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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/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.)