Differential Immunogenicity and Lung Disease-Inducing Potential of Mycobacterium immunogenum Genotypes and Impact of Co-Exposure with Pseudomonas: Optimizing a Mouse Model of Chronic Hypersensitivity Pneumonitis.

Mycobacterium immunogenum (MI) colonizing metalworking fluids (MWFs) has been associated with chronic hypersensitivity pneumonitis (HP) in machinists. However, it is etiologically unclear why only certain mycobacteria-contaminated fluids induce this interstitial lung disease. We hypothesized that this may be due to differential immunogenicity and the HP-inducing potential of MI strains/genotypes as well as the confounding effect of co-inhaled endotoxin-producers. To test this hypothesis, we optimized a chronic HP mouse model in terms of MI antigen dose, timepoint of sacrifice, and form of antigen (cell lysates vs. live cells) and compared six different field-isolated MI strains. Overall, MJY10 was identified as the most immunogenic and MJY4 (or MJY13) as the least immunogenic genotype based on lung pathoimmunological changes as well as Th1 cellular response (IFN-γ release). Infection with MI live cells induced a more severe phenotype than MI cell lysate. Co-exposure with Pseudomonas fluorescens caused a greater degree of lung innate immune response and granuloma formation but a diminished adaptive (Th1) immune response (IFN-γ) in the lung and spleen. In summary, this study led to the first demonstration of differential immunogenicity and the disease-inducing potential of field strains of MI and an interfering effect of the co-contaminating Pseudomonas. The improved chronic MI-HP mouse model and the identified polar pair of MI strains will facilitate future diagnostic and therapeutic research on this poorly understood environmental lung disease.

Temporal relationships between Staphylococcus aureus colonization, filaggrin expression, and pediatric atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is characterized by Staphylococcus aureus (S. aureus) colonization. Longitudinal early life data delineating relationships of S. aureus colonization, barrier function, and AD outcomes are lacking. We define longitudinal S. aureus endotypes and AD pathogenesis in early life. METHODS: We defined longitudinal S. aureus skin colonization phenotypes across two annual visits (non-colonized: V1- V2- , early transient: V1+ V2- , late-onset: V1- V2+ , persistent: V1+ V2+ ) in the Mechanisms of Progression of Atopic Dermatitis to Asthma in Children cohort. We analyzed AD severity, sensitization, and skin barrier function across phenotypes, and performed mediation analyses between colonization and FLG expression. RESULTS: Persistent S. aureus colonization was associated with increased SCORAD at V1 (33.5 vs. 19.0, p = .004) and V2 (40.1 vs.16.9, p < .001), and lower non-lesional (NL) FLG at V2 (1.77 vs. 4.09, p = .029) compared to the non-colonized phenotype, with early transient and late-onset colonization as intermediate phenotypes. Children colonized at V2 demonstrated a decrease in NL-FLG expression from V1 to V2 compared to those non-colonized at V2 (p = .0012), who maintained expression. This effect remained significant even after adjusting for V1 colonization and SCORAD (p = .011). CONCLUSIONS: Our findings are the first to present longitudinal quantitative FLG expression and S. aureus skin colonization in early life and suggest that a decrease in NL-FLG drives later colonization. Hence, therapies to maintain NL-FLG expression may prevent S. aureus colonization. Further, a longitudinal AD endotype of persistent colonization is characterized by increased AD severity, sensitization, and decreasing NL-FLG.

Multi-omics profiling approach in food allergy.

The prevalence of food allergy (FA) among children is increasing, affecting nearly 8% of children, and FA is the most common cause of anaphylaxis and anaphylaxis-related emergency department visits in children. Importantly, FA is a complex, multi-system, multifactorial disease mediated by food-specific immunoglobulin E (IgE) and type 2 immune responses and involving environmental and genetic factors and gene-environment interactions. Early exposure to external and internal environmental factors largely influences the development of immune responses to allergens. Genetic factors and gene-environment interactions have established roles in the FA pathophysiology. To improve diagnosis and identification of FA therapeutic targets, high-throughput omics approaches have emerged and been applied over the past decades to screen for potential FA biomarkers, such as genes, transcripts, proteins, and metabolites. In this article, we provide an overview of the current status of FA omics studies, namely genomic, transcriptomic, epigenomic, proteomic, exposomic, and metabolomic. The current development of multi-omics integration of FA studies is also briefly discussed. As individual omics technologies only provide limited information on the multi-system biological processes of FA, integration of population-based multi-omics data and clinical data may lead to robust biomarker discovery that could translate into advances in disease management and clinical care and ultimately lead to precision medicine approaches.

Vitamin D, skin filaggrin, allergic sensitization, and race.

BACKGROUND: In addition to its involvement in both the innate and adaptive immune systems, vitamin D has been found to affect keratinocyte function and proliferation, suggesting a possible role for vitamin D in cutaneous allergic sensitization. OBJECTIVE: To explore the role of circulating vitamin D levels in allergic sensitization. METHODS: Serum 25-hydroxyvitamin D (25(OH)D) levels were measured in a subset of children (N = 323) enrolled in the Mechanisms of Progression of Atopic Dermatitis to Asthma in Children cohort, a prospective early life cohort of children with atopic dermatitis. Allergic sensitization was determined using skin prick testing, and FLG expression in the keratinocytes was measured by quantitative polymerase chain reaction. Multiple Poisson regression was used to evaluate interaction effects between serum 25(OH)D levels and FLG expression with sensitization load as the outcome. RESULTS: Black participants had significantly lower mean levels of serum 25(OH)D compared with non-Black participants (29.3 vs 32.9 ng/mL; P < .001). FLG expression and sensitization load were negatively correlated in non-Black participants with 25(OH)D levels less than 27.2 ng/mL (Rho = -0.45; P = .02). No association between FLG expression and sensitization load was found in Black participants or participants with 25(OH)D levels greater than or equal to 27.2 ng/mL. Multiple Poisson regression models confirmed that 25(OH)D levels interact with FLG expression to affect sensitization load in non-Black participants. CONCLUSION: Despite lower vitamin D levels in Black participants, sensitization load was associated with nonlesional skin FLG expression in non-Black, but not Black, children with low vitamin D levels. Thus, a complex interplay of factors determines the impact of vitamin D on allergic sensitization.

Multi-Omics Profiling Approach to Asthma: An Evolving Paradigm.

Asthma is a complex multifactorial and heterogeneous respiratory disease. Although genetics is a strong risk factor of asthma, external and internal exposures and their interactions with genetic factors also play important roles in the pathophysiology of asthma. Over the past decades, the application of high-throughput omics approaches has emerged and been applied to the field of asthma research for screening biomarkers such as genes, transcript, proteins, and metabolites in an unbiased fashion. Leveraging large-scale studies representative of diverse population-based omics data and integrating with clinical data has led to better profiling of asthma risk. Yet, to date, no omic-driven endotypes have been translated into clinical practice and management of asthma. In this article, we provide an overview of the current status of omics studies of asthma, namely, genomics, transcriptomics, epigenomics, proteomics, exposomics, and metabolomics. The current development of the multi-omics integrations of asthma is also briefly discussed. Biomarker discovery following multi-omics profiling could be challenging but useful for better disease phenotyping and endotyping that can translate into advances in asthma management and clinical care, ultimately leading to successful precision medicine approaches.

TSLP disease-associated genetic variants combined with airway TSLP expression influence asthma risk.

BACKGROUND: Thymic stromal lymphopoietin (TSLP) is an epithelial-derived cytokine important in initiation of allergic inflammation. Single nucleotide polymorphisms (SNPs) in TSLP are associated with asthma, yet studies have shown inconsistent associations between circulating TSLP and asthma. Studies that integrate the combined effects of TSLP genotype, TSLP mRNA, circulating TSLP levels, and asthma outcome are lacking. OBJECTIVES: This study sought to recruit a novel cohort based on asthma-relevant TSLP SNPs and determine their impact on TSLP mRNA expression and TSLP circulating protein levels, and their individual and combined effects on asthma. METHODS: This study developed an algorithm to prioritize TSLP SNPs and recruited 51 carriers and noncarriers based on TSLP genotypes. TSLP mRNA was quantified in nasal epithelial cells and circulating TSLP levels in plasma. This study determined the associations of defined TSLP risk genotypes and/or TSLP mRNA and protein levels with asthma. RESULTS: TSLP mRNA expression, but not circulating TSLP, was significantly increased in people who are asthmatic compared with in people who are nonasthmatic (P = .007; odds ratio, 1.44). Notably, 90% of children with the defined TSLP risk genotypes and high nasal TSLP mRNA expression (top tertile) had asthma compared with 40% of subjects without risk genotypes and with low TSLP expression (bottom tertile) (P = .024). No association between circulating TSLP and asthma was observed. CONCLUSIONS: Collectively, these data suggest childhood asthma is modified by the combined effects of TSLP genotype and TSLP expression in the nasal epithelium. The increased asthma risk likely manifests when genetic variation enables expression quantitative trait loci in the TSLP locus to elevate TSLP. It is important to consider both biomarkers when factoring asthma risk.

Genetics of Food Allergy.

The risk factors for food allergy (FA) include both genetic variants and environmental factors. Advances using both candidate-gene association studies and genome-wide approaches have led to the identification of FA-associated genes involved in immune responses and skin barrier functions. Epigenetic changes have also been associated with the risk of FA. In this chapter, we outline current understanding of the genetics, epigenetics and the interplay with environmental risk factors associated with FA. Future studies of gene-environment interactions, gene-gene interactions, and multi-omics integration may help shed light on the mechanisms of FA, and lead to improved diagnostic and treatment strategies.

Resolving Clinical Phenotypes into Endotypes in Allergy: Molecular and Omics Approaches.

Allergic diseases are highly complex with respect to pathogenesis, inflammation, and response to treatment. Current efforts for allergic disease diagnosis have focused on clinical evidence as a binary outcome. Although outcome status based on clinical phenotypes (observable characteristics) is convenient and inexpensive to measure in large studies, it does not adequately provide insight into the complex molecular determinants of allergic disease. Individuals with similar clinical diagnoses do not necessarily have similar disease etiologies, natural histories, or responses to treatment. This heterogeneity contributes to the ineffective response to treatment leading to an annual estimated cost of $350 billion in the USA alone. There has been a recent focus to deconvolute the clinical heterogeneity of allergic diseases into specific endotypes using molecular and omics approaches. Endotypes are a means to classify patients based on the underlying pathophysiological mechanisms involving distinct functions or treatment response. The advent of high-throughput molecular omics, immunophenotyping, and bioinformatics methods including machine learning algorithms is facilitating the development of endotype-based diagnosis. As we move to the next decade, we should truly start treating clinical endotypes not clinical phenotype. This review highlights current efforts taking place to improve allergic disease endotyping via molecular omics profiling, immunophenotyping, and machine learning approaches in the context of precision diagnostics in allergic diseases. Graphical Abstract.

Second-hand smoke and NFE2L2 genotype interaction increases paediatric asthma risk and severity.

BACKGROUND: Second-hand smoke (SHS) exposure is associated with paediatric asthma, and oxidative stress is believed to play a role in mediating this association. The nuclear factor (erythroid-derived 2)-like 2 (NFE2L2) is important for the defence against oxidative stress. OBJECTIVE: To explore interactions between NFE2L2 genotype and SHS exposure in paediatric asthma risk. METHODS: We used a genotyped subset of patients of European ancestry (N = 669, median age at enrolment = 6.8 years) enrolled in the clinical cohort Greater Cincinnati Pediatric Clinic Repository as the study population, and a population-based paediatric cohort (N = 791) to replicate our findings. History of asthma diagnosis was obtained from medical records, and SHS exposure was obtained from questionnaires. Four NFE2L2 tagging SNPs were included in the analysis, and interactions between SHS and NFE2L2 genotype were evaluated using logistic regression. RESULTS: Three of the analysed SNPs, rs10183914, rs1806649 and rs2886161, interacted significantly with SHS exposure to increase asthma risk (p ≤ .02). The interaction was replicated in an independent cohort for rs10183914 (p = .04). Interactions between SHS exposure and NFE2L2 genotype were also associated with an increased risk of hospitalization (p = .016). In stratified analyses, NFE2L2 genotype was associated with daily asthma symptoms in children with SHS exposure (OR = 3.1; p = .048). No association was found in children without SHS exposure. Examination of publicly available chromatin immunoprecipitation followed by sequencing (ChIP-seq) data sets confirmed the presence of active histone marks and binding sites for particular transcription factors overlapping the coordinates for the significantly associated SNPs. CONCLUSIONS AND CLINICAL RELEVANCE: Our study provides evidence that NFE2L2 genotype interacts with SHS exposure to affect both asthma risk and severity in children and identifies a population of children at increased risk of asthma development.

Disease-associated KIF3A variants alter gene methylation and expression impacting skin barrier and atopic dermatitis risk.

Single nucleotide polymorphisms (SNPs) in the gene encoding kinesin family member 3A, KIF3A, have been associated with atopic dermatitis (AD), a chronic inflammatory skin disorder. We find that KIF3A SNP rs11740584 and rs2299007 risk alleles create cytosine-phosphate-guanine sites, which are highly methylated and result in lower KIF3A expression, and this methylation is associated with increased transepidermal water loss (TEWL) in risk allele carriers. Kif3aK14∆/∆ mice have increased TEWL, disrupted junctional proteins, and increased susceptibility to develop AD. Thus, KIF3A is required for skin barrier homeostasis whereby decreased KIF3A skin expression causes disrupted skin barrier function and promotes development of AD.

Identification of two early life eczema and non-eczema phenotypes with high risk for asthma development.

BACKGROUND: The "atopic march" has been considered a linear progression starting with eczema and culminating with development of asthma. Not all asthma cases, however, are preceded by eczema, and not all children with eczema go on to develop asthma. OBJECTIVE: The aim of this study was to explore the impact of allergic sensitization patterns on the association between early eczema and later childhood asthma. Given the numerous reported associations of the ciliary gene KIF3A with the atopic march, we also examined the impact of KIF3A risk allele rs12186803 on our analyses. METHODS: We studied 505 participants in the Cincinnati Childhood Allergy and Air Pollution Study (CCAAPS), a prospective birth cohort, with longitudinal eczema and asthma outcomes as well as prospective data regarding timing of sensitization to foods and aeroallergens. KIF3A genotypes were available on all children. RESULTS: Two high-risk groups were identified: one with and one without early eczema. The high-risk group with early eczema was more likely to be sensitized to food allergens, while the group without early eczema was more likely to be polysensitized to aeroallergens. The KIF3A rs12186803 risk allele interacted with food sensitization to increase asthma risk in children with eczema (P = 0.02). In children without eczema, asthma was associated with the interaction between rs12186803 and aeroallergen sensitization (P = 0.007). CONCLUSIONS & CLINICAL RELEVANCE: KIF3A interacted differentially with sensitization pattern to increase the risk of asthma in two high-risk groups of children with and without early eczema. Given the reported role of KIF3A in epithelial cell functioning, the results add evidence to the hypothesis that an impaired epithelial barrier is a key aspect in the development of allergic disease.

NFAT-regulated cytokine gene expression during tacrolimus therapy early after renal transplantation.

AIMS: Despite pharmacokinetic monitoring of calcineurin inhibitors, the long-term outcome after transplantation (Tx) is still hampered by the side effects of these drugs. The aim of the present study was to characterize nuclear factor of activated T cells (NFAT)-regulated gene expression as a potential pharmacodynamic biomarker for further individualization of tacrolimus (Tac) therapy. METHODS: In 29 renal allograft recipients, samples were drawn once pre-Tx, and before and 1.5 h after Tac dosing at approximately 1 week, 6 weeks and 1 year post-Tx. Tac concentrations were measured by immunoassay, while the expression of genes encoding NFAT-regulated cytokines [interleukin 2 (IL2), interferon gamma (IFNG), colony stimulating factor 2 (CSF2)] and cytochrome P450 3A5 (CYP3A5) genotyping were determined by real-time polymerase chain reaction. RESULTS: The cytokine response after Tac dosing varied up to 46-fold between patients and changed significantly with time post-engraftment. Tac concentrations 1.5 h postdose (C1.5 ) >15 µg l-1 were associated with strong cytokine inhibition and residual gene expression (RGE) ≤10%, while lower Tac C1.5 resulted in more variable responses (RGE 2.5-68.7%). Patients with ongoing subclinical acute rejection (n = 5) demonstrated limited cytokine inhibition (RGE 39.7-72.6%), while patients with polyoma virus viraemia (n = 3) had relatively strong inhibition of cytokines (RGE 2.5-32.5%). By contrast, there was no association between Tac exposure and rejection or viraemia. CONCLUSIONS: The findings of our study support the potential of NFAT-regulated gene expression measurements as a pharmacodynamic tool for additional monitoring of Tac therapy, especially in the context of overimmunosuppression and viraemia.

Early immunopathological events in acute model of mycobacterial hypersensitivity pneumonitis in mice.

Prolonged exposure to antigens of non-tuberculous mycobacteria species colonizing industrial metalworking fluid (MWF), particularly Mycobacterium immunogenum (MI), has been implicated in chronic forms of hypersensitivity pneumonitis (HP) in machinists based on epidemiology studies and long-term exposure of mouse models. However, a role of short-term acute exposure to these antigens has not been described in the context of acute forms of HP. This study investigated short-term acute exposure of mice to MI cell lysate (or live cell suspension) via oropharyngeal aspiration. The results showed there was a dose- and time-dependent increase (peaking at 2 h post-instillation) in lung immunological responses in terms of the pro- (TNFα, IL-6, IL-1ß) and anti-inflammatory (IL-10) cytokines. Bronchoalveolar lavage and histology showed neutrophils as the predominant infiltrating cell type, with lymphocytes <5% at all timepoints or concentrations. Granulomatous inflammation peaked between 8 and 24 h post-exposure, and resolved by 96 h. Live bacterial challenge, typically encountered in real-world exposures, showed no significant differences from bacterial lysate except for induction of appreciable levels of interferon (IFN)-γ, implying additional immunogenic potential. Collectively, the short-term mycobacterial challenge in mice led to a transient early immunopathologic response, with little adaptive immunity, which is consistent with events associated with human acute forms of HP. Screening of MWF-originated mycobacterial genotypes/variants (six of MI, four of M. chelonae, two of M. abscessus) showed both inter- and intra-species differences, with MI genotype MJY10 being the most immunogenic. In conclusion, this study characterized the first short-term mycobacterial exposure mouse model that mimics acute HP in machinists; this could serve as a potentially useful model for rapid screening of field MWF-associated mycobacteria for routine and timely occupational risk assessment and for investigating early biomarkers and mechanisms of this understudied immune lung disease.

Resolving the etiology of atopic disorders by using genetic analysis of racial ancestry.

Atopic dermatitis (AD), food allergy, allergic rhinitis, and asthma are common atopic disorders of complex etiology. The frequently observed atopic march from early AD to asthma, allergic rhinitis, or both later in life and the extensive comorbidity of atopic disorders suggest common causal mechanisms in addition to distinct ones. Indeed, both disease-specific and shared genomic regions exist for atopic disorders. Their prevalence also varies among races; for example, AD and asthma have a higher prevalence in African Americans when compared with European Americans. Whether this disparity stems from true genetic or race-specific environmental risk factors or both is unknown. Thus far, the majority of the genetic studies on atopic diseases have used populations of European ancestry, limiting their generalizability. Large-cohort initiatives and new analytic methods, such as admixture mapping, are currently being used to address this knowledge gap. Here we discuss the unique and shared genetic risk factors for atopic disorders in the context of ancestry variations and the promise of high-throughput "-omics"-based systems biology approach in providing greater insight to deconstruct their genetic and nongenetic etiologies. Future research will also focus on deep phenotyping and genotyping of diverse racial ancestry, gene-environment, and gene-gene interactions.

Association of Streptomyces community composition determined by PCR-denaturing gradient gel electrophoresis with indoor mold status.

Both Streptomyces species and mold species have previously been isolated from moisture-damaged building materials; however, an association between these two groups of microorganisms in indoor environments is not clear. In this study, we used a culture-independent method, PCR-denaturing gradient gel electrophoresis (PCR-DGGE), to investigate the composition of the Streptomyces community in house dust. Twenty-three dust samples each from two sets of homes categorized as high-mold and low-mold based on mold-specific quantitative PCR analysis were used in the study. Taxonomic identification of prominent bands was performed by cloning and sequencing. Associations between DGGE amplicon band intensities and home mold status were assessed using univariate analyses as well as multivariate recursive partitioning (decision trees) to test the predictive value of combinations of bands intensities. In the final classification tree, a combination of two bands was significantly associated with mold status of the home (p = 0.001). The sequence corresponding to one of the bands in the final decision tree matched a group of Streptomyces species that included Streptomyces coelicolor and Streptomyces sampsonii, both of which have been isolated from moisture-damaged buildings previously. The closest match for the majority of sequences corresponding to a second band consisted of a group of Streptomyces species that included Streptomyces hygroscopicus, an important producer of antibiotics and immunosuppressors. Taken together, the study showed that DGGE can be a useful tool for identifying bacterial species that may be more prevalent in mold-damaged buildings.

Microbial content of household dust associated with exhaled NO in asthmatic children.

Exhaled nitric oxide (eNO) is increasingly used as a non-invasive measure of airway inflammation. Despite this, little information exists regarding the potential effects of indoor microbial components on eNO. We determined the influence of microbial contaminants in house dust and other indoor environmental characteristics on eNO levels in seven-year-olds with and without a physician-diagnosis of asthma. The study included 158 children recruited from a birth cohort study, and 32 were physician-diagnosed as asthmatic. The relationship between eNO levels and exposures to home dust streptomycetes, endotoxin, and molds was investigated. Streptomycetes and endotoxin were analyzed both as loads and concentrations in separate models. Dog, cat, and dust mite allergens also were evaluated. In the multivariate exposure models, high streptomycetes loads and concentrations were significantly associated with a decrease in eNO levels in asthmatic (p<0.001) but not in healthy children. The presence of dog allergen, however, was associated with increased levels of eNO (p=0.001). Dust endotoxin was not significant. The relationship between eNO and indoor exposure to common outdoor molds was u-shaped. In non-asthmatic children, none of the exposure variables was significantly associated with eNO levels. To our knowledge, this is the first study demonstrating a significant association between microbial components in the indoor environment and eNO levels in asthmatic children. This study demonstrates the importance of simultaneously assessing multiple home exposures of asthmatic children to better understand opposing effects. Common components of the indoor Streptomyces community may beneficially influence airway inflammation.

Hypoxic conditions induce a cancer-like phenotype in human breast epithelial cells.

INTRODUCTION: Solid tumors are less oxygenated than their tissue of origin. Low intra-tumor oxygen levels are associated with worse outcome, increased metastatic potential and immature phenotype in breast cancer. We have reported that tumor hypoxia correlates to low differentiation status in breast cancer. Less is known about effects of hypoxia on non-malignant cells. Here we address whether hypoxia influences the differentiation stage of non-malignant breast epithelial cells and potentially have bearing on early stages of tumorigenesis. METHODS: Normal human primary breast epithelial cells and immortalized non-malignant mammary epithelial MCF-10A cells were grown in a three-dimensional overlay culture on laminin-rich extracellular matrix for up to 21 days at normoxic or hypoxic conditions. Acinar morphogenesis and expression of markers of epithelial differentiation and cell polarization were analyzed by immunofluorescence, immunohistochemistry, qPCR and immunoblot. RESULTS: In large ductal carcinoma in situ patient-specimens, we find that epithelial cells with high HIF-1α levels and multiple cell layers away from the vasculature are immature compared to well-oxygenated cells. We show that hypoxic conditions impaired acinar morphogenesis of primary and immortalized breast epithelial cells grown ex vivo on laminin-rich matrix. Normoxic cultures formed polarized acini-like spheres with the anticipated distribution of marker proteins associated with mammary epithelial polarization e.g. α6-integrin, laminin 5 and Human Milk Fat Globule/MUC1. At hypoxia, cells were not polarized and the sub-cellular distribution pattern of the marker proteins rather resembled that reported in vivo in breast cancer. The hypoxic cells remained in a mitotic state, whereas proliferation ceased with acinar morphogenesis at normoxia. We found induced expression of the differentiation repressor ID1 in the undifferentiated hypoxic MCF-10A cell structures. Acinar morphogenesis was associated with global histone deacetylation whereas the hypoxic breast epithelial cells showed sustained global histone acetylation, which is generally associated with active transcription and an undifferentiated proliferative state.

ZIP14 and ZIP8 zinc/bicarbonate symporters in Xenopus oocytes: characterization of metal uptake and inhibition.

The highly conserved human and mouse SLC39A14 and SLC39A8 genes encode the ZIP14 and ZIP8 transporters, respectively-functioning as divalent cation/bicarbonate symporters and expressed in dozens of tissues. Due to alternative splicing of exons 4, human and mouse SLC39A14 genes each encode two distinct gene products, whereas SLC39A8 produces a single product. This lab previously noted that ZIP14A and ZIP14B show highly variable expression in different cell types, suggesting differences in metal uptake function. We ligated mouse ZIP14A, ZIP14B and ZIP8 cDNA coding regions into the Xenopus-specific vector pXFRM, transcribed these in vitro, and microinjected the capped RNAs into Xenopus oocytes. K(m) and V(max) values for Cd, Zn and Fe uptake were determined. Electrogenicity studies using a potassium gradient confirmed that (just as we found previously for ZIP8) ZIP14A- and ZIP14B-mediated divalent Cd- or Zn-bicarbonate complexes are electroneutral. Competitive inhibition of Cd and Zn uptake with ten additional divalent cations showed a unique gradient of patterns for each of ZIP14A, ZIP14B and ZIP8. ZIP14 proteins are prominent in the gastrointestinal tract and ZIP8 protein is located on the surface of renal proximal tubular epithelial cells. It is known that renal Fanconi syndrome can be caused by five nonessential heavy metals: Cd(2+), Hg(2+), Pb(2+), Pt(2+) and U(2+). In the present study we show that these five divalent cations are usually competitors of ZIP14- and/or ZIP8-mediated Zn uptake; our data thus support the possible involvement of intestinal ZIP14 for uptake of these five metals into the body and ZIP8 for efficient uptake into the kidney.