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1.  Iron Supplementation in Suckling Piglets: How to Correct Iron Deficiency Anemia without Affecting Plasma Hepcidin Levels 
PLoS ONE  2013;8(5):e64022.
The aim of the study was to establish an optimized protocol of iron dextran administration to pig neonates, which better meets the iron demand for erythropoiesis. Here, we monitored development of red blood cell indices, plasma iron parameters during a 28-day period after birth (till the weaning), following intramuscular administration of different concentrations of iron dextran to suckling piglets. To better assess the iron status we developed a novel mass spectrometry assay to quantify pig plasma levels of the iron-regulatory peptide hormone hepcidin-25. This hormone is predominantly secreted by the liver and acts as a negative regulator of iron absorption and reutilization. The routinely used protocol with high amount of iron resulted in the recovery of piglets from iron deficiency but also in strongly elevated plasma hepcidin-25 levels. A similar protocol with reduced amounts of iron improved hematological status of piglets to the same level while plasma hepcidin-25 levels remained low. These data show that plasma hepcidin-25 levels can guide optimal dosing of iron treatment and pave the way for mixed supplementation of piglets starting with intramuscular injection of iron dextran followed by dietary supplementation, which could be efficient under condition of very low plasma hepcidin-25 level.
doi:10.1371/journal.pone.0064022
PMCID: PMC3667775  PMID: 23737963
2.  Iron Homeostasis in Mother and Child during Placental Malaria Infection 
In malaria-endemic areas, iron deficiency and placental Plasmodium falciparum infection commonly coexist. In primigravidae and their newborns, hepcidin and other iron parameters were evaluated in groups and classified according to placental P. falciparum and maternal anemia status. Mothers had relatively high hepcidin levels considering their low iron status. In cord blood, levels of hepcidin, hemoglobin, and other iron parameters were also similar for groups. We conclude that maternal hepcidin is not significantly altered as a function of placental infection and/or anemia. Importantly, fetal hemoglobin and iron status were also unaffected, regardless of the presence of placental infection or maternal anemia.
doi:10.4269/ajtmh.2011.10-0250
PMCID: PMC3005511  PMID: 21212218
3.  Inter-ethnic differences in genetic variants within the transmembrane protease, serine 6 (TMPRSS6) gene associated with iron status indicators: a systematic review with meta-analyses 
Genes & Nutrition  2014;10(1):442.
Transmembrane protease, serine 6 (TMPRSS6), is likely to be involved in iron metabolism through its pleiotropic effect on hepcidin concentrations. Recently, genome-wide association studies have identified common variants in the TMPRSS6 gene to be linked to anaemia and low iron status. To get a more precise evaluation of identified TMPRSS6 single nucleotide polymorphism associations with iron status in cohorts of differing continental ancestry, we conducted a systematic review with meta-analyses. We searched the literature using HuGE Navigator, Pubmed and Scopus databases for primarily genome-wide association studies using TMPRSS6 as a free term. Fixed-effects meta-analysis was used to obtain summary estimates of associations. Eleven studies comprised Caucasian populations, four included an Asian population and one study included an African-American population. Differences in minor allele frequencies of 8 TMPRSS6 SNPs (rs855791, rs4820268, rs2111833, rs1421312, rs228921, rs228918, rs228919 and rs575620) across ethnic groups were observed, with the MAF of rs855791 significantly higher in Asian populations than in Caucasians (0.55 vs 0.42, P < 0.0001). In the meta-analysis, the A allele of rs855791 was associated with lower Hb and ferritin concentrations in all populations. This allele was also associated with increased serum transferrin receptor and transferrin concentrations. We observed similar associations for the G allele in rs4820268. Clear disparities in associations were found for the African-American population, although not statistically significant. Associations between TMPRSS6 SNPs and anaemia are consistent across Caucasian and Asian populations. This study highlights the need to conduct studies in African populations where iron deficiency is of utmost public health significance.
doi:10.1007/s12263-014-0442-2
PMCID: PMC4240767  PMID: 25416640
Systematic review; Meta-analyses; TMPRSS6; Iron status
4.  Iron-Induced Virulence of Salmonella enterica Serovar Typhimurium at the Intestinal Epithelial Interface Can Be Suppressed by Carvacrol 
Oral iron therapy can increase the abundance of bacterial pathogens, e.g., Salmonella spp., in the large intestine of African children. Carvacrol is a natural compound with antimicrobial activity against various intestinal bacterial pathogens, among which is the highly prevalent Salmonella enterica serovar Typhimurium. This study aimed to explore a presumed interaction between carvacrol and bacterial iron handling and to assess the potential of carvacrol in preventing the increase of bacterial pathogenicity during high iron availability. S. Typhimurium was cultured with increasing concentrations of iron and carvacrol to study the effects of these combined interventions on growth, adhesion to intestinal epithelial cells, and iron uptake/influx in both bacterial and epithelial cells. In addition, the ability of carvacrol to remove iron from the high-affinity ligand transferrin and an Fe-dye complex was examined. Carvacrol retarded growth of S. Typhimurium at all iron conditions. Furthermore, iron-induced epithelial adhesion was effectively reduced by carvacrol at high iron concentrations. The reduction of growth and virulence by carvacrol was not paralleled by a change in iron uptake or influx into S. Typhimurium. In contrast, bioavailability of iron for epithelial cells was moderately decreased under these conditions. Further, carvacrol was shown to lack the properties of an iron binding molecule; however, it was able to weaken iron-ligand interactions by which it may possibly interfere with bacterial virulence. In conclusion, our in vitro data suggest that carvacrol has the potential to serve as a novel dietary supplement to prevent pathogenic overgrowth and colonization in the large intestine during oral iron therapy.
doi:10.1128/AAC.02060-13
PMCID: PMC3957860  PMID: 24379194
5.  A seven day running training period increases basal urinary hepcidin levels as compared to cycling 
Background
This investigation compared the effects of an extended period of weight-bearing (running) vs. non-weight-bearing (cycling) exercise on hepcidin production and its implications for iron status.
Methods
Ten active males performed two separate exercise training blocks with either running (RTB) or cycling (CTB) as the exercise mode. Each block consisted of five training sessions (Day 1, 2, 4, 5, 6) performed over a seven day period that were matched for exercise intensity. Basal venous blood samples were obtained on Day 1 (D1), and on Recovery Days 3 (R3) and 7 (R7) to assess iron status, while basal and 3 h post-exercise urinary hepcidin levels were measured on D1, D2, D6, as well as R3 and R7 (basal levels only) for each condition.
Results
Basal urinary hepcidin levels were significantly elevated (p ≤ 0.05) at D2, R3 and R7 as compared to D1 in RTB. Furthermore, 3 h post-exercise urinary hepcidin levels on D1 were also significantly higher in RTB compared to CTB (p ≤ 0.05). In CTB, urinary hepcidin levels were not statistically different on D1 as compared to R7. Iron parameters were not significantly different at D1 compared to R3 and R7 during both conditions.
Conclusions
These results suggest that basal hepcidin levels may increase over the course of an extended training program, especially if a weight-bearing exercise modality is undertaken. However, despite any variations in hepcidin production, serum iron parameters in both RTB and CTB were unaffected, possibly due to the short duration of each training block. In comparing running to cycling, non-weight-bearing activity may require more training sessions, or sessions of extended duration, before any significant changes in basal hepcidin levels appear. Chronic elevations in hepcidin levels may help to explain the high incidence of iron deficiency in athletes.
doi:10.1186/1550-2783-11-14
PMCID: PMC3991905  PMID: 24716892
Iron deficiency; Weight-bearing exercise; Non-weight-bearing exercise; Cytokines
6.  Iron Status and the Acute Post-Exercise Hepcidin Response in Athletes 
PLoS ONE  2014;9(3):e93002.
This study explored the relationship between serum ferritin and hepcidin in athletes. Baseline serum ferritin levels of 54 athletes from the control trial of five investigations conducted in our laboratory were considered; athletes were grouped according to values <30 μg/L (SF<30), 30–50 μg/L (SF30–50), 50–100 μg/L (SF50–100), or >100 μg/L (SF>100). Data pooling resulted in each athlete completing one of five running sessions: (1) 8×3 min at 85% vVO2peak; (2) 5×4 min at 90% vVO2peak; (3) 90 min continuous at 75% vVO2peak; (4) 40 min continuous at 75% vVO2peak; (5) 40 min continuous at 65% vVO2peak. Athletes from each running session were represented amongst all four groups; hence, the mean exercise duration and intensity were not different (p>0.05). Venous blood samples were collected pre-, post- and 3 h post-exercise, and were analysed for serum ferritin, iron, interleukin-6 (IL-6) and hepcidin-25. Baseline and post-exercise serum ferritin levels were different between groups (p<0.05). There were no group differences for pre- or post-exercise serum iron or IL-6 (p>0.05). Post-exercise IL-6 was significantly elevated compared to baseline within each group (p<0.05). Pre- and 3 h post-exercise hepcidin-25 was sequentially greater as the groups baseline serum ferritin levels increased (p<0.05). However, post-exercise hepcidin levels were only significantly elevated in three groups (SF30–50, SF50–100, and SF>100; p<0.05). An athlete's iron stores may dictate the baseline hepcidin levels and the magnitude of post-exercise hepcidin response. Low iron stores suppressed post-exercise hepcidin, seemingly overriding any inflammatory-driven increases.
doi:10.1371/journal.pone.0093002
PMCID: PMC3965532  PMID: 24667393
7.  Left Ventricular Mass in Dialysis Patients, Determinants and Relation with Outcome. Results from the COnvective TRansport STudy (CONTRAST) 
PLoS ONE  2014;9(2):e84587.
Background and Objectives
Left ventricular mass (LVM) is known to be related to overall and cardiovascular mortality in end stage kidney disease (ESKD) patients. The aims of the present study are 1) to determine whether LVM is associated with mortality and various cardiovascular events and 2) to identify determinants of LVM including biomarkers of inflammation and fibrosis.
Design, Setting, Participants, & Measurements
Analysis was performed with data of 327 ESKD patients, a subset from the CONvective TRAnsport STudy (CONTRAST). Echocardiography was performed at baseline. Cox regression analysis was used to assess the relation of LVM tertiles with clinical events. Multivariable linear regression models were used to identify factors associated with LVM.
Results
Median age was 65 (IQR: 54–73) years, 203 (61%) were male and median LVM was 227 (IQR: 183–279) grams. The risk of all-cause mortality (hazard ratio (HR) = 1.73, 95% CI: 1.11–2.99), cardiovascular death (HR = 3.66, 95% CI: 1.35–10.05) and sudden death (HR = 13.06; 95% CI: 6.60–107) was increased in the highest tertile (>260grams) of LVM. In the multivariable analysis positive relations with LVM were found for male gender (B = 38.8±10.3), residual renal function (B = 17.9±8.0), phosphate binder therapy (B = 16.9±8.5), and an inverse relation for a previous kidney transplantation (B = −41.1±7.6) and albumin (B = −2.9±1.1). Interleukin-6 (Il-6), high-sensitivity C-reactive protein (hsCRP), hepcidin-25 and connective tissue growth factor (CTGF) were not related to LVM.
Conclusion
We confirm the relation between a high LVM and outcome and expand the evidence for increased risk of sudden death. No relationship was found between LVM and markers of inflammation and fibrosis.
Trial Registration
Controlled-Trials.com ISRCTN38365125
doi:10.1371/journal.pone.0084587
PMCID: PMC3914777  PMID: 24505249
8.  Low Hepcidin Levels in Severely Anemic Malawian Children with High Incidence of Infectious Diseases and Bone Marrow Iron Deficiency 
PLoS ONE  2013;8(12):e78964.
Introduction
A reliable diagnostic biomarker of iron status is required for severely anemic children living in malarious areas because presumptive treatment with iron may increase their infection risk if they are not iron deficient. Current biomarkers are limited because they are altered by host inflammation. In this study hepcidin concentrations were assessed in severely anemic children living in a highly malarious area of Malawi and evaluated against bone marrow iron in order to determine the usefulness of hepcidin as a point of care test.
Methods
207 severely anemic children were assessed for levels of hepcidin, ferritin, serum transferrin receptor, erythropoietin, hematological indices, C-reactive protein, interleukin-6, malaria parasites and HIV infection. Deficiency of bone marrow iron stores was graded and erythroblast iron incorporation estimated. Interaction of covariates was assessed by structural-equation-modeling.
Results and Conclusion
Hepcidin was a poor predictor of bone marrow iron deficiency (sensitivity 66.7%; specificity 48.5%), and of iron incorporation (sensitivity 54.2%; specificity 61.8%), and therefore would have limitations as a point of care test in this category of children. As upregulation of hepcidin by inflammation and iron status was blunted by erythropoietin in this population, enhanced iron absorption through the low hepcidin values may increase infection risk. Current recommendations to treat all severely anemic children living in malarious areas with iron should therefore be reconsidered.
doi:10.1371/journal.pone.0078964
PMCID: PMC3855157  PMID: 24339866
9.  Improved Mass Spectrometry Assay For Plasma Hepcidin: Detection and Characterization of a Novel Hepcidin Isoform 
PLoS ONE  2013;8(10):e75518.
Mass spectrometry (MS)-based assays for the quantification of the iron regulatory hormone hepcidin are pivotal to discriminate between the bioactive 25-amino acid form that can effectively block the sole iron transporter ferroportin and other naturally occurring smaller isoforms without a known role in iron metabolism. Here we describe the design, validation and use of a novel stable hepcidin-25+40 isotope as internal standard for quantification. Importantly, the relative large mass shift of 40 Da makes this isotope also suitable for easy-to-use medium resolution linear time-of-flight (TOF) platforms. As expected, implementation of hepcidin-25+40 as internal standard in our weak cation exchange (WCX) TOF MS method yielded very low inter/intra run coefficients of variation. Surprisingly, however, in samples from kidney disease patients, we detected a novel peak (m/z 2673.9) with low intensity that could be identified as hepcidin-24 and had previously remained unnoticed due to peak interference with the formerly used internal standard. Using a cell-based bioassay it was shown that synthetic hepcidin-24 was, like the -22 and -20 isoforms, a significantly less potent inducer of ferroportin degradation than hepcidin-25. During prolonged storage of plasma at room temperature, we observed that a decrease in plasma hepcidin-25 was paralleled by an increase in the levels of the hepcidin-24, -22 and -20 isoforms. This provides first evidence that all determinants for the conversion of hepcidin-25 to smaller inactive isoforms are present in the circulation, which may contribute to the functional suppression of hepcidin-25, that is significantly elevated in patients with renal impairment. The present update of our hepcidin TOF MS assay together with improved insights in the source and preparation of the internal standard, and sample stability will further improve our understanding of circulating hepcidin and pave the way towards further optimization and standardization of plasma hepcidin assays.
doi:10.1371/journal.pone.0075518
PMCID: PMC3790851  PMID: 24124495
10.  CSK regulatory polymorphism is associated with systemic lupus erythematosus and influences B cell signaling and activation 
Nature genetics  2012;44(11):1227-1230.
C-src tyrosine kinase, Csk, physically interacts with the intracellular phosphatase Lyp (PTPN22) and can modify the activation state of downstream Src kinases, such as Lyn, in lymphocytes. We identified an association of Csk with systemic lupus erythematosus (SLE) and refined its location to an intronic polymorphism rs34933034 (OR 1.32, p = 1.04 × 10−9). The risk allele is associated with increased CSK expression and augments inhibitory phosphorylation of Lyn. In carriers of the risk allele, B cell receptor (BCR)-mediated activation of mature B cells, as well as plasma IgM, are increased. Moreover, the fraction of transitional B cells is doubled in the cord blood of carriers of the risk allele compared to non-risk haplotypes due to an expansion of the late transitional cells, a stage targeted by selection mechanisms. This suggests that the Lyp-Csk complex increases susceptibility to lupus at multiple maturation and activation points of B cells.
doi:10.1038/ng.2439
PMCID: PMC3715052  PMID: 23042117
11.  Tubular reabsorption and local production of urine hepcidin-25 
BMC Nephrology  2013;14:70.
Background
Hepcidin is a central regulator of iron metabolism. Serum hepcidin levels are increased in patients with renal insufficiency, which may contribute to anemia. Urine hepcidin was found to be increased in some patients after cardiac surgery, and these patients were less likely to develop acute kidney injury. It has been suggested that urine hepcidin may protect by attenuating heme-mediated injury, but processes involved in urine hepcidin excretion are unknown.
Methods
To assess the role of tubular reabsorption we compared fractional excretion (FE) of hepcidin-25 with FE of β2-microglobulin (β2m) in 30 patients with various degrees of tubular impairment due to chronic renal disease. To prove that hepcidin is reabsorbed by the tubules in a megalin-dependent manner, we measured urine hepcidin-1 in wild-type and kidney specific megalin-deficient mice. Lastly, we evaluated FE of hepcidin-25 and β2m in 19 patients who underwent cardiopulmonary bypass surgery. Hepcidin was measured by a mass spectrometry assay (MS), whereas β2m was measured by ELISA.
Results
In patients with chronic renal disease, FE of hepcidin-25 was strongly correlated with FE of β2m (r = 0.93, P <0.01). In megalin-deficient mice, urine hepcidin-1 was 7-fold increased compared to wild-type mice (p < 0.01) indicating that proximal tubular reabsorption occurs in a megalin- dependent manner. Following cardiac surgery, FE of hepcidin-25 increased despite a decline in FE of β2m, potentially indicating local production at 12–24 hours.
Conclusions
Hepcidin-25 is reabsorbed by the renal tubules and increased urine hepcidin-25 levels may reflect a reduction in tubular uptake. Uncoupling of FE of hepcidin-25 and β2m in cardiac surgery patients suggests local production.
doi:10.1186/1471-2369-14-70
PMCID: PMC3623618  PMID: 23531037
AKI; β2-microglobulin; Hepcidin; Megalin; Kidney tubules
12.  Common sequence variants on 2p15 and Xp11.22 confer susceptibility to prostate cancer 
Gudmundsson, Julius | Sulem, Patrick | Rafnar, Thorunn | Bergthorsson, Jon T | Manolescu, Andrei | Gudbjartsson, Daniel | Agnarsson, Bjarni A | Sigurdsson, Asgeir | Benediktsdottir, Kristrun R | Blondal, Thorarinn | Jakobsdottir, Margret | Stacey, Simon N | Kostic, Jelena | Kristinsson, Kari T | Birgisdottir, Birgitta | Ghosh, Shyamali | Magnusdottir, Droplaug N | Thorlacius, Steinunn | Thorleifsson, Gudmar | Zheng, S Lilly | Sun, Jielin | Chang, Bao-Li | Elmore, J Bradford | Breyer, Joan P | McReynolds, Kate M | Bradley, Kevin M | Yaspan, Brian L | Wiklund, Fredrik | Stattin, Par | Lindström, Sara | Adami, Hans-Olov | McDonnell, Shannon K | Schaid, Daniel J | Cunningham, Julie M | Wang, Liang | Cerhan, James R | St Sauver, Jennifer L | Isaacs, Sara D | Wiley, Kathleen E | Partin, Alan W | Walsh, Patrick C | Polo, Sonia | Ruiz-Echarri, Manuel | Navarrete, Sebastian | Fuertes, Fernando | Saez, Berta | Godino, Javier | Weijerman, Philip C | Swinkels, Dorine W | Aben, Katja K | Witjes, J Alfred | Suarez, Brian K | Helfand, Brian T | Frigge, Michael L | Kristjansson, Kristleifur | Ober, Carole | Jonsson, Eirikur | Einarsson, Gudmundur V | Xu, Jianfeng | Gronberg, Henrik | Smith, Jeffrey R | Thibodeau, Stephen N | Isaacs, William B | Catalona, William J | Mayordomo, Jose I | Kiemeney, Lambertus A | Barkardottir, Rosa B | Gulcher, Jeffrey R | Thorsteinsdottir, Unnur | Kong, Augustine | Stefansson, Kari
Nature genetics  2008;40(3):281-283.
We conducted a genome-wide SNP association study on prostate cancer on over 23,000 Icelanders, followed by a replication study including over 15,500 individuals from Europe and the United States. Two newly identified variants were shown to be associated with prostate cancer: rs5945572 on Xp11.22 and rs721048 on 2p15 (odds ratios (OR) = 1.23 and 1.15; P = 3.9 × 10−13 and 7.7 × 10−9, respectively). The 2p15 variant shows a significantly stronger association with more aggressive, rather than less aggressive, forms of the disease.
doi:10.1038/ng.89
PMCID: PMC3598012  PMID: 18264098
13.  Genome-wide meta-analysis of common variant differences between men and women 
Boraska, Vesna | Jerončić, Ana | Colonna, Vincenza | Southam, Lorraine | Nyholt, Dale R. | William Rayner, Nigel | Perry, John R.B. | Toniolo, Daniela | Albrecht, Eva | Ang, Wei | Bandinelli, Stefania | Barbalic, Maja | Barroso, Inês | Beckmann, Jacques S. | Biffar, Reiner | Boomsma, Dorret | Campbell, Harry | Corre, Tanguy | Erdmann, Jeanette | Esko, Tõnu | Fischer, Krista | Franceschini, Nora | Frayling, Timothy M. | Girotto, Giorgia | Gonzalez, Juan R. | Harris, Tamara B. | Heath, Andrew C. | Heid, Iris M. | Hoffmann, Wolfgang | Hofman, Albert | Horikoshi, Momoko | Hua Zhao, Jing | Jackson, Anne U. | Hottenga, Jouke-Jan | Jula, Antti | Kähönen, Mika | Khaw, Kay-Tee | Kiemeney, Lambertus A. | Klopp, Norman | Kutalik, Zoltán | Lagou, Vasiliki | Launer, Lenore J. | Lehtimäki, Terho | Lemire, Mathieu | Lokki, Marja-Liisa | Loley, Christina | Luan, Jian'an | Mangino, Massimo | Mateo Leach, Irene | Medland, Sarah E. | Mihailov, Evelin | Montgomery, Grant W. | Navis, Gerjan | Newnham, John | Nieminen, Markku S. | Palotie, Aarno | Panoutsopoulou, Kalliope | Peters, Annette | Pirastu, Nicola | Polašek, Ozren | Rehnström, Karola | Ripatti, Samuli | Ritchie, Graham R.S. | Rivadeneira, Fernando | Robino, Antonietta | Samani, Nilesh J. | Shin, So-Youn | Sinisalo, Juha | Smit, Johannes H. | Soranzo, Nicole | Stolk, Lisette | Swinkels, Dorine W. | Tanaka, Toshiko | Teumer, Alexander | Tönjes, Anke | Traglia, Michela | Tuomilehto, Jaakko | Valsesia, Armand | van Gilst, Wiek H. | van Meurs, Joyce B.J. | Smith, Albert Vernon | Viikari, Jorma | Vink, Jacqueline M. | Waeber, Gerard | Warrington, Nicole M. | Widen, Elisabeth | Willemsen, Gonneke | Wright, Alan F. | Zanke, Brent W. | Zgaga, Lina | Boehnke, Michael | d'Adamo, Adamo Pio | de Geus, Eco | Demerath, Ellen W. | den Heijer, Martin | Eriksson, Johan G. | Ferrucci, Luigi | Gieger, Christian | Gudnason, Vilmundur | Hayward, Caroline | Hengstenberg, Christian | Hudson, Thomas J. | Järvelin, Marjo-Riitta | Kogevinas, Manolis | Loos, Ruth J.F. | Martin, Nicholas G. | Metspalu, Andres | Pennell, Craig E. | Penninx, Brenda W. | Perola, Markus | Raitakari, Olli | Salomaa, Veikko | Schreiber, Stefan | Schunkert, Heribert | Spector, Tim D. | Stumvoll, Michael | Uitterlinden, André G. | Ulivi, Sheila | van der Harst, Pim | Vollenweider, Peter | Völzke, Henry | Wareham, Nicholas J. | Wichmann, H.-Erich | Wilson, James F. | Rudan, Igor | Xue, Yali | Zeggini, Eleftheria
Human Molecular Genetics  2012;21(21):4805-4815.
The male-to-female sex ratio at birth is constant across world populations with an average of 1.06 (106 male to 100 female live births) for populations of European descent. The sex ratio is considered to be affected by numerous biological and environmental factors and to have a heritable component. The aim of this study was to investigate the presence of common allele modest effects at autosomal and chromosome X variants that could explain the observed sex ratio at birth. We conducted a large-scale genome-wide association scan (GWAS) meta-analysis across 51 studies, comprising overall 114 863 individuals (61 094 women and 53 769 men) of European ancestry and 2 623 828 common (minor allele frequency >0.05) single-nucleotide polymorphisms (SNPs). Allele frequencies were compared between men and women for directly-typed and imputed variants within each study. Forward-time simulations for unlinked, neutral, autosomal, common loci were performed under the demographic model for European populations with a fixed sex ratio and a random mating scheme to assess the probability of detecting significant allele frequency differences. We do not detect any genome-wide significant (P < 5 × 10−8) common SNP differences between men and women in this well-powered meta-analysis. The simulated data provided results entirely consistent with these findings. This large-scale investigation across ∼115 000 individuals shows no detectable contribution from common genetic variants to the observed skew in the sex ratio. The absence of sex-specific differences is useful in guiding genetic association study design, for example when using mixed controls for sex-biased traits.
doi:10.1093/hmg/dds304
PMCID: PMC3471397  PMID: 22843499
14.  Hepcidin-25 in Chronic Hemodialysis Patients Is Related to Residual Kidney Function and Not to Treatment with Erythropoiesis Stimulating Agents 
PLoS ONE  2012;7(7):e39783.
Hepcidin-25, the bioactive form of hepcidin, is a key regulator of iron homeostasis as it induces internalization and degradation of ferroportin, a cellular iron exporter on enterocytes, macrophages and hepatocytes. Hepcidin levels are increased in chronic hemodialysis (HD) patients, but as of yet, limited information on factors associated with hepcidin-25 in these patients is available. In the current cross-sectional study, potential patient-, laboratory- and treatment-related determinants of serum hepcidin-20 and -25, were assessed in a large cohort of stable, prevalent HD patients. Baseline data from 405 patients (62% male; age 63.7±13.9 [mean SD]) enrolled in the CONvective TRAnsport STudy (CONTRAST; NCT00205556) were studied. Predialysis hepcidin concentrations were measured centrally with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Patient-, laboratory- and treatment related characteristics were entered in a backward multivariable linear regression model. Hepcidin-25 levels were independently and positively associated with ferritin (p<0.001), hsCRP (p<0.001) and the presence of diabetes (p = 0.02) and inversely with the estimated glomerular filtration rate (p = 0.01), absolute reticulocyte count (p = 0.02) and soluble transferrin receptor (p<0.001). Men had lower hepcidin-25 levels as compared to women (p = 0.03). Hepcidin-25 was not associated with the maintenance dose of erythropoiesis stimulating agents (ESA) or iron therapy. In conclusion, in the currently studied cohort of chronic HD patients, hepcidin-25 was a marker for iron stores and erythropoiesis and was associated with inflammation. Furthermore, hepcidin-25 levels were influenced by residual kidney function. Hepcidin-25 did not reflect ESA or iron dose in chronic stable HD patients on maintenance therapy. These results suggest that hepcidin is involved in the pathophysiological pathway of renal anemia and iron availability in these patients, but challenges its function as a clinical parameter for ESA resistance.
doi:10.1371/journal.pone.0039783
PMCID: PMC3396629  PMID: 22808058
15.  Surface-Affinity Profiling To Identify Host-Pathogen Interactions ▿ †  
Infection and Immunity  2011;79(12):4777-4783.
Proteolytic treatment of intact bacterial cells has proven to be a convenient approach for the identification of surface-exposed proteins. This class of proteins directly interacts with the outside world, for instance, during adherence to human epithelial cells. Here, we aimed to identify host receptor proteins by introducing a preincubation step in which bacterial cells were first allowed to capture human proteins from epithelial cell lysates. Using Streptococcus gallolyticus as a model bacterium, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of proteolytically released peptides yielded the identification of a selective number of human epithelial proteins that were retained by the bacterial surface. Of these potential receptors for bacterial interference, (cyto)keratin-8 (CK8) was verified as the most significant hit, and its surface localization was investigated by subcellular fractionation and confocal microscopy. Interestingly, bacterial enolase could be assigned as an interaction partner of CK8 by MS/MS analysis of cross-linked protein complexes and complementary immunoblotting experiments. As surface-exposed enolase has a proposed role in epithelial adherence of several Gram-positive pathogens, its interaction with CK8 seems to point toward a more general virulence mechanism. In conclusion, our study shows that surface-affinity profiling is a valuable tool to identify novel adhesin-receptor pairs, which advocates its application in other hybrid biological systems.
doi:10.1128/IAI.05572-11
PMCID: PMC3232642  PMID: 21947776
16.  Candidate Gene Sequencing of SLC11A2 and TMPRSS6 in a Family with Severe Anaemia: Common SNPs, Rare Haplotypes, No Causative Mutation 
PLoS ONE  2012;7(4):e35015.
Background
Iron-refractory iron deficiency anaemia (IRIDA) is a rare disorder which was linked to mutations in two genes (SLC11A2 and TMPRSS6). Common polymorphisms within these genes were associated with serum iron levels. We identified a family of Serbian origin with asymptomatic non-consanguineous parents with three of four children presenting with IRIDA not responding to oral but to intravenous iron supplementation. After excluding all known causes responsible for iron deficiency anaemia we searched for mutations in SLC11A2 and TMPRSS6 that could explain the severe anaemia in these children.
Methodology/Results
We sequenced the exons and exon–intron boundaries of SLC11A2 and TMPRSS6 in all six family members. Thereby, we found seven known and fairly common SNPs, but no new mutation. We then genotyped these seven SNPs in the population-based SAPHIR study (n = 1,726) and performed genetic association analysis on iron and ferritin levels. Only two SNPs, which were top-hits from recent GWAS on iron and ferritin, exhibited an effect on iron and ferritin levels in SAPHIR. Six SAPHIR participants carrying the same TMPRSS6 genotypes and haplotype-pairs as one anaemic son showed lower ferritin and iron levels than the average. One individual exhibiting the joint SLC11A2/TMPRSS6 profile of the anaemic son had iron and ferritin levels lying below the 5th percentile of the population's iron and ferritin level distribution. We then checked the genotype constellations in the Nijmegen Biomedical Study (n = 1,832), but the profile of the anaemic son did not occur in this population.
Conclusions
We cannot exclude a gene-gene interaction between SLC11A2 and TMPRSS6, but we can also not confirm it. As in this case candidate gene sequencing did not reveal causative rare mutations, the samples will be subjected to whole exome sequencing.
doi:10.1371/journal.pone.0035015
PMCID: PMC3324414  PMID: 22509377
17.  Iron Availability Increases the Pathogenic Potential of Salmonella Typhimurium and Other Enteric Pathogens at the Intestinal Epithelial Interface 
PLoS ONE  2012;7(1):e29968.
Recent trials have questioned the safety of untargeted oral iron supplementation in developing regions. Excess of luminal iron could select for enteric pathogens at the expense of beneficial commensals in the human gut microflora, thereby increasing the incidence of infectious diseases. The objective of the current study was to determine the effect of high iron availability on virulence traits of prevalent enteric pathogens at the host-microbe interface. A panel of enteric bacteria was cultured under iron-limiting conditions and in the presence of increasing concentrations of ferric citrate to assess the effect on bacterial growth, epithelial adhesion, invasion, translocation and epithelial damage in vitro. Translocation and epithelial integrity experiments were performed using a transwell system in which Caco-2 cells were allowed to differentiate to a tight epithelial monolayer mimicking the intestinal epithelial barrier. Growth of Salmonella typhimurium and other enteric pathogens was increased in response to iron. Adhesion of S. typhimurium to epithelial cells markedly increased when these bacteria were pre-incubated with increasing iron concentration (P = 0.0001), whereas this was not the case for the non-pathogenic Lactobacillus plantarum (P = 0.42). Cellular invasion and epithelial translocation of S. typhimurium followed the trend of increased adhesion. Epithelial damage was increased upon incubation with S. typhimurium or Citrobacter freundii that were pre-incubated under iron-rich conditions. In conclusion, our data fit with the consensus that oral iron supplementation is not without risk as iron could, in addition to inducing pathogenic overgrowth, also increase the virulence of prevalent enteric pathogens.
doi:10.1371/journal.pone.0029968
PMCID: PMC3260200  PMID: 22272265
18.  Hepcidin in anemia of chronic heart failure 
American journal of hematology  2011;86(1):107-109.
Anemia is a common finding among patients with chronic heart failure. Although co-morbidities, such as kidney failure, might contribute to the pathogenesis of anemia, many patients with heart failure do not have any other obvious etiology for their anemia. We investigated whether anemia in heart failure is associated with an elevation in hepcidin concentration.
We used time-of-flight mass spectrometry to measure hepcidin concentration in urine and serum samples of patients with heart failure and in control subjects. We found that the concentration of hepcidin was lower in urine samples of patients with heart failure compared to those of control subjects. Serum hepcidin was also reduced in heart failure but was not significantly lower than that in controls. There were no significant differences between hepcidin levels in patients with heart failure and anemia compared to patients with heart failure and normal hemoglobin. We concluded that hepcidin probably does not play a major role in pathogenesis of anemia in patients with chronic heart failure.
doi:10.1002/ajh.21902
PMCID: PMC3076004  PMID: 21080339
Anemias; Cytokines; Iron
19.  Intra-graft expression of genes involved in iron homeostasis predicts the development of operational tolerance in human liver transplantation 
Following organ transplantation, lifelong immunosuppressive therapy is required to prevent the host immune system from destroying the allograft. This can cause severe side effects and increased recipient morbidity and mortality. Complete cessation of immunosuppressive drugs has been successfully accomplished in selected transplant recipients, providing proof of principle that operational allograft tolerance is attainable in clinical transplantation. The intra-graft molecular pathways associated with successful drug withdrawal, however, are not well defined. In this study, we analyzed sequential blood and liver tissue samples collected from liver transplant recipients enrolled in a prospective multicenter immunosuppressive drug withdrawal clinical trial. Before initiation of drug withdrawal, operationally tolerant and non-tolerant recipients differed in the intra-graft expression of genes involved in the regulation of iron homeostasis. Furthermore, as compared with non-tolerant recipients, operationally tolerant patients exhibited higher serum levels of hepcidin and ferritin and increased hepatocyte iron deposition. Finally, liver tissue gene expression measurements accurately predicted the outcome of immunosuppressive withdrawal in an independent set of patients. These results point to a critical role for iron metabolism in the regulation of intra-graft alloimmune responses in humans and provide a set of biomarkers to conduct drug-weaning trials in liver transplantation.
doi:10.1172/JCI59411
PMCID: PMC3248302  PMID: 22156196
20.  Increased Exposure to Bacterial Antigen RpL7/L12 in Early Stage Colorectal Cancer Patients 
Cancer  2010;116(17):4014-4022.
Background
Intestinal bacteria have been implicated in colorectal cancer pathology for a long time and a large number of reports point to a close linkage between Streptococcus bovis biotype I (recently renamed Streptococcus gallolyticus) infections and tumors of the human colon. This work aims to investigate the humoral immune response to this bacterium during different stages of colorectal cancer.
Method
The presence of serum antibodies against S. bovis antigen RpL7/L12, previously assigned as a potential diagnostic antigen, was evaluated in Dutch (n=209) and American (n=112) populations using a newly developed ELISA assay.
Results
The analyses consistently showed that an immune response against this bacterial antigen was increased in polyp patients and stage I/II colorectal cancer patients as compared to asymptomatic individuals. This was not paralleled by increased antibody production to endotoxin, an intrinsic cell wall component of the majority of intestinal bacteria, which implicates that the humoral immune response against RpL7/L12 is not a general phenomenon induced by the loss of colonic barrier function. Notably, increased anti-RpL7/L12 levels were not or only mildly detected in late stage colorectal cancer patients having lymph node or distant metastasis.
Conclusion
These findings are indicative for an increased exposure to antigen RpL7/L12 during early stages of colon carcinogenesis and suggest that intestinal bacteria, such as S. bovis, constitute a risk factor for the progression of pre-malignant lesions into early stage carcinomas. Clearly, the current findings emphasize the necessity for further studies on the possible etiologic relationship between intestinal bacteria and human colorectal cancer.
doi:10.1002/cncr.25212
PMCID: PMC2930125  PMID: 20564125
Colorectal Cancer; Bacterial Infection; Humoral Immune Response; Intestinal Bacteria; Streptococcus bovis; Streptococcus gallolyticus; Carcinogenesis
21.  Mass Spectrometry Analysis of Hepcidin Peptides in Experimental Mouse Models 
PLoS ONE  2011;6(3):e16762.
The mouse is a valuable model for unravelling the role of hepcidin in iron homeostasis, however, such studies still report hepcidin mRNA levels as a surrogate marker for bioactive hepcidin in its pivotal function to block ferroportin-mediated iron transport. Here, we aimed to assess bioactive mouse Hepcidin-1 (Hep-1) and its paralogue Hepcidin-2 (Hep-2) at the peptide level. To this purpose, fourier transform ion cyclotron resonance (FTICR) and tandem-MS was used for hepcidin identification, after which a time-of-flight (TOF) MS-based methodology was exploited to routinely determine Hep-1 and -2 levels in mouse serum and urine. This method was biologically validated by hepcidin assessment in: i) 3 mouse strains (C57Bl/6; DBA/2 and BABL/c) upon stimulation with intravenous iron and LPS, ii) homozygous Hfe knock out, homozygous transferrin receptor 2 (Y245X) mutated mice and double affected mice, and iii) mice treated with a sublethal hepatotoxic dose of paracetamol. The results showed that detection of Hep-1 was restricted to serum, whereas Hep-2 and its presumed isoforms were predominantly present in urine. Elevations in serum Hep-1 and urine Hep-2 upon intravenous iron or LPS were only moderate and varied considerably between mouse strains. Serum Hep-1 was decreased in all three hemochromatosis models, being lowest in the double affected mice. Serum Hep-1 levels correlated with liver hepcidin-1 gene expression, while acute liver damage by paracetamol depleted Hep-1 from serum. Furthermore, serum Hep-1 appeared to be an excellent indicator of splenic iron accumulation. In conclusion, Hep-1 and Hep-2 peptide responses in experimental mouse agree with the known biology of hepcidin mRNA regulators, and their measurement can now be implemented in experimental mouse models to provide novel insights in post-transcriptional regulation, hepcidin function, and kinetics.
doi:10.1371/journal.pone.0016762
PMCID: PMC3050808  PMID: 21408141
22.  Inflammation-induced hepcidin-25 is associated with the development of anemia in septic patients: an observational study 
Critical Care  2011;15(1):R9.
Introduction
Anemia is a frequently encountered problem during inflammation. Hepcidin is an interleukin-6 (IL-6)-induced key modulator of inflammation-associated anemia. Human sepsis is a prototypical inflammatory syndrome, often complicated by the development of anemia. However, the association between inflammation, hepcidin release and anemia has not been demonstrated in this group of patients. Therefore, we explored the association between hepcidin and sepsis-associated anemia.
Methods
92 consecutive patients were enrolled after presentation on the emergency ward of a university hospital with sepsis, indicated by the presence of a proven or suspected infection and ≥ 2 extended systemic inflammatory response syndrome (SIRS) criteria. Blood was drawn at day 1, 2 and 3 after admission for the measurement of IL-6 and hepcidin-25. IL-6 levels were correlated with hepcidin concentrations. Hemoglobin levels and data of blood transfusions during 14 days after hospitalisation were retrieved and the rate of hemoglobin decrease was correlated to hepcidin levels.
Results
53 men and 39 women with a mean age of 53.3 ± 1.8 yrs were included. Hepcidin levels were highest at admission (median[IQR]): 17.9[10.1 to 28.4]nmol/l and decreased to normal levels in most patients within 3 days (9.5[3.4 to 17.9]nmol/l). Hepcidin levels increased with the number of extended SIRS criteria (P = 0.0005). Highest IL-6 levels were measured at admission (125.0[46.3 to 330.0]pg/ml) and log-transformed IL-6 levels significantly correlated with hepcidin levels at admission (r = 0.28, P = 0.015), day 2 (r = 0.51, P < 0.0001) and day 3 (r = 0.46, P < 0.0001). Twelve patients received one or more blood transfusions during the first 2 weeks of admission, not related to active bleeding. These patients had borderline significant higher hepcidin level at admission compared to non-transfused patients (26.9[17.2 to 53.9] vs 17.9[9.9 to 28.8]nmol/l, P = 0.052). IL-6 concentrations did not differ between both groups. Correlation analyses showed significant associations between hepcidin levels on day 2 and 3 and the rate of decrease in hemoglobin (Spearman's r ranging from -0.32, P = 0.03 to -0.37, P = 0.016, respectively).
Conclusions
These data suggest that hepcidin-25 may be an important modulator of anemia in septic patients with systemic inflammation.
doi:10.1186/cc9408
PMCID: PMC3222038  PMID: 21219610
23.  Surface-Exposed Histone-Like Protein A Modulates Adherence of Streptococcus gallolyticus to Colon Adenocarcinoma Cells ▿  
Infection and Immunity  2009;77(12):5519-5527.
Streptococcus gallolyticus (formerly known as Streptococcus bovis biotype I) is a low-grade opportunistic pathogen which is considered to be associated with colon cancer. It is thought that colon polyps or tumors are the main portal of entry for this bacterium and that heparan sulfate proteoglycans (HSPGs) at the colon tumor cell surface are involved in bacterial adherence during the first stages of infection. In this study, we have shown that the histone-like protein A (HlpA) of S. gallolyticus is a genuine anchorless bacterial surface protein that binds to lipoteichoic acid (LTA) of the gram-positive cell wall in a growth phase-dependent manner. In addition, HlpA was shown to be one of the major heparin-binding proteins of S. gallolyticus able to bind to the HSPG-expressing colon tumor cell lines HCT116 and HT-29. Strikingly, although wild-type levels of HlpA appeared to contribute to adherence, coating of additional HlpA at the bacterial surface resulted in reduced binding to colon tumor cells. This may be explained by the fact that heparan sulfate and LTA compete for the same binding site in HlpA. Altogether, this study implies that HlpA serves as a fine-tuning factor for bacterial adherence.
doi:10.1128/IAI.00384-09
PMCID: PMC2786452  PMID: 19752027
24.  A Novel Immunological Assay for Hepcidin Quantification in Human Serum 
PLoS ONE  2009;4(2):e4581.
Background
Hepcidin is a 25-aminoacid cysteine-rich iron regulating peptide. Increased hepcidin concentrations lead to iron sequestration in macrophages, contributing to the pathogenesis of anaemia of chronic disease whereas decreased hepcidin is observed in iron deficiency and primary iron overload diseases such as hereditary hemochromatosis. Hepcidin quantification in human blood or urine may provide further insights for the pathogenesis of disorders of iron homeostasis and might prove a valuable tool for clinicians for the differential diagnosis of anaemia. This study describes a specific and non-operator demanding immunoassay for hepcidin quantification in human sera.
Methods and Findings
An ELISA assay was developed for measuring hepcidin serum concentration using a recombinant hepcidin25-His peptide and a polyclonal antibody against this peptide, which was able to identify native hepcidin. The ELISA assay had a detection range of 10–1500 µg/L and a detection limit of 5.4 µg/L. The intra- and interassay coefficients of variance ranged from 8–15% and 5–16%, respectively. Mean linearity and recovery were 101% and 107%, respectively. Mean hepcidin levels were significantly lower in 7 patients with juvenile hemochromatosis (12.8 µg/L) and 10 patients with iron deficiency anemia (15.7 µg/L) and higher in 7 patients with Hodgkin lymphoma (116.7 µg/L) compared to 32 age-matched healthy controls (42.7 µg/L).
Conclusions
We describe a new simple ELISA assay for measuring hepcidin in human serum with sufficient accuracy and reproducibility.
doi:10.1371/journal.pone.0004581
PMCID: PMC2640459  PMID: 19238200
25.  An Insight into the Relationships between Hepcidin, Anemia, Infections and Inflammatory Cytokines in Pediatric Refugees: A Cross-Sectional Study 
PLoS ONE  2008;3(12):e4030.
Background
Hepcidin, a key regulator of iron homeostasis, is increased in response to inflammation and some infections, but the in vivo role of hepcidin, particularly in children with iron deficiency anemia (IDA) is unclear. We investigated the relationships between hepcidin, cytokines and iron status in a pediatric population with a high prevalence of both anemia and co-morbid infections.
Methodology/Principal Findings
African refugee children <16 years were consecutively recruited at the initial post-resettlement health check with 181 children meeting inclusion criteria. Data on hematological parameters, cytokine levels and co-morbid infections (Helicobacter pylori, helminth and malaria) were obtained and urinary hepcidin assays performed. The primary outcome measure was urinary hepcidin levels in children with and without iron deficiency (ID) and/or ID anaemia (IDA). The secondary outcome measures included were the relationship between co-morbid infections and (i) ID and IDA, (ii) urinary hepcidin levels and (iii) cytokine levels. IDA was present in 25/181 (13.8%). Children with IDA had significantly lower hepcidin levels (IDA median hepcidin 0.14 nmol/mmol Cr (interquartile range 0.05–0.061) versus non-IDA 2.96 nmol/mmol Cr, (IQR 0.95–6.72), p<0.001). Hemoglobin, log-ferritin, iron, mean cell volume (MCV) and transferrin saturation were positively associated with log-hepcidin levels (log-ferritin beta coefficient (β): 1.30, 95% CI 1.02 to 1.57) and transferrin was inversely associated (β: −0.12, 95% CI −0.15 to −0.08). Cytokine levels (including IL-6) and co-morbid infections were not associated with IDA or hepcidin levels.
Conclusions/Significance
This is the largest pediatric study of the in vivo associations between hepcidin, iron status and cytokines. Gastro-intestinal infections (H. pylori and helminths) did not elevate urinary hepcidin or IL-6 levels in refugee children, nor were they associated with IDA. Longitudinal and mechanistic studies of IDA will further elucidate the role of hepcidin in paediatric iron regulation.
doi:10.1371/journal.pone.0004030
PMCID: PMC2603326  PMID: 19107209

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