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Objective: The opportunistic pathogen Streptococcus gallolyticus is one of the few intestinal bacteria that has been consistently linked to colorectal cancer (CRC). This study aimed to identify novel S. gallolyticus -induced pathways in colon epithelial cells that could further explain how S. gallolyticus contributes to CRC development., Design and Results: Transcription profiling of in vitro cultured CRC cells that were exposed to S. gallolyticus revealed the specific induction of oxidoreductase pathways. Most prominently, CYP1A and ALDH1 genes that encode phase I biotransformation enzymes were responsible for the detoxification or bio-activation of toxic compounds. A common feature is that these enzymes are induced through the Aryl hydrocarbon receptor (AhR). Using the specific inhibitor CH223191, we showed that the induction of CYP1A was dependent on the AhR both in vitro using multiple CRC cell lines as in vivo using wild-type C57bl6 mice colonized with S. gallolyticus . Furthermore, we showed that CYP1 could also be induced by other intestinal bacteria and that a yet unidentified diffusible factor from the S. galloltyicus secretome (SGS) induces CYP1A enzyme activity in an AhR-dependent manner. Importantly, priming CRC cells with SGS increased the DNA damaging effect of the polycyclic aromatic hydrocarbon 3-methylcholanthrene., Conclusion: This study shows that gut bacteria have the potential to modulate the expression of biotransformation pathways in colonic epithelial cells in an AhR-dependent manner. This offers a novel theory on the contribution of intestinal bacteria to the etiology of CRC by modifying the capacity of intestinal epithelial or (pre-)cancerous cells to (de)toxify dietary components, which could alter intestinal susceptibility to DNA damaging events., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Taddese, Roelofs, Draper, Wu, Wu, Swinkels, Tjalsma and Boleij.)

Antibodies to Streptococcus gallolyticus subspecies gallolyticus (SGG) have been associated with colorectal cancer (CRC). Because SGG may correlate with impaired gut epithelia, we assessed the association of antibodies to bacterial flagellin C (FliC), a measure potentially related to this impairment, with CRC and the CRC-specific interaction with antibodies to SGG proteins. Antibodies to FliC and SGG pilus proteins Gallo2178 and Gallo2179 were measured in two independent studies, a combined study from Nijmegen and Detroit (93 CRC cases, 74 controls) and a replication data set including 576 cases and 576 controls from the Spanish multicenter multicase-control study (MCC-Spain). Logistic regression was applied to assess whether antibodies to FliC were associated with CRC and modified the association of antibodies to SGG proteins with CRC. Antibodies to FliC were associated with those to SGG Gallo2178 among CRC cases, resulting in an interaction in the association of antibodies to Gallo2178 with CRC (p = 0.007). This association was only present among individuals with high antibody responses to FliC (OR: 2.42, 95% CI: 1.45-4.06). In conclusion, our findings suggest that colorectal tumorigenesis could be accompanied by an impaired integrity of the epithelium that could result in associated increased antibody responses to bacterial proteins.

The colonic opportunist Streptococcus gallolyticus subspecies gallolyticus (SGG) is potentially associated with colorectal cancer (CRC). Large-scale seroepidemiological data for SGG antibodies and their possible association with CRC is currently missing. Associations between CRC and antibody responses to SGG were examined in 576 CRC cases and 576 controls matched by sex, age and province from a population-based multicase-control project (MCC-Spain). MCC-Spain was conducted between 2008 and 2013 in 12 Spanish provinces. Antibody responses to recombinant affinity-purified SGG pilus proteins Gallo1569, 2039, 2178 and 2179 were analysed by multiplex serology. Polyomavirus (PyV) JC VP1 and PyV 6 VP1 proteins served as disease-specificity controls. In the control population, antibody responses to pilus proteins were mostly weak. Antibody responses to individual pilus proteins Gallo2039 (OR: 1.58, 95% CI: 1.09-2.28), Gallo2178 (OR: 1.58, 95% CI: 1.09-2.30) and Gallo2179 (OR: 1.45, 95% CI: 1.00-2.11) were significantly associated with CRC risk. The association was stronger for positivity to two or more pilus proteins of Gallo1569, Gallo2178 and Gallo2179 (OR:1.93, 95% CI: 1.04-3.56) and for double-positivity to Gallo2178 and Gallo2179 (OR: 3.54, 95% CI: 1.49-8.44). The association between SGG infection and CRC risk was stronger among individuals younger than 65 years. For the first time we demonstrated a statistically significant association of exposure to SGG antigens and CRC in a large seroepidemiological study. These results should stimulate further studies on the role of SGG in CRC pathogenesis., (© 2015 UICC.)

Oral iron administration in African children can increase the risk for infections. However, it remains unclear to what extent supplementary iron affects the intestinal microbiome. We here explored the impact of iron preparations on microbial growth and metabolism in the well-controlled TNO's in vitro model of the large intestine (TIM-2). The model was inoculated with a human microbiota, without supplementary iron, or with 50 or 250 μmol/L ferrous sulfate, 50 or 250 μmol/L ferric citrate, or 50 μmol/L hemin. High resolution responses of the microbiota were examined by 16S rDNA pyrosequencing, microarray analysis, and metagenomic sequencing. The metabolome was assessed by fatty acid quantification, gas chromatography-mass spectrometry (GC-MS), and (1)H-NMR spectroscopy. Cultured intestinal epithelial Caco-2 cells were used to assess fecal water toxicity. Microbiome analysis showed, among others, that supplementary iron induced decreased levels of Bifidobacteriaceae and Lactobacillaceae, while it caused higher levels of Roseburia and Prevotella. Metagenomic analyses showed an enrichment of microbial motility-chemotaxis systems, while the metabolome markedly changed from a saccharolytic to a proteolytic profile in response to iron. Branched chain fatty acids and ammonia levels increased significantly, in particular with ferrous sulfate. Importantly, the metabolite-containing effluent from iron-rich conditions showed increased cytotoxicity to Caco-2 cells. Our explorations indicate that in the absence of host influences, iron induces a more hostile environment characterized by a reduction of microbes that are generally beneficial, and increased levels of bacterial metabolites that can impair the barrier function of a cultured intestinal epithelial monolayer.

Iron supplements acutely increase hepcidin, but the duration and magnitude of the increase, its dose dependence, and its effects on subsequent iron absorption have not been characterized in humans. Better understanding of these phenomena might improve oral iron dosing schedules. We investigated whether the acute iron-induced increase in hepcidin influences iron absorption of successive daily iron doses and twice-daily iron doses. We recruited 54 nonanemic young women with plasma ferritin ≤20 µg/L and conducted: (1) a dose-finding investigation with 40-, 60-, 80-, 160-, and 240-mg labeled Fe as [(57)Fe]-, [(58)Fe]-, or [(54)Fe]-FeSO4 given at 8:00 am fasting on 1 or on 2 consecutive days (study 1, n = 25; study 2, n = 16); and (2) a study giving three 60-mg Fe doses (twice-daily dosing) within 24 hours (study 3, n = 13). In studies 1 and 2, 24 hours after doses ≥60 mg, serum hepcidin was increased (P < .01) and fractional iron absorption was decreased by 35% to 45% (P < .01). With increasing dose, fractional absorption decreased (P < .001), whereas absolute absorption increased (P < .001). A sixfold increase in iron dose (40-240 mg) resulted in only a threefold increase in iron absorbed (6.7-18.1 mg). In study 3, total iron absorbed from 3 doses (2 mornings and an afternoon) was not significantly greater than that from 2 morning doses. Providing lower dosages (40-80 mg Fe) and avoiding twice-daily dosing maximize fractional absorption. The duration of the hepcidin response supports alternate day supplementation, but longer-term effects of these schedules require further investigation. These clinical trials were registered at www.ClinicalTrials.gov as #NCT01785407 and #NCT02050932., (© 2015 by The American Society of Hematology.)

Orally administrated iron is suspected to increase susceptibility to enteric infections among children in infection endemic regions. Here we investigated the effect of dietary iron on the pathology and local immune responses in intestinal infection models. Mice were held on iron-deficient, normal iron, or high iron diets and after 2 weeks they were orally challenged with the pathogen Citrobacter rodentium. Microbiome analysis by pyrosequencing revealed profound iron- and infection-induced shifts in microbiota composition. Fecal levels of the innate defensive molecules and markers of inflammation lipocalin-2 and calprotectin were not influenced by dietary iron intervention alone, but were markedly lower in mice on the iron-deficient diet after infection. Next, mice on the iron-deficient diet tended to gain more weight and to have a lower grade of colon pathology. Furthermore, survival of the nematode Caenorhabditis elegans infected with Salmonella enterica serovar Typhimurium was prolonged after iron deprivation. Together, these data show that iron limitation restricts disease pathology upon bacterial infection. However, our data also showed decreased intestinal inflammatory responses of mice fed on high iron diets. Thus additionally, our study indicates that the effects of iron on processes at the intestinal host-pathogen interface may highly depend on host iron status, immune status, and gut microbiota composition., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Objective: To describe the aetiology of anaemia in pregnant Ugandan women and explore Fe deficiency and common infections as contributors to anaemia in this population., Design: Cross-sectional study in which Hb, ferritin, transferrin receptor (sTfR), C-reactive protein, α-1 acid glycoprotein, hepcidin, malaria, hookworm infestation, syphilis and Helicobacter pylori infection were assessed., Setting: Antenatal care clinic at Kawempe Health Centre, Kampala, Uganda., Subjects: HIV-negative women (n 151) in their first or second pregnancy at 10-16 weeks' gestation., Results: The prevalence of anaemia was 29·1 %. Fe deficiency was 40·4 % and 14·6 % based on ferritin 8·3 μg/ml. The prevalence of Fe-deficiency anaemia was 9·3 % based on ferritin 8·3 μg/ml. Hepcidin concentration was positively correlated with ferritin concentration (n 151, r=0·578, P1 g/l and/or C-reactive protein >5 mg/l. Malaria parasitaemia (OR=6·85; 95 % CI 1·25, 37·41, P=0·026) and Fe deficiency defined using sTfR (OR=5·58; 95 % CI 1·26, 24·80, P=0·024) were independently and positively associated with anaemia. Population-attributable risk factors for anaemia for raised C-reactive protein, Fe deficiency defined by sTfR >8·3 μg/ml and presence of malaria parasites were 41·6 (95 % CI 11·1, 72·2) %, 13·5 (95 % CI 2·0, 25·0) % and 12·0 (95 % CI 1·4, 22·6) %, respectively., Conclusions: Infections and inflammation are of greater significance than Fe deficiency in the aetiology of anaemia in pregnant Ugandan women during the first trimester.

Background: In-home iron fortification for infants in developing countries is recommended for control of anaemia, but low absorption typically results in >80% of the iron passing into the colon. Iron is essential for growth and virulence of many pathogenic enterobacteria. We determined the effect of high and low dose in-home iron fortification on the infant gut microbiome and intestinal inflammation., Methods: We performed two double-blind randomised controlled trials in 6-month-old Kenyan infants (n=115) consuming home-fortified maize porridge daily for 4 months. In the first, infants received a micronutrient powder (MNP) containing 2.5 mg iron as NaFeEDTA or the MNP without iron. In the second, they received a different MNP containing 12.5 mg iron as ferrous fumarate or the MNP without the iron. The primary outcome was gut microbiome composition analysed by 16S pyrosequencing and targeted real-time PCR (qPCR). Secondary outcomes included faecal calprotectin (marker of intestinal inflammation) and incidence of diarrhoea. We analysed the trials separately and combined., Results: At baseline, 63% of the total microbial 16S rRNA could be assigned to Bifidobacteriaceae but there were high prevalences of pathogens, including Salmonella Clostridium difficile, Clostridium perfringens, and pathogenic Escherichia coli. Using pyrosequencing, +FeMNPs increased enterobacteria, particularly Escherichia/Shigella (p=0.048), the enterobacteria/bifidobacteria ratio (p=0.020), and Clostridium (p=0.030). Most of these effects were confirmed using qPCR; for example, +FeMNPs increased pathogenic E. coli strains (p=0.029). +FeMNPs also increased faecal calprotectin (p=0.002). During the trial, 27.3% of infants in +12.5 mgFeMNP required treatment for diarrhoea versus 8.3% in -12.5 mgFeMNP (p=0.092). There were no study-related serious adverse events in either group., Conclusions: In this setting, provision of iron-containing MNPs to weaning infants adversely affects the gut microbiome, increasing pathogen abundance and causing intestinal inflammation., Trial Registration Number: NCT01111864., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Background: In sub-Saharan Africa, parasitic diseases and low bioavailable iron intake are major causes of anemia. Anemia results from inflammation, preventing iron recycling and decreasing dietary iron absorption. Hookworm, Plasmodium, and Schistosoma infections contribute to anemia, but their influence on dietary iron absorption and recycling is unknown., Objective: The objective was to measure inflammation biomarkers, hepcidin, iron absorption, and utilization pre- and posttreatment in children with afebrile malaria, hookworm, and Schistosoma haematobium infection., Design: Ivorian children aged 11-17 y with afebrile Plasmodium falciparum (n = 17), hookworm (n = 16), or S. haematobium infection (n = 8) consumed a syrup containing 3 mg ⁵⁷Fe as ferrous sulfate and received an intravenous infusion of 50 μg ⁵⁸Fe as ferrous citrate. Children were treated for their respective infection, and the iron studies were repeated 4 wk later. Iron and inflammation biomarkers and hepcidin were measured., Results: Geometric mean iron absorptions in the afebrile malaria and hookworm groups were 12.9% and 32.2% (P < 0.001) before treatment and 23.6% and 30.0% (P = 0.113) after treatment, respectively. Treatment of afebrile malaria reduced inflammation (P < 0.001) and serum hepcidin (P = 0.004) and improved iron absorption (P = 0.003). Treatment of hookworm infection neither affected inflammation biomarkers nor altered iron absorption. Similarly, there was a lack of treatment effects in the S. haematobium-infected group; however, the small sample size limits conclusions. Geometric mean iron utilization ranged between 79.1% and 88.0% in the afebrile malaria and hookworm groups with no significant differences pre- and posttreatment., Conclusions: In school-age children, hookworm infection does not produce inflammation or increase serum hepcidin, and it does not influence iron absorption or utilization. In contrast, afebrile malaria causes inflammation, increases hepcidin, and reduces iron absorption but not utilization. These findings provide insights into the iron metabolism and the etiology of anemia in parasitic infections., (© 2015 American Society for Nutrition.)

Background/aims: Innately low hepcidin levels lead to iron overload in HFE-associated hereditary haemochromatosis., Methods: This study compared hepcidin and non-transferrin bound iron (NTBI) levels in untreated iron-loaded and non-iron-loaded C282Y homozygotes to levels in C282Y/H63D compound heterozygotes and individuals with other HFE genotypes associated with less risk of iron overload., Results: As the genotypic risk for iron overload increased, transferrin saturation and serum NTBI levels increased while serum hepcidin levels decreased. Overweight and obese male C282Y homozygotes had significantly higher hepcidin levels than male C282Y homozygotes with a normal BMI. Pearson product-moment analysis showed that serum hepcidin levels significantly correlated with HFE status, serum ferritin, age, NTBI, transferrin saturation, gender and BMI. Subsequent multiple regression analysis showed that HFE status and serum ferritin were significant independent correlates of serum hepcidin levels., Conclusions: In summary, this study has shown that while serum ferritin and HFE status are the most important determinants of hepcidin levels, factors such age, gender, BMI, transferrin saturation and NTBI all interact closely in the matrix of homeostatic iron balance., (© 2014 S. Karger AG, Basel.)

Objectives: The post-exercise interleukin-6 (IL-6) and hepcidin response was investigated during the hormone-deplete and hormone-replete phases of an estradiol and progestogen regulated oral contraceptive cycle (OCC)., Design: Counterbalanced, repeated measures cross-over study., Methods: Ten active female monophasic oral contraceptive pill (OCP) users completed two 40 min treadmill running trials at 75% of their pre-determined peak oxygen uptake velocity (vVO2peak). These trials were randomly performed in two specific phases of the OCC: (a) Day 2-4, representing a hormone-free withdrawal period (D-0); (b) Day 12-14, representing the end of the first week of active hormone therapy (D+7). Venous blood samples were drawn pre-, post- and 3h post-exercise., Results: In both trials, serum IL-6 was significantly elevated (p<0.05) immediately post-exercise, while serum hepcidin was significantly elevated (p<0.05) 3h post-exercise, with no significant differences recorded between trials., Conclusions: These findings suggest that exercise performed during the different phases (D-0 vs. D+7) of a monophasic OCP regulated cycle does not alter exercise induced IL-6 or hepcidin production. As such, future studies looking to investigate similar variables post-exercise, may not need to 'control' for different phases of the OCC, provided participants are current monophasic OCP users., (Copyright © 2013 Sports Medicine Australia. Published by Elsevier Ltd. All rights reserved.)

Background: The value of ferritin in the diagnosis of iron deficiency is limited in patients with CF since it increases in the presence of inflammation. We hypothesized that the soluble transferrin receptor (sTfR) and hepcidin may provide more information than ferritin in assessing iron status in children with CF., Methods: We analyzed sTfR and hepcidin in relation to conventional iron status indicators in 49 children with CF., Results: We found no differences in sTfR concentration between children with and those without ID. sTfR concentrations were within the normal range in all children. Hepcidin concentrations were low, and concentrations below the limit of detection were observed in 25% of the clinically stable children., Conclusion: The sTfR is not useful to determine the iron status in this population, whereas hepcidin might serve as an early indicator of deficient iron stores in children with CF., (Copyright © 2014 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

Iron is abundantly present on earth, essential for most microorganisms and crucial for human health. Human iron deficiency that is nevertheless highly prevalent in developing regions of the world can be effectively treated by oral iron administration. Accumulating evidence indicates that excess of unabsorbed iron that enters the colonic lumen causes unwanted side effects at the intestinal host-microbiota interface. The chemical properties of iron, the luminal environment and host iron withdrawal mechanisms, especially during inflammation, can turn the intestine in a rather stressful milieu. Certain pathogenic enteric bacteria can, however, deal with this stress at the expense of other members of the gut microbiota, while their virulence also seems to be stimulated in an iron-rich intestinal environment. This review covers the multifaceted aspects of nutritional iron stress with respect to growth, composition, metabolism and pathogenicity of the gut microbiota in relation to human health. We aim to present an unpreceded view on the dynamic effects and impact of oral iron administration on intestinal host-microbiota interactions to provide leads for future research and other applications., (© 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Background: The diagnostic use of hepcidin is limited by the absence of standardization and lack of age-specific reference ranges in children in particular. The aim of this study was to determine reference ranges of serum hepcidin in healthy children aged 0.5-3 y using mass spectometry (MS) and a commercial immunochemical (IC) assay, and to investigate its association with other indicators of iron status and inflammation., Methods: We included 400 healthy children aged 0.5-3 y. We constructed reference ranges for MS-hepcidin and IC-hepcidin concentrations using the median, P2.5, and P97.5 in a normative population of 219 children with no anemia, no infection and/or inflammation, and no iron deficiency., Results: Median concentrations (P2.5-P97.5) of MS-hepcidin and IC-hepcidin were 3.6 nmol/l (0.6-13.9 nmol/l) and 7.9 nmol/l (1.9-28.6 nmol/l), respectively. We found a good correlation between both methods. However, MS-hepcidin was consistently lower than IC-hepcidin. Hepcidin correlated with ferritin and C-reactive protein., Conclusion: We provide reference ranges for hepcidin for an MS and commercial IC method. Absolute values between assays differed significantly, but hepcidin concentrations obtained by MS and IC methods correlate with each other, and both correlate with ferritin and CRP.

Aim: Iron deficiency is an important child health problem. Its diagnosis in areas of high infection exposure remains complicated as inflammation may interfere with the accuracy of peripheral iron markers. With this study, we aimed to validate the conventional iron markers and two novel iron markers, hepcidin and Red blood cell Size Factor (RSf), against the reference standard of iron status, bone marrow iron, in children living in an infectious setting., Methods: We compared ferritin, soluble transferrin receptor, Soluble Transferrin Log-Ferritin Index (sTfR-F), mean cellular volume, mean cellular haemoglobin concentration, hepcidin and RSf, against bone marrow iron in 87 healthy Malawian children (6-66 months) scheduled for elective surgery., Results: Of all children, 44.8% had depleted bone marrow iron stores. Using optimised cut-offs, ferritin (<18 µg/L) and sTfR-F (>1.85) best predicted depleted iron stores with a sensitivity/specificity of 73.7%/77.1% and 72.5%/75.0%, respectively. Hepcidin (<1.4 nmol/L) was a moderate sensitive marker (73.0%) although specificity was 54.2%; RSf poorly predicted depleted iron stores., Conclusions: We provide the first bone marrow-validated data on peripheral iron markers in African children, and showed ferritin and sTfR-F best predicted iron status. Using appropriately defined cut-offs, these indicators can be applied in surveillance and research. As their accuracy is limited for clinical purposes, more reliable iron biomarkers are still required in African children., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Purpose: To assess the influence of a simulated altitude exposure (~2,900 m above sea level) for a 3 h recovery period following intense interval running on post-exercise inflammation, serum iron, ferritin, erythropoietin, and hepcidin response., Methods: In a cross-over design, ten well-trained male endurance athletes completed two 8 × 3 min interval running sessions at 85 % of their maximal aerobic velocity on a motorized treadmill, before being randomly assigned to either a hypoxic (HYP: F IO2 ~0.1513) or a normoxic (NORM: F IO2 0.2093) 3 h recovery period. Venous blood was collected pre- and immediately post-exercise, and after 3 and 24 h of recovery. Blood was analyzed for interleukin-6, serum iron, ferritin, erythropoietin, and hepcidin., Results: Interleukin-6 was significantly elevated (p < 0.01) immediately post-exercise compared to baseline (NORM: 1.08 ± 0.061 to 3.12 ± 1.80) (HYP: 1.32 ± 0.86 to 2.99 ± 2.02), but was not different between conditions. Hepcidin levels were significantly elevated (p < 0.01) at 3 h post-exercise for both conditions when compared to baseline (NORM: 3.25 ± 1.23 to 7.40 ± 4.00) (HYP: 3.24 ± 1.94 to 5.42 ± 3.20), but were significantly lower (p < 0.05) in the HYP trial compared to NORM. No significant differences existed between HYP and NORM for erythropoietin, serum iron, or ferritin., Conclusion: Simulated altitude exposure (~2,900 m) for 3 h following intense interval running attenuates the peak hepcidin levels recorded at 3 h post-exercise. Consequently, a hypoxic recovery after exercise may be useful for athletes with compromised iron status to potentially increase acute dietary iron absorption.

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.

Anemia is a frequent sign in patients with diffuse large B-cell lymphoma (DLBCL) at diagnosis. We determined erythropoietin, hepcidin and interleukin-6 (IL-6) in plasma samples of 53 patients with DLBCL. The majority of patients (40/53, 75%) showed defective endogenous erythropoietin production, in particular when anemia was present (p = 0.01). Hepcidin plasma levels were significantly higher in patients compared to controls (p = 0.006), particularly in those with characteristics associated with a more active disease, including elevated lactate dehydrogenase (LDH) (p = 0.0004), B-symptoms (p = 0.07) and an age-adjusted international prognostic index (IPI) score > 1 (p = 0.01). Hepcidin levels correlated strongly to ferritin (r = 0.77, p < 0.0001) and weakly to IL-6 concentrations (r = 0.30, p = 0.03), but not to hemoglobin values. IL-6 inversely correlated to hemoglobin values in both univariate and multivariate analysis (p = 0.04), including hepcidin and erythropoietin as variables. Our findings suggest that elevated hepcidin levels and inadequate erythropoietin response are frequent in DLBCL, but elevated IL-6 plays the major role for the development of anemia.

The association of colorectal neoplasia (CRN) with Streptococcus bovis biotype I (SBI) infection is well recognized. However, this is not the case for Streptococcus bovis biotype II (SBII). We conducted this study in order to analyze the relationship between SBII and CRN. We analyzed all cases of bacteremia due to SBI (n = 99) and SBII (n = 36) diagnosed in our hospital (during the period 1988-2011) that were followed up with colonoscopy. In addition, we reviewed the literature (during the period 1982-2011) to select all cases of infection of SB that had undergone colonoscopy or other adequate form of colorectal examination. A multivariate analysis was performed to detect CRN risk factors in patients infected with SB. From the 223 cases of SB infection included in the analysis (135 from our institution and 88 from the literature review), 159 were due to SBI and 64 were caused by SBII. As compared with SBI, the SBII cases had a lower frequency of CRN (27 % vs. 67 %, p <0.001), advanced adenomas (8 % vs. 29 %, p <0.01), and carcinomas (6 % vs. 21 %, p <0.01). In a multivariate analysis, and after adjusting for age, sex, type of infection, and biotype, SBII infection was not associated with CRN: odds ratio (OR) = 0.17; 95 % confidence interval (CI) = 0.09 to 0.33. The only factor independently associated with CRN was SBI infection: OR = 5.7; 95 % CI = 3.0 to 10.9. The prevalence of CRN in patients infected with SBII is significantly lower than patients with SBI and does not appear to be higher than the CRN prevalence among the general population.

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.

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.

Unlabelled: Human iron homeostasis is regulated by intestinal iron transport, hepatic hepcidin release, and signals from pathways that consume or supply iron. The aim of this study was to characterize the adaptation of iron homeostasis under hypoxia in mountaineers at the levels of (1) hepatic hepcidin release, (2) intestinal iron transport, and (3) systemic inflammatory and erythropoietic responses. Twenty-five healthy mountaineers were studied. Blood samples and duodenal biopsies were taken at baseline of 446 m as well as on day 2 (MG2) and 4 (MG4) after rapid ascent to 4559 m. Divalent metal-ion transporter 1 (DMT-1), ferroportin 1 (FP-1) messenger RNA (mRNA), and protein expression were analyzed in biopsy specimens by quantitative reverse-transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. Serum hepcidin levels were analyzed by mass spectrometry. Serum iron, ferritin, transferrin, interleukin (IL)-6, and C-reactive protein (CRP) were quantified by standard techniques. Serum erythropoietin and growth differentiation factor 15 (GDF15) levels were measured by enzyme-linked immunosorbent assay (ELISA). Under hypoxia, erythropoietin peaked at MG2 (P < 0.001) paralleled by increased GDF15 on MG2 (P < 0.001). Serum iron and ferritin levels declined rapidly on MG2 and MG4 (P < 0.001). Duodenal DMT-1 and FP-1 mRNA expression increased up to 10-fold from baseline on MG2 and MG4 (P < 0.001). Plasma CRP increased on MG2 and MG4, while IL-6 only increased on MG2 (P < 0.001). Serum hepcidin levels decreased at high altitude on MG2 and MG4 (P < 0.001)., Conclusion: This study in healthy volunteers showed that under hypoxemic conditions hepcidin is repressed and duodenal iron transport is rapidly up-regulated. These changes may increase dietary iron uptake and allow release of stored iron to ensure a sufficient iron supply for hypoxia-induced compensatory erythropoiesis., (© 2013 by the American Association for the Study of Liver Diseases.)

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.

Bacteria constitute about 90% of all cells in the human body. The densest and most complex bacterial community is in the large intestine. This population is quite stable in healthy intestines, but intestinal disease distorts the ecological balance and induces dysbiosis. Results of studies have indicated that the epithelial and metabolic changes that occur with colorectal cancer provide a competitive advantage to a subset of intestinal bacteria. Strikingly, however, Streptococcus gallolyticus gallolyticus (previously known as Streptococcus bovis biotype I) is one of the very few opportunistic pathogens that has been clinically linked to colonic malignant diseases. In this Personal View we describe how S. gallolyticus gallolyticus exploits its unique range of virulence features to cause infections in patients with colorectal cancer. We postulate that distinct virulence factors on one hand enable this bacterium to establish a symptomatic infection in susceptible individuals, and on the other hand make its ability to do this dependent on pre-existing colonic abnormalities. We believe that our current reconstruction of this route of infection aids understanding of how S. gallolyticus gallolyticus infections can be best exploited for early detection of colorectal cancer., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

In the past 8 years, there has been renewed interest in the role of iron in both acute kidney injury (AKI) and chronic kidney disease (CKD). In patients with kidney diseases, renal tubules are exposed to a high concentration of iron owing to increased glomerular filtration of iron and iron-containing proteins, including haemoglobin, transferrin and neutrophil gelatinase-associated lipocalin (NGAL). Levels of intracellular catalytic iron may increase when glomerular and renal tubular cells are injured. Reducing the excessive luminal or intracellular levels of iron in the kidney could be a promising approach to treat AKI and CKD. Understanding the role of iron in kidney injury and as a therapeutic target requires insight into the mechanisms of iron metabolism in the kidney, the role of endogenous proteins involved in iron chelation and transport, including hepcidin, NGAL, the NGAL receptor and divalent metal transporter 1, and iron-induced toxic effects. This Review summarizes emerging knowledge, which suggests that complex mechanisms of iron metabolism exist in the kidney, modulated directly or indirectly by cellular iron content, inflammation, ischaemia and oxidative stress. The potential exists for prevention and treatment of iron-induced kidney injury by customized iron removal or relocation, aided by detailed insight into the underlying pathological mechanisms.

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.

The effect of exercise modality and intensity on Interleukin-6 (IL-6), iron status, and hepcidin levels was investigated. Ten trained male triathletes performed 4 exercise trials including low-intensity continuous running (L-R), low-intensity continuous cycling (L-C), high-intensity interval running (H-R), and high-intensity interval cycling (H-C). Both L-R and L-C consisted of 40 min continuous exercise performed at 65% of peak running velocity (vVO2peak) and cycling power output (pVO2peak), while H-R and H-C consisted of 8 × 3-min intervals performed at 85% vVO2peak and pVO2peak. Venous blood samples were drawn pre-, post-, and 3 hr postexercise. Significant increases in postexercise IL-6 were seen within each trial (p < .05) and were significantly greater in H-R than L-R (p < .05). Hepcidin levels were significantly elevated at 3 hr postexercise within each trial (p < .05). Serum iron levels were significantly elevated (p < .05) immediately postexercise in all trials except L-C. These results suggest that, regardless of exercise mode or intensity, postexercise increases in IL-6 may be expected, likely influencing a subsequent elevation in hepcidin. Regardless, the lack of change in postexercise serum iron levels in L-C may indicate that reduced hemolysis occurs during weight-supported, low-intensity activity.

Background: The iron-regulating hormone hepcidin is a promising biomarker in the diagnosis of iron disorders. Concentrations of hepcidin have been shown to increase during the day in individuals who are following a regular diet. It is currently unknown whether these increases are determined by an innate rhythm or by other factors. We aimed to assess the effect of dietary iron on hepcidin concentrations during the day., Methods: Within a 7-day interval, 32 volunteers received an iron-deficient diet on 1 day and the same diet supplemented with 65 mg ferrous fumarate at 0815 and 1145 on another day. Blood was drawn to assess ferritin, hepcidin-25, and transferrin saturation (TS) throughout both days at 4 time points between 0800 (fasted) and 1600. A linear mixed model for repeated data was used to analyze the effect of iron intake on TS and hepcidin concentrations., Results: Baseline values of hepcidin at 0800 correlated significantly with ferritin (r = 0.61). During the day of an iron-deficient diet the mean TS was similar both in men and in women, whereas hepcidin increased. During the day with iron supplementation the mean TS was significantly higher both in men and in women, and the mean hepcidin was moderately but significantly higher in women (1.0 nmol/L, 95% CI, 0.2-1.8) but not in men (0.0 nmol/L, 95% CI, -0.8 to 0.8)., Conclusions: Our data demonstrate that ferritin sets the basal hepcidin concentrations and suggest that innate diurnal rhythm rather than dietary iron mediates the daily hepcidin variations. These findings will be useful for optimizing sampling protocols and will facilitate the interpretation of hepcidin as an iron biomarker.

The colonic mucosa is in constant physical interaction with a dense and complex bacterial community that comprises health-promoting and pathogenic microbes. Here, we highlight important clinical studies and experimental models that have linked the intestinal microbiota to the development of colorectal cancer (CRC). Moreover, we use recently published metatranscriptome sequencing data to test whether potentially carcinogenic toxin genes exhibit higher expression levels in human CRC tissue compared to adjacent non-malignant mucosa. Our analyses show a large variation in expression of toxin(-related) genes from different species. Surprisingly, Enterobacterial toxins were among the highest expressed, while Enterobacteria were not among the most abundant species in these samples. Although we can differentiate on- and off-tumour sites based on toxin reads, the read depth profiles are quite similar and show only limited coverage of the toxin genes. Thus, extended metagenomic studies are needed to obtain a high-resolution picture of host-pathogen interactions during human CRC., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background: Many countries in the nutrition transition have high rates of iron deficiency (ID) and overweight (OW). ID is more common in OW children; this may be due to adiposity-related inflammation reducing iron absorption., Objective: We investigated whether weight status predicts response to oral iron supplementation in ID South African children., Design: A placebo-controlled trial of oral iron supplementation (50 mg, 4 × weeks for 8.5 months) was done in ID 6- to 11-year-old children (n=321); 28% were OW or obese. BMI-for-age z-scores (BAZ), hepcidin (in a sub-sample), hemoglobin, serum ferritin (SF), transferrin receptor (TfR), zinc protoporphyrin (ZnPP) and C-reactive protein (CRP) were measured; body iron was calculated from the SF to TfR ratio., Results: At baseline, BAZ correlated with CRP (r=0.201, P<0.001) and CRP correlated with hepcidin (r=0.384, P<0.001). Normal weight children supplemented with iron had significantly lower TfR concentrations at endpoint than the OW children supplemented with iron and the children receiving placebo. Higher BAZ predicted higher TfR (β=0.232, P<0.001) and lower body iron (β=-0.090, P=0.016) at endpoint, and increased the odds ratio (OR) for remaining ID at endpoint in both the iron and placebo groups (iron: OR 2.31, 95% CI: 1.13, 4.73; placebo: OR 1.78, 95% CI: 1.09, 2.91). In the children supplemented with iron, baseline hepcidin and BAZ were significant predictors of endpoint TfR, with a trend towards a hepcidin × BAZ interaction (P=0.058)., Conclusion: South African children with high BAZ have a two-fold higher risk of remaining ID after iron supplementation. This may be due to their higher hepcidin concentrations reducing iron absorption. Thus, the current surge in OW in rapidly developing countries may undercut efforts to control anemia in vulnerable groups. The trial is registered at clinicaltrials.gov as NCT01092377.

Smoking and high red meat intake have been associated with colorectal cancer (CRC) risk. Increased iron exposure may be a common factor, favoring the colonization of certain bacterial pathogens that preferentially grow in an iron-rich luminal environment. We analyzed the data from a population-based case-control study of CRC and measured antibody levels against flagelin of Salmonella (FliC), one of the irontrophic bacteria, in 2 independent blood collections. The risk of CRC synergistically increased by combined exposures to heme iron intake and pack-yr (PY) of cigarette smoking (P value for the interaction = 0.039 on the continuous scale). There was a marginally significant interaction between heme iron intake and PY in increasing FliC antibody in the U.S. control subjects (P = 0.055), although no iron or smoking data were available for Dutch samples. Furthermore, FliC antibody levels were significantly higher in patients with colorectal polyps and cancer than in controls in both Dutch (3.93 vs. 2.23) (P = 0.014) and U.S. samples (6.65 vs. 4.37) (P < 0.001). Potential roles of iron from cigarette smoking and dietary heme in CRC through altering irontrophic luminal bacterial population may warrant further investigation.

Hepcidin regulation by competing stimuli such as infection and iron deficiency has not been studied in infants and it's yet unknown whether hepcidin regulatory pathways are fully functional in infants. In this cross-sectional study including 339 Kenyan infants aged 6.0±1.1 months (mean±SD), we assessed serum hepcidin-25, biomarkers of iron status and inflammation, and fecal calprotectin. Prevalence of inflammation, anemia, and iron deficiency was 31%, 71%, 26%, respectively. Geometric mean (±SD) serum hepcidin was 6.0 (±3.4) ng/mL, and was significantly lower in males than females. Inflammation (C-reactive protein and interleukin-6) and iron status (serum ferritin, zinc protoporphyrin and soluble transferrin receptor) were significant predictors of serum hepcidin, explaining nearly 60% of its variance. There were small, but significant differences in serum hepcidin comparing iron deficient anemic (IDA) infants without inflammation to iron-deficient anemic infants with inflammation (1.2 (±4.9) vs. 3.4 (±4.9) ng/mL; P<0.001). Fecal calprotectin correlated with blood/mucus in the stool but not with hepcidin. Similarly, the gut-linked cytokines IL-12 and IL-17 did not correlate with hepcidin. We conclude that hepcidin regulatory pathways are already functional in infancy, but serum hepcidin alone may not clearly discriminate between iron-deficient anemic infants with and without infection. We propose gender-specific reference values for serum hepcidin in iron-replete infants without inflammation.

One of the few bacteria that have been consistently linked to colorectal cancer (CRC) is the opportunistic pathogen Streptococcus gallolyticus. Infections with this bacterium are generally regarded as an indicator for colonic malignancy, while the carriage rate of this bacterium in the healthy large intestine is relatively low. We speculated that the physiological changes accompanying the development of CRC might favor the colonization of this bacterium. To investigate whether colon tumor cells can support the survival of S. gallolyticus, this bacterium was grown in spent medium of malignant colonocytes to simulate the altered metabolic conditions in the CRC microenvironment. These in vitro simulations indicated that S. gallolyticus had a significant growth advantage in these spent media, which was not observed for other intestinal bacteria. Under these conditions, bacterial responses were profiled by proteome analysis and metabolic shifts were analyzed by (1)H-NMR-spectroscopy. In silico pathway analysis of the differentially expressed proteins and metabolite analysis indicated that this advantage resulted from the increased utilization of glucose, glucose derivates, and alanine. Together, these data suggest that tumor cell metabolites facilitate the survival of S. gallolyticus, favoring its local outgrowth and providing a possible explanation for the specific association of S. gallolyticus with colonic malignancy.

Measurements of the iron regulatory hormone hepcidin by various methodologies and laboratories are not harmonized. As a result different numeric results are obtained for the same clinical sample. We investigated whether better agreement between plasma hepcidin methods can be achieved by harmonization. Native plasma pools (n = 11) of a variety of hepcidin concentrations and blank plasma spiked with three different quantities of synthetic hepcidin-25 purchased from two different commercial sources (n = 6), were distributed in duplicate among 21 methods worldwide. We assessed commutability by comparing results from synthetic hepcidin with those from native samples in various method couples by Bland-Altman plots. Methods differed substantially in absolute values and reproducibility. For the majority of methods we found that samples with synthetic hepcidin-25 were noncommutable with the native samples. In an attempt to harmonize by using native hepcidin results, we selected two methods that showed good mutual agreement of native results and calculated consensus values as the medians for the 11 duplicate native samples obtained by these two methods. Finally, we constructed algorithms enabling the laboratories to calculate the hepcidin consensus (HEPCON) value using their own native hepcidin results. We found that the use of these algorithms substantially reduced the between-method CV. Until commutable materials are defined, hepcidin harmonization can be achieved by exploiting specific algorithms, allowing each lab to report their native hepcidin concentrations in HEPCON values. This study represents the first step toward harmonization of plasma hepcidin methods and facilitates aggregation of hepcidin data from different research investigations., (Copyright © 2012 Wiley Periodicals, Inc.)

A healthy human body contains at least tenfold more bacterial cells than human cells and the most abundant and diverse microbial community resides in the intestinal tract. Intestinal health is not only maintained by the human intestine itself and by dietary factors, but is also largely supported by this resident microbial community. Conversely, however, a large body of evidence supports a relationship between bacteria, bacterial activities and human colorectal cancer. Symbiosis in this multifaceted organ is thus crucial to maintain a healthy balance within the host-diet-microbiota triangle and accordingly, changes in any of these three factors may drive a healthy situation into a state of disease. In this review, the factors that sustain health or drive this complex intestinal system into dysbiosis are discussed. Emphasis is on the role of the intestinal microbiota and related mechanisms that can drive the initiation and progression of sporadic colorectal cancer (CRC). These mechanisms comprise the induction of pro-inflammatory and pro-carcinogenic pathways in epithelial cells as well as the production of (geno)toxins and the conversion of pro-carcinogenic dietary factors into carcinogens. A thorough understanding of these processes will provide leads for future research and may ultimately aid in development of new strategies for CRC diagnosis and prevention., (© 2012 The Authors. Biological Reviews © 2012 Cambridge Philosophical Society.)

Purpose: To investigate the effects of acute exercise on serum hepcidin and iron (sFe) in active women. Changes in interleukin-6 (IL-6), hepcidin, ferritin, and sFe in response to 2 different exercise durations were compared., Methods: Twelve women age 19-32 yr performed 2 treadmill runs (60 and 120 min) at 65% of VO2max. Blood samples were obtained before, immediately after, and 3, 6, 9, and 24 hr after exercise. Two-way repeated-measures ANOVA was conducted to examine changes in measured variables. Significance was accepted at p < .05., Results: Significant effects for trial were observed for hepcidin (60 min: 1.15 ± 0.48 nmol/L; 120 min: 2.28 ± 1.44 nmol/L) and for time, with hepcidin significantly increased 3 hr postexercise in both trials (60 min: 3 hr - 1.99 ± 2.00 nmol/L; 120 min: 3 hr - 4.60 ± 4.61 nmol/L). Significant main effects for time occurred for sFe, ferritin, and IL-6. sFe was significantly decreased 9 hr postexercise compared with 3 and 24 hr postexercise. IL-6 was significantly increased immediately postexercise., Conclusions: Both runs resulted in significant increases in hepcidin 3 hr after exercise. Increases in hepcidin were preceded by significant increases in IL-6 immediately postexercise and followed by significant decreases in sFe 9 hr postexercise. It was concluded that endurance exercise increases the production of hepcidin, which affects sFe. The 2-hr exercise bout stimulated greater changes in serum hepcidin than the 1-hr bout.

Streptococcus gallolyticus subsp. gallolyticus (previously called Streptococcus bovis biotype I) infections have long been associated with colorectal cancer (CRC). This work aimed to investigate the CRC-associated humoral immune response to four pilus proteins of this bacterium by newly developed ELISAs. Pilus proteins are interesting diagnostic targets as they are the building blocks of pilin-like structures that mediate bacterial virulence and are readily exposed to the host immune system upon infection. The presence of serum antibodies against these pilus proteins was evaluated in Dutch and American populations. These analyses showed that an immune response to these antigens was specific for clinical S. gallolyticus subsp. gallolyticus infections, but that increased serum antibody titers to multiple pilus proteins in single individuals were rarely observed. However, a multiplex approach based on antibody titers against any of these four antigens resulted in assay sensitivities between 16% and 43% for the detection of early-stage CRC. Together these findings underscore the potential of a multi-antigen approach to complement diagnosis of S. gallolyticus subsp. gallolyticus-associated CRC., (©2011 AACR.)

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.

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.

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.

Background: The peptide hormone hepcidin plays a central role in regulating dietary iron absorption and body iron distribution. Many human diseases are associated with alterations in hepcidin concentrations. The measurement of hepcidin in biological fluids is therefore a promising tool in the diagnosis and management of medical conditions in which iron metabolism is affected., Content: We describe hepcidin structure, kinetics, function, and regulation. We moreover explore the therapeutic potential for modulating hepcidin expression and the diagnostic potential for hepcidin measurements in clinical practice., Summary: Cell-culture, animal, and human studies have shown that hepcidin is predominantly synthesized by hepatocytes, where its expression is regulated by body iron status, erythropoietic activity, oxygen tension, and inflammatory cytokines. Hepcidin lowers serum iron concentrations by counteracting the function of ferroportin, a major cellular iron exporter present in the membrane of macrophages, hepatocytes, and the basolateral site of enterocytes. Hepcidin is detected in biologic fluids as a 25 amino acid isoform, hepcidin-25, and 2 smaller forms, i.e., hepcidin-22 and -20; however, only hepcidin-25 has been shown to participate in the regulation of iron metabolism. Reliable assays to measure hepcidin in blood and urine by use of immunochemical and mass spectrometry methods have been developed. Results of proof-of-principle studies have highlighted hepcidin as a promising diagnostic tool and therapeutic target for iron disorders. However, before hepcidin measurements can be used in routine clinical practice, efforts will be required to assess the relevance of hepcidin isoform measurements, to harmonize the different assays, to define clinical decision limits, and to increase assay availability for clinical laboratories.

Background: Streptococcus bovis has long been associated with colorectal cancer (CRC). However, not all genospecies are as closely related to CRC. With this systematic review, we aim to increase the awareness of the association between S. bovis biotype I (Streptococcus gallolyticus) and CRC and urge for uniform molecular microbiological classification., Methods: In January 2011, the PubMed database was searched for all studies that investigated the association between S. bovis, infective endocarditis (IE), and CRC. A total of 191 studies were screened for eligibility and yielded 52 case reports and 31 case series, of which 11 were used for meta-analysis on the association between S. bovis biotype, IE, and adenomas/carcinomas (CRC)., Results: Among the S. bovis-infected patients who underwent colonic evaluation, the median percentage of patients who had concomitant adenomas/carcinomas was 60% (interquartile range, 22%), which largely exceeds the disease rate reported in the general asymptomatic population. Meta-analysis showed that patients with S. bovis biotype I infection had a strongly increased risk of having CRC (pooled odds ratio [OR], 7.26; 95% confidence interval [CI], 3.94-13.36) and IE (pooled OR, 16.61; 95% CI, 8.85-31.16), compared with S. bovis biotype II-infected patients. Notably, CRC occurred more often among patients with S. bovis IE than among patients with S. bovis infection at other sites (pooled OR, 3.72; 95% CI, 2.03-6.81)., Conclusions: Our meta-analysis clearly indicates that S. bovis should no longer be regarded as a single species in clinical practice, because S. gallolyticus (S. bovis biotype I) infection, in particular, has an unambiguous association with CRC.

Background and Aims: Increased ferritin and body iron stores are frequently observed in nonalcoholic fatty liver disease (NAFLD), associated with heightened susceptibility to vascular damage. Conflicting data have been reported on the role of iron in atherosclerosis, with recent data suggesting that excess iron induces vascular damage by increasing levels of the hormone hepcidin, which would determine iron trapping into macrophages, oxidative stress, and promotion of transformation into foam cells. Aim of this study was to investigate the relationship between iron status and cardiovascular damage in NAFLD., Methods and Results: Vascular damage was evaluated by common carotid arteries intima-media thickness (CC-IMT) measurement and plaque detection by ecocolor-doppler ultrasonography in 506 patients with clinical and ultrasonographic diagnosis of NAFLD, hemochromatosis gene (HFE) mutations by restriction analysis in 342 patients. Serum hepcidin-25 was measured by time-of-flight mass spectrometry in 143 patients. At multivariate analysis CC-IMT was associated with systolic blood pressure, glucose, LDL cholesterol, abdominal circumference, age, and ferritin (p=0.048). Carotid plaques were independently associated with age, ferritin, glucose, and hypertension. Ferritin reflected iron stores and metabolic syndrome components, but not inflammation or liver damage. Hyperferritinemia was associated with increased vascular damage only in patients with HFE genotypes associated with hepcidin upregulation by iron stores (p<0.0001), and serum hepcidin-25 was independently associated with carotid plaques (p=0.05)., Conclusion: Ferritin levels, reflecting iron stores, are independent predictors of vascular damage in NAFLD. The mechanism may involve upregulation of hepcidin by increased iron stores in patients not carrying HFE mutations, and iron compartmentalization into macrophages., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Background: The prevalence of Streptococcus gallolyticus subsp gallolyticus ( Streptococcus bovis biotype I) endocarditis is in general low but very often linked to colorectal cancer. Therefore, this study aimed to reveal the virulence characteristics that distinguish this opportunistic pathogen from a panel of (closely related) intestinal bacteria., Methods: The route of infection was reconstructed in vitro with adhesion, invasion, and translocation assays on differentiated Caco-2 cells. Furthermore, cellular immune responses upon infection and bacterial biofilm formation were analyzed in a comparative manner., Results: S. gallolyticus subsp gallolyticus strains were demonstrated to have a relative low adhesiveness and could not internalize epithelial cells. However, these bacteria were uniquely able to paracellularly cross a differentiated epithelium without inducing epithelial interleukin 8 or 1β responses. Importantly, they had an outstanding ability to form biofilms on collagen-rich surfaces, which in vivo are found at damaged heart valves and (pre)cancerous sites with a displaced epithelium., Conclusions: Together, these data show that S. gallolyticus subsp gallolyticus has a unique repertoire of virulence factors that facilitate infection through (pre)malignant colonic lesions and subsequently can provide this bacterium with a competitive advantage in (1) evading the innate immune system and (2) forming resistant vegetations at collagen-rich sites in susceptible patients with colorectal cancer., (© The Author 2011. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved.)

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.

Multiple factors drive the progression from healthy mucosa towards sporadic colorectal carcinomas and accumulating evidence associates intestinal bacteria with disease initiation and progression. Therefore, the aim of this study was to provide a first high-resolution map of colonic dysbiosis that is associated with human colorectal cancer (CRC). To this purpose, the microbiomes colonizing colon tumor tissue and adjacent non-malignant mucosa were compared by deep rRNA sequencing. The results revealed striking differences in microbial colonization patterns between these two sites. Although inter-individual colonization in CRC patients was variable, tumors consistently formed a niche for Coriobacteria and other proposed probiotic bacterial species, while potentially pathogenic Enterobacteria were underrepresented in tumor tissue. As the intestinal microbiota is generally stable during adult life, these findings suggest that CRC-associated physiological and metabolic changes recruit tumor-foraging commensal-like bacteria. These microbes thus have an apparent competitive advantage in the tumor microenvironment and thereby seem to replace pathogenic bacteria that may be implicated in CRC etiology. This first glimpse of the CRC microbiome provides an important step towards full understanding of the dynamic interplay between intestinal microbial ecology and sporadic CRC, which may provide important leads towards novel microbiome-related diagnostic tools and therapeutic interventions.

Background: Iron deficiency anemia (IDA) affects many young women in sub-Saharan Africa. Its etiology is multifactorial, but the major cause is low dietary iron bioavailability exacerbated by parasitic infections such as malaria., Objective: We investigated whether asymptomatic Plasmodium falciparum parasitemia in Beninese women would impair absorption of dietary iron or utilization of circulating iron., Design: Iron absorption and utilization from an iron-fortified sorghum-based meal were estimated by using oral and intravenous isotope labels in 23 afebrile women with a positive malaria smear (asexual P. falciparum parasitemia; > 500 parasites/μL blood). The women were studied while infected, treated, and then restudied 10 d after treatment. Iron status, hepcidin, and inflammation indexes were measured before and after treatment., Results: Treatment reduced low-grade inflammation, as reflected by decreases in serum ferritin, C-reactive protein, interleukin-6, interleukin-8, and interleukin-10 (P < 0.05); this was accompanied by a reduction in median serum hepcidin of ≈ 50%, from 2.7 to 1.4 nmol/L (P < 0.005). Treatment decreased serum erythropoietin and growth differentiation factor 15 (P < 0.05). Clearance of parasitemia increased geometric mean dietary iron absorption (from 10.2% to 17.6%; P = 0.008) but did not affect systemic iron utilization (85.0% compared with 83.1%; NS)., Conclusions: Dietary iron absorption is reduced by ≈ 40% in asymptomatic P. falciparum parasitemia, likely because of low-grade inflammation and its modulation of circulating hepcidin. Because asymptomatic parasitemia has a protracted course and is very common in malarial areas, this effect may contribute to IDA and blunt the efficacy of iron supplementation and fortification programs. This trial was registered at clinicaltrials.gov as NCT01108939.