Culture of a Gastric Non‐Helicobacter pylori Helicobacter from the Stomach of a 14‐Year‐Old Girl

Helicobacter felis belongs to the fastidious gastric non‐Helicobacter pylori helicobacter species that are typically found in the stomach of cats and dogs. These bacteria have the potential to colonize the human stomach and are then associated with gastritis, gastroduodenal ulcers, and MALT lymphoma. Strains cultured from the human stomach are rare. Here, we present the first isolation of H. felis from a gastric biopsy specimen of a 14‐year‐old girl who presented with persistent epigastric pain. The strain was cultured using our routine protocol for H. pylori and identified by phylogenetic analyses of partial urease AB and gyrB gene sequences.     

Cloning, expression and sequencing of Helicobacter felis urease genes

Urease genes from Helicobacter felis were cloned and expressed in Escherichia coli cells. A genomic bank of Sau3A-digested H. felis chromosomal DNA was created using a cosmid vector. Cosmid clones were screened for urease activity following subculture on a nitrogen-limiting medium. Subcloning of DNA from an urease-positive cosmid cione led to the construction of plLL205 (9.5 kb) which conferred a urease activity of 1.2±0.5 μmole urea min^-1 mg^-1 bacterial protein to E. coli HB101 bacteria grown on a nitrogen-limiting medium. Random mutagenesis using a MiniTn3-Km transposable element permitted the identification of three DNA regions on plLL205 which were necessary for the expression of an urease-positive phenotype in E. coii clones. To localize the putative structural genes of H. felis on plLL205, extracts of clones harbouring the mutated copies of the plasmid were analysed by Western blotting with anti-H. felis rabbit serum. One mutant cione did not synthesize the putative UreB subunit of H. felis urease and it was postulated that the transposable element had disrupted the corresponding structural gene. By sequencing the DNA region adjacent to the transposon insertion site two open reading frames, designated ureA and ureB, were identified. The polypeptides encoded by these genes had caicuiated moiecuiar masses of 26 074 and 61 663 Da, respectively, and shared 73.5% and 88.2% identity with the corresponding gene products of Helicobacter pylori urease.     

The Interaction of Helicobacter pylori with the Adherent Mucus Gel Layer Secreted by Polarized HT29-MTX-E12 Cells

Helicobacter pylori colonises the gastric mucosa of humans. The majority of organisms live in mucus. These organisms are an important reservoir for infection of the underlying epithelium. Cell culture models for H. pylori infection do not normally possess a mucus layer. The interaction of H. pylori with TFF1, a member of the trefoil factor family found in gastric mucin, is mediated by lipopolysaccharide. To test the hypothesis that the interaction of H. pylori with TFF1 promotes mucus colonization we characterised the interaction of H. pylori with a mucus secreting cell line, HT29-MTX-E12. An isogenic mutant of H. pylori with truncated core oligosaccharides was produced and binding to TFF1 and ability to colonise HT29-MTX-E12 cells determined. The adherent mucus layer of HT29-MTX-E12 cells contained the gastric mucin MUC5AC and trefoil factors, TFF1 and TFF3. H. pylori was found within the mucus layer in discrete clusters and in close association with TFF1. It also interacted with the membrane bound mucin MUC1 and replicated when co-cultured with the cells. An isogenic mutant of H. pylori with a truncated LPS core did not interact with TFF1, and colonization of HT29-MTX-E12 cells was reduced compared to the wild-type strain (p<0.05). Preincubation of cells with wild type LPS but not with truncated LPS resulted in reduced colonization by H. pylori. These results demonstrate that the interaction of TFF1 with H. pylori is important for colonization of gastric mucus and the core oligosaccharide of H. pylori LPS is critical for this interaction to occur. HT29-MTX-E12 cells are a useful system with which to study the interaction of bacteria with mucosal surfaces and the effect of such interactions on mediating colonization.     

Helicobacter felis�CAssociated Gastric Disease in Microbiota-Restricted Mice

Human Helicobacter pylori infection leads to multiple pathological consequences, including gastritis and adenocarcinoma. Although this association has led to the classification of H. pylori as a type 1 carcinogen, it is not clear if additional nonhelicobacter gastric microbiota play a role in these diseases. In this study, we utilized either specific pathogen-free C57BL/6 mice (B6.SPF) or mice colonized with altered Schaedler flora (B6.ASF) to evaluate the role of nonhelicobacter gastric microbiota in disease development after Helicobacter felis infection. Despite similar histological changes, H. felis persisted in B6.ASF stomachs, while H. felis could no longer be detected in the majority of B6.SPF mice. The B6.SPF mice also acquired multiple Lactobacillus spp. in their stomachs after H. felis infection. Our data indicate that potential mechanisms responsible for the ineffective H. felis clearance in the B6.ASF model include the absence of new gastric microbiota to compete for the gastric niche, the lack of expression of new gastric mucins, and a reduced ratio of H. felis–specific IgG2c:IgG1 serum antibodies. These data suggest that although H. felis is sufficient to initiate gastric inflammation and atrophy, bacterial eradication and the systemic immune response to infection are significantly influenced by pre-existing and acquired gastric microbiota.     

Use of selected lactic acid bacteria in the eradication of Helicobacter pylori infection

Helicobacter pylori is among the major pathogenic bacteria that cause chronic gastritis and peptic ulcer disease and is related to the development of gastric cancer. Several chemicals, including antibiotics, have been used to eradicate H. pylori; however, they do not always curb the infection. Ten representative type strains of lactic acid bacteria (LAB) were screened for antagonism toward H. pylori via inhibition of urease activity. Strains inhibiting the binding of H. pylori to human gastric cell line cells and suppressing H. pylori-induced interleukin-8 (IL-8) production were also screened. Of these, Pediococcus pentosaseus (SL4), which inhibited the adhesion of H. pylori to MKN-45 gastric cancer cells, Bifidobacterium longum (BG7), with urease inhibiting activity, and Lactococcus lactis (SL3), and Enterococcus faecalis (SL5), which suppressed H. pylori-induced IL-8 production within MKN-45 and AGS cells, were selected. In mouse model, these LAB stains in combination significantly suppressed IL-8 levels in serum. Gastric pH also recovered to normal values after the administration of these LAB. These stains effectively suppressed H. pylori viability, although not to the extent of antibiotic treatment. When used as probiotics, LAB may help decrease the occurrence of gastritis and reduce the risk of H. pylori infection without, inducing side effects.     

Streptococcus mitis Induces Conversion of Helicobacter pylori to Coccoid Cells during Co-Culture In Vitro

Helicobacter pylori (H. pylori) is a major gastric pathogen that has been associated with humans for more than 60,000 years. H. pylori causes different gastric diseases including dyspepsia, ulcers and gastric cancers. Disease development depends on several factors including the infecting H. pylori strain, environmental and host factors. Another factor that might influence H. pylori colonization and diseases is the gastric microbiota that was overlooked for long because of the belief that human stomach was a hostile environment that cannot support microbial life. Once established, H. pylori mainly resides in the gastric mucosa and interacts with the resident bacteria. How these interactions impact on H. pylori-caused diseases has been poorly studied in human. In this study, we analyzed the interactions between H. pylori and two bacteria, Streptocccus mitis and Lactobacillus fermentum that are present in the stomach of both healthy and gastric disease human patients. We have found that S. mitis produced and released one or more diffusible factors that induce growth inhibition and coccoid conversion of H. pylori cells. In contrast, both H. pylori and L. fermentum secreted factors that promote survival of S. mitis during the stationary phase of growth. Using a metabolomics approach, we identified compounds that might be responsible for the conversion of H. pylori from spiral to coccoid cells. This study provide evidences that gastric bacteria influences H. pylori physiology and therefore possibly the diseases this bacterium causes.     

Comparison of the Caco-2, HT-29 and the mucus-secreting HT29-MTX intestinal cell models to investigate Salmonella adhesion and invasion

Human intestinal cell models are widely used to study host-enteric pathogen interactions, with different cell lines exhibiting specific characteristics and functions in the gut epithelium. In particular, the presence of mucus may play an important role in adhesion and invasion of pathogens. The aim of this study was to evaluate the suitability of the mucus-secreting HT29-MTX intestinal epithelial cell model to test adhesion and invasion of Salmonella strains and compare with data obtained with the more commonly used Caco-2 and HT-29 models. Adhesion of Salmonella to HT29-MTX cell model was significantly higher, likely due to high adhesiveness to mucins present in the native human mucus layer covering the whole cell surface, compared to the non- and low-mucus producing Caco-2 and HT-29 cell models, respectively. In addition, invasion percentages of some clinical Salmonella strains to HT29-MTX cultures were remarkably higher than to Caco-2 and HT-29 cells suggesting that these Salmonellae have subverted the mucus to enhance pathogenicity. The transepithelial electrical resistances of the infected HT29-MTX cell model decreased broadly and were highly correlated with invasion ability of the strain. Staining of S. Typhimurium-infected cell epithelium confirmed the higher invasion by Salmonella and subsequent disruption of tight junctions of HT29-MTX cell model compared with the Caco-2 and HT-29 cell models. Data from this study suggest that the HT29-MTX cell model, with more physiologically relevant characteristics with the mucus layer formation, could be better suited for studying cells–pathogens interactions.     

Maintenance of Helicobacter pylori cultures in agar stabs

Background: Helicobacter pylori requires frequent passage at 37 °C with reduced oxygen tension to maintain viability, and recovery from frozen stocks can be unpredictable and slow. Agar stab cultures were assessed as a possible means of maintaining viability without the need to passage every 4–7 days. Materials and Methods: Agar stabs prepared from either Brucella or Brain Heart Infusion media were inoculated deeply with H. pylori strains or H. felis and grown under varying conditions for up to 13 weeks. Subcultures were prepared from these stabs at various intervals to test for viability. Results: Established cultures in agar stabs failed to survive at room temperature but did survive at 37 °C with 10% CO₂ for up to 56 days. H. felis remained viable for up to 28 days. No difference was observed between the two media formulations. Conclusion: H. pylori grown in agar stabs remains viable for prolonged periods of time without the need to subculture and may represent an improved method for storing H. pylori for infrequent use.     

In Vitro and In Vivo Inhibition of Helicobacter pylori by Lactobacillus casei Strain Shirota

We studied the potential inhibitory effect of Lactobacillus casei strain Shirota (from the fermented milk product Yakult [Yakult Ltd., Tokyo, Japan]) on Helicobacter pylori by using (i) in vitro inhibition assays with H. pylori SS1 (Sydney strain 1) and nine H. pylori clinical isolates and (ii) the in vivo H. pylori SS1 mouse model of infection over a period of 9 months. In vitro activity against H. pylori SS1 and all of the clinical isolates was observed in the presence of viable L. casei strain Shirota cells but not in the cell-free culture supernatant, although there was profound inhibition of urease activity. In vivo experiments were performed by oral administration of L. casei strain Shirota in the water supply over a period of 9 months to 6-week-old C57BL/6 mice previously infected with H. pylori SS1 (study group; n = 25). Appropriate control groups of H. pylori-infected but untreated animals (n = 25) and uninfected animals given L. casei strain Shirota (n = 25) also were included in the study. H. pylori colonization and development of gastritis were assessed at 1, 2, 3, 6, and 9 months postinfection. A significant reduction in the levels of H. pylori colonization was observed in the antrum and body mucosa in vivo in the lactobacillus-treated study group, as assessed by viable cultures, compared to the levels in the H. pylori-infected control group. This reduction was accompanied by a significant decline in the associated chronic and active gastric mucosal inflammation observed at each time point throughout the observation period. A trend toward a decrease in the anti-H. pylori immunoglobulin G response was measured in the serum of the animals treated with lactobacillus, although this decrease was not significant.     

3-Arylpropionylhydroxamic acid derivatives as Helicobacter pylori urease inhibitors: Synthesis,molecular docking and biological evaluation

Helicobacter pylori urease is involved in several physiologic responses such as stomach and duodenal ulcers, adenocarcinomas and stomach lymphomas. Thus, inhibition of urease is taken for a good chance to treat H. pylori-caused infections, we have therefore focused our efforts on seeking novel urease inhibitors. Here, a series of arylpropionylhydroxamic acids were synthesized and evaluated for urease inhibition. Out of these compounds, 3-(2-benzyloxy-5-chlorophenyl)-3-hydroxypropionylhydroxamic acid (d24) was the most active inhibitor with IC₅₀ of 0.15 ± 0.05 μM, showing a mixed inhibition with both competitive and uncompetitive aspects. Non-linear fitting of kinetic data gives kinetics parameters of 0.13 and 0.12 μg·mL⁻¹ for K_i and K_i′, respectively. The plasma protein binding assays suggested that d24 exhibited moderate binding to human and rabbit plasma proteins.     

Properties of the P-Type ATPases Encoded by the copAP Operons of Helicobacter pylori and Helicobacter felis

The cop operons of Helicobacter pylori and Helicobacter felis were cloned by gene library screening. Both operons contain open reading frames for a P-type ion pump (CopA) with homology to Cd²⁺ and Cu²⁺ ATPases and a putative ion binding protein (CopP), the latter representing a CopZ homolog of the copYZAB operon of Enterococcus hirae. The predicted CopA ATPases contained an N-terminal GMXCXXC ion binding motif and a membrane-associated CPC sequence. A synthetic N-terminal peptide of the H. pylori CopA ATPase bound to Cu²⁺ specifically, and gene disruption mutagenesis of CopA resulted in an enhanced growth sensitivity of H. pylori to Cu²⁺ but not to other divalent cations. As determined experimentally, H. pylori CopA contains four pairs of transmembrane segments (H1 to H8), with the ATP binding and phosphorylation domains lying between H6 and H7, as found for another putative transition metal pump of H. pylori (K. Melchers, T. Weitzenegger, A. Buhmann, W. Steinhilber, G. Sachs, and K. P. Schäfer, J. Biol. Chem. 271:446–457, 1996). The corresponding transmembrane segments of the H. felis CopA pump were identified by hydrophobicity analysis and via sequence similarity. To define functional domains, similarly oriented regions of the two enzymes were examined for sequence identity. Regions with high degrees of identity included the N-terminal Cu²⁺ binding domain, the regions of ATP binding and phosphorylation in the energy transduction domain, and a transport domain consisting of the last six transmembrane segments with conserved cysteines in H4, H6, and H7. The data suggest that H. pylori and H. felis employ conserved mechanisms of ATPase-dependent copper resistance.     

Immunological features and efficacy of a multi-epitope vaccine CTB-UE against H. pylori in BALB/c mice model

Epitope vaccine is a promising option for prophylactic and therapeutic vaccination against Helicobacter pylori infection. Urease is an essential virulence factor and colonization factor for H. pylori. In this study, we constructed a multi-epitope vaccine named CTB-UE with mucosal adjuvant cholera toxin B subunit (CTB) and tandem copies of Th and B cell epitopes from H. pylori urease A and B subunits. The immunogenicity, specificity, ability to induce neutralizing antibodies against H. pylori urease, and prophylactic and therapeutic efficacy of the CTB-UE vaccine were evaluated in BALB/c mice model after purification. The experimental results indicated that CTB-UE could induce comparatively high levels of specific antibodies against native H. pylori urease, UreA, UreB, or the selected B cell epitopes UreA_183–203 and UreB_327–334 involved with the active site of urease and showed an effectively inhibitory effect on the enzymatic activity of urease. Besides, oral prophylactic or therapeutic immunization with CTB-UE significantly decreased H. pylori colonization compared with oral immunization with rUreB or PBS, and the protection was correlated with antigen-specific CD4⁺ T cells and IgG, IgA, and mucosal sIgA antibody responses. This CTB-UE vaccine may be a promising vaccine candidate for the control of H. pylori infection.     

In vitro and in vivo effects of the Mongolian drug Amu‐ru 7 on Helicobacter pylori growth and viability

Amu‐ru 7, a Mongolian folk medicine, is used to treat digestive diseases such as gastritis and gastric and duodenal ulcers. We examined the effect of Amu‐ru 7 on the growth and viability of Helicobacter pylori in vivo and in vitro. By the agar dilution method, the MIC of Amu‐ru 7 for H. pylori strains was shown to be 100–200 μg/mL with a MIC₉₀ of 200 μg/mL. Two hundred micrograms per milliliter of Amu‐ru 7 exhibited potent bactericidal activity against H. pylori in the stationary phase of growth 6 hr after treatment. Amu‐ru 7 inhibited the growth of both AMPC‐resistant and CAM‐resistant strains, and also had a combined effect with AMPC on AMPC‐resistant strain 403. The Amu‐ru 7 inhibited biofilm formation by H. pylori and induced morphological changes, such as bleb‐like formation and shortening of the cell. Although colonization of the stomach of the Mongolian gerbil by H. pylori was not cured by treatment with Amu‐ru 7, both the mean number of H. pylori colonized and the colonization rate were decreased in Amu‐ru 7 treated gerbils. These results suggest the effectiveness Amu‐ru 7 as an adjunct therapy for eradication therapies consisting of a PPI combined with antibiotics.     

Effect of Weissella confusa Strain PL9001 on the Adherence and Growth of Helicobacter pylori

Viable and nonviable Weissella confusa strain PL9001 inhibited the binding of Helicobacter pylori to human gastric-cell line MKN-45 cells by more than 90%. Spent culture supernatant of PL9001 rapidly decreased the viability of H. pylori, rupturing cell walls. The results suggest that PL9001 is a probiotic that can reduce the infectivity and persistence of H. pylori.     

A Novel System of Cytoskeletal Elements in the Human Pathogen Helicobacter pylori

Pathogenicity of the human pathogen Helicobacter pylori relies upon its capacity to adapt to a hostile environment and to escape from the host response. Therefore, cell shape, motility, and pH homeostasis of these bacteria are specifically adapted to the gastric mucus. We have found that the helical shape of H. pylori depends on coiled coil rich proteins (Ccrp), which form extended filamentous structures in vitro and in vivo, and are differentially required for the maintenance of cell morphology. We have developed an in vivo localization system for this pathogen. Consistent with a cytoskeleton-like structure, Ccrp proteins localized in a regular punctuate and static pattern within H. pylori cells. Ccrp genes show a high degree of sequence variation, which could be the reason for the morphological diversity between H. pylori strains. In contrast to other bacteria, the actin-like MreB protein is dispensable for viability in H. pylori, and does not affect cell shape, but cell length and chromosome segregation. In addition, mreB mutant cells displayed significantly reduced urease activity, and thus compromise a major pathogenicity factor of H. pylori. Our findings reveal that Ccrp proteins, but not MreB, affect cell morphology, while both cytoskeletal components affect the development of pathogenicity factors and/or cell cycle progression.     

The CrdRS (HP1365-HP1364) Two-Component System Is Not Involved in pH-Responsive Gene Regulation in the <Emphasis Type="Italic">Helicobacter pylori</Emphasis> Strains 26695 and G27

The human gastric pathogen Helicobacter pylori is extremely well adapted to the highly acidic conditions encountered in the stomach. The pronounced acid resistance of H. pylori relies mainly on the ammonia-producing enzyme urease. However, urease-independent mechanisms are likely to contribute to acid adaptation. pH-responsive gene regulation in this organism is mediated by a two-component system (HP0166-HP0165) designated ArsRS and the metal-dependent regulators NikR and Fur. Recently, it was reported that another two-component system termed CrdRS (HP1365-HP1364) is required for pH-responsive regulation of the major acid-resistance systems in the H. pylori strain J99. By the analysis of crdRS null mutants of the H. pylori strains 26695 and G27, we show that low pH induction of both the urease and the amidase genes occurs in the absence of crdRS in these strains, suggesting substantial strain-specific differences in the regulation of a major virulence determinant.     

Inhibitory effect of Lactobacillus acidophilus on Helicobacter hepaticus in vitro

Helicobacter hepaticus and Helicobacter pylori both belong to Helicobacter species. Strains of Lactobacillus acidophilus, including L4 and L6, have shown significant inhibitory effects on H. pylori. Based on this phenomenon, we aim to investigate the inhibitory effect of L. acidophilus on H. hepaticus. Both standard and isolated H. hepaticus strains were grown under microaerophilic conditions at 37 °C in the presence of L. acidophilus supernatant, or lactic acid. The diameters of the inhibition zones were measured on the solid culture media. In liquid culture, the cell concentrations were measured and the urease activity was determined by phenol red staining. Sixteen strains of L. acidophilus isolated from human feces (named as L1–L16) showed anti-H. hepaticus effects. Two of them (L4 and L6) exhibited the most apparent effects on H. hepaticus inhibition. The L. acidophilus supernatant of L4 and L6 significantly increased the diameters of the inhibition zones compared with that of the lactic acid control (P < 0.05). The inhibitory role of L. acidophilus supernatant was independent of the pH value of solution (P > 0.05). Moreover, in liquid culture, L. acidophilus supernatant significantly reduced the cell growth rate and the urease activity of H. hepaticus cells in a time-dependent pattern (P < 0.05 compared with lactic acid control). No obvious difference was observed between the standard and isolated strain of H. hepaticus (P > 0.05). Our results indicate that L. acidophilus can decrease the viability and urease activity of H. hepaticus in vitro and this inhibition is independent of pH levels. This provides evidence for developing novel approaches for the prevention and treatment of H. hepaticus infection.     

Antagonistic Activity of Lactobacillus acidophilus LB against Intracellular Salmonella enterica Serovar Typhimurium Infecting Human Enterocyte-Like Caco-2/TC-7 Cells

To gain further insight into the mechanism by which lactobacilli develop antimicrobial activity, we have examined how Lactobacillus acidophilus LB inhibits the promoted cellular injuries and intracellular lifestyle of Salmonella enterica serovar Typhimurium SL1344 infecting the cultured, fully differentiated human intestinal cell line Caco-2/TC-7. We showed that the spent culture supernatant of strain LB (LB-SCS) decreases the number of apical serovar Typhimurium-induced F-actin rearrangements in infected cells. LB-SCS treatment efficiently decreased transcellular passage of S. enterica serovar Typhimurium. Moreover, LB-SCS treatment inhibited intracellular growth of serovar Typhimurium, since treated intracellular bacteria displayed a small, rounded morphology resembling that of resting bacteria. We also showed that LB-SCS treatment inhibits adhesion-dependent serovar Typhimurium-induced interleukin-8 production.     

Potential antigen candidates for subunit vaccine development against Helicobacter pylori infection

Helicobacter pylori (H. pylori) is a resident bacterium in the stomach that accounts for 75% cases of gastric cancer. In this review, we comprehensively studied published papers on H. pylori vaccines using Google Scholar and NCBI databases to gather information about vaccines against H. pylori. Considering the pivotal roles of the enzyme urease (in production of NH₃ and neutralization of the acidic medium of the stomach), cytotoxin-associated gene A, and vacuolating cytotoxin A proteins in H. pylori infection, they could be the best candidates for the construction of recombinant vaccines. The outer membrane porins (Hop), blood group antigen-binding adhesin (BabA), sialic acid-binding adhesin (SabA), and outer inflammatory protein A, play significant roles in binding of bacterium to human gastric tissues, and because binding is the first step in bacterial fixation and colonization, these antigens also can be considered as suitable candidates for designing vaccines. Likely, other significant bacterial antigens, such as NapA (chemotactic factor for recruitment of human neutrophils and monocytes to the site of infection), duodenal ulcer promoting protein A (to promote duodenal ulcer), and Hsp60 (as a molecular chaperon for activation of urease enzyme), can be used in the construction of subunit vaccines. New vaccines in use currently, such as DNA vaccines and subunit vaccines, can efficiently replace the dead and attenuated vaccines. Nonetheless, the results show that urease enzyme is most used compared with bacterial components in the designing and construction of recombinant vaccines. The BabA and SabA antigens belong to the outer membrane porins family in H. pylori and are required for binding and fixation of the bacterium to the human gastric tissues.     

Characterization of Caco-2 and HT29-MTX cocultures in an in vitro digestion/cell culture model used to predict iron bioavailability

Cocultures of two human cell lines, Caco-2 and HT29-MTX mucus-producing cells, have been incorporated into an in vitro digestion/cell culture model used to predict iron bioavailability. A range of different foods were subjected to in vitro digestion, and iron bioavailability from digests was assessed with Caco-2, Caco-2 overlaid with porcine mucin, HT29-MTX or cocultures of Caco-2 and HT29-MTX at varying ratios. It was found that increasing the ratio of HT29-MTX cells decreased the amount of ferritin formed and resulted in an overall decline in the ability of the model to detect differences in iron bioavailability. At the physiologically relevant ratios of 90% Caco-2/10% HT29-MTX and 75% Caco-2/25% HT29-MTX, however, a mucus layer completely covered the cell monolayer and the in vitro digestion model was nearly as responsive to changes in sample iron bioavailability as pure Caco-2 cultures. The in vitro digestion/Caco-2 cell culture model correlates well with human iron bioavailability studies, but, as mucus appears to play a role in iron absorption, the addition of a physiologically realistic mucus layer and goblet-type cells to this model may give more accurate iron bioavailability predictions.