or high-dose erythropoietin.

Inhibition of hepcidin expression by erythropoietic signals is of great physiological importance; however, the inhibitory pathways remain poorly understood. To investigate (i) the direct effect of erythropoietin (Epo) and (ii) the contribution of putative mediators on hepcidin repression, healthy volunteers were injected with a single dose of Epo, either low (63 IU/kg, n = 8) or high (400 IU/kg, n = 6). Low-dose Epo provoked hepcidin down-modulation within 24 h; the effect was not immediate as hepcidin circadian variations were still present following injection. High-dose Epo induced no additional effect on the hepcidin response, that is hepcidin diurnal fluctuations were not abolished in spite of extremely high Epo levels. We did not find significant changes in putative mediators of hepcidin repression, such as transferrin saturation, soluble transferrin receptor, or growth differentiation factor 15. Furthermore, the potential hepcidin inhibitor, soluble hemojuvelin, was found unaltered by Epo stimulation. This finding was consistent with the absence of signs of iron deficiency observed at the level of skeletal muscle tissue. Our data suggest that hepcidin repression by erythropoietic signals in humans may not be controlled directly by Epo, but mediated by a still undefined factor.

Increased serum hepcidin levels in subjects with the metabolic syndrome: a population study.

The recent discovery of hepcidin, the key iron regulatory hormone, has changed our view of iron metabolism, which in turn is long known to be linked with insulin resistant states, including type 2 diabetes mellitus and the Metabolic Syndrome (MetS). Serum ferritin levels are often elevated in MetS (Dysmetabolic hyperferritinemia--DHF), and are sometimes associated with a true mild-to-moderate hepatic iron overload (dysmetabolic iron overload syndrome--DIOS). However, the pathophysiological link between iron and MetS remains unclear. This study was aimed to investigate, for the first time, the relationship between MetS and hepcidin at population level. We measured serum hepcidin levels by Mass Spectrometry in 1,391 subjects from the Val Borbera population, and evaluated their relationship with classical MetS features. Hepcidin levels increased significantly and linearly with increasing number of MetS features, paralleling the trend of serum ferritin. In multivariate models adjusted for relevant variables including age, C-Reactive Protein, and the HFE C282Y mutation, ferritin was the only significant independent predictor of hepcidin in males, while in females MetS was also independently associated with hepcidin. Overall, these data indicate that the fundamental iron regulatory feedback is preserved in MetS, i.e. that hepcidin tends to progressively increase in response to the increase of iron stores. Due to recently discovered pleiotropic effects of hepcidin, this may worsen insulin resistance and contribute to the cardiovascular complications of MetS.

Serum levels of the hepcidin-20 isoform in a large general population: the Val Borbera study.

Hepcidin, a 25 amino-acid liver hormone, has recently emerged as the key regulator of iron homeostasis. Proteomic studies in limited number of subjects have shown that biological fluids can also contain truncated isoforms, whose role remains to be elucidated. We report, for the first time, data about serum levels of the hepcidin-20 isoform (hep-20) in a general population, taking advantage of the Val Borbera (VB) study where hepcidin-25 (hep-25) was measured by SELDI-TOF-MS. Detectable amount of hep-20 were found in sera from 854 out of 1577 subjects (54.2%), and its levels were about 14% of hep-25 levels. A small fraction of subjects (n=30, 1.9%) had detectable hep-20 but undetectable hep-25. In multivariate regression models, significant predictors of hep-20 were hep-25 and age in males, and hep-25, age, serum ferritin and body mass index in females. Of note, the hep-25:hep-20 ratio was not constant in the VB population, but increased progressively with increasing ferritin levels. This is not consistent with the simplistic view of hep-20 as a mere catabolic byproduct of hep-25. Although a possible active regulation of hep-20 production needs further confirmation, our results may also have implications for immunoassays for serum hepcidin based on antibodies lacking specificity for hep-25. This article is part of a Special Issue entitled: Integrated omics.

Hepcidin levels and their determinants in different types of myelodysplastic syndromes.

Iron overload may represent an additional clinical problem in patients with Myelodysplastic Syndromes (MDS), with recent data suggesting prognostic implications. Beyond red blood cells transfusions, dysregulation of hepcidin, the key iron hormone, may play a role, but studies until now have been hampered by technical problems. Using a recently validated assay, we measured serum hepcidin in 113 patients with different MDS subtypes. Mean hepcidin levels were consistently heterogeneous across different MDS subtypes, with the lowest levels in refractory anemia with ringed sideroblasts (RARS, 1.43 nM) and the highest in refractory anemia with excess blasts (RAEB, 11.3 nM) or in chronic myelomonocytic leukemia (CMML, 10.04 nM) (P = 0.003 by ANOVA). MDS subtypes remained significant predictors of hepcidin in multivariate analyses adjusted for ferritin and transfusion history. Consistently with current knowledge on hepcidin action/regulation, RARS patients had the highest levels of toxic non-transferrin-bound-iron, while RAEB and CMML patients had substantial elevation of C-Reactive Protein as compared to other MDS subtypes, and showed lost of homeostatic regulation by iron. Growth differentiation factor 15 did not appear as a primary hepcidin regulator in this series. If confirmed, these results may help to calibrate future treatments with chelating agents and/or hepcidin modulators in MDS patients.

TMPRSS6 rs855791 modulates hepcidin transcription in vitro and serum hepcidin levels in normal individuals.

The iron hormone hepcidin is inhibited by matriptase-2 (MT2), a liver serine protease encoded by the TMPRSS6 gene. Cleaving the bone morphogenetic protein (BMP) coreceptor hemojuvelin (HJV), MT2 impairs the BMP/son of mothers against decapentaplegic homologs (SMAD) signaling pathway, down-regulates hepcidin, and facilitates iron absorption. TMPRSS6 inactivation causes iron-deficiency anemia refractory to iron administration both in humans and mice. Genome-wide association studies have shown that the SNP rs855791, which causes the MT2 V736A amino acid substitution, is associated with variations of serum iron, transferrin saturation, hemoglobin, and erythrocyte traits. In the present study, we show that, in vitro, MT2 736(A) inhibits hepcidin more efficiently than 736(V). Moreover, in a genotyped population, after exclusion of samples with iron deficiency and inflammation, hepcidin, hepcidin/transferrin saturation, and hepcidin/ferritin ratios were significantly lower and iron parameters were consistently higher in homozygotes 736(A) than in 736(V). Our results indicate that rs855791 is a TMPRSS6 functional variant and strengthen the idea that even a partial inability to modulate hepcidin influences iron parameters and, indirectly, erythropoiesis.

Association of HFE and TMPRSS6 genetic variants with iron and erythrocyte parameters is only in part dependent on serum hepcidin concentrations.

BACKGROUND: Hepcidin is the main regulator of iron homeostasis: inappropriate production of hepcidin results in iron overload or iron deficiency and anaemia. AIMS: To study variation of serum hepcidin concentration in a normal population. RESULTS: Hepcidin showed age and sex dependent variations that correlated with ferritin but not with serum iron and transferrin saturation. The size of the study population was underpowered to find genome wide significant associations with hepcidin concentrations but it allowed to show that association with serum iron, transferrin saturation and erythrocyte traits of common DNA variants in HFE (rs1800562) and TMPRSS6 (rs855791) genes is not exclusively dependent on hepcidin values. When multiple interactions between environmental factors, the iron parameters and hepcidin were taken into account, the HFE variant, and to lesser extent the TMPRSS6 variant, were associated with ferritin and with hepcidin normalised to ferritin (the hepcidin/ferritin ratio). CONCLUSIONS: The results suggest a mutual control of serum hepcidin and ferritin concentrations, a mechanism relevant to the pathophysiology of HFE haemochromatosis, and demonstrate that the HFE rs1800562 C282Y variant exerts a direct pleiotropic effect on the iron parameters, in part independent of hepcidin.

Proteomic analysis of Arabidopsis halleri shoots in response to the heavy metals cadmium and zinc and rhizosphere microorganisms.

Arabidopsis halleri has the rare ability to colonize heavy metal-polluted sites and is an emerging model for research on adaptation and metal hyperaccumulation. The aim of this study was to analyze the effect of plant-microbe interaction on the accumulation of cadmium (Cd) and zinc (Zn) in shoots of an ecotype of A. halleri grown in heavy metal-contaminated soil and to compare the shoot proteome of plants grown solely in the presence of Cd and Zn or in the presence of these two metals and the autochthonous soil rhizosphere-derived microorganisms. The results of this analysis emphasized the role of plant-microbe interaction in shoot metal accumulation. Differences in protein expression pattern, identified by a proteomic approach involving 2-DE and MS, indicated a general upregulation of photosynthesis-related proteins in plants exposed to metals and to metals plus microorganisms, suggesting that metal accumulation in shoots is an energy-demanding process. The analysis also showed that proteins involved in plant defense mechanisms were downregulated indicating that heavy metals accumulation in leaves supplies a protection system and highlights a cross-talk between heavy metal signaling and defense signaling.