Novel Bacteroides Vulgatus strain protects against gluten-induced break of human celiac gut epithelial homeostasis: a pre-clinical proof-of-concept study.

BACKGROUND AND AIMS: We have identified a decreased abundance of microbial species known to have a potential anti-inflammatory, protective effect in subjects that developed Celiac Disease (CeD) compared to those who did not. We aim to confirm the potential protective role of one of these species, namely Bacteroides vulgatus, and to mechanistically establish the effect of bacterial bioproducts on gluten-dependent changes on human gut epithelial functions. METHODS: We identified, isolated, cultivated, and sequenced a unique novel strain (20220303-A2) of B. vulgatus found only in control subjects. Using a human gut organoid system developed from pre-celiac patients, we monitored epithelial phenotype and innate immune cytokines at baseline, after exposure to gliadin, or gliadin plus B. vulgatus cell free supernatant (CFS). RESULTS: Following gliadin exposure, we observed increases in epithelial cell death, epithelial monolayer permeability, and secretion of pro-inflammatory cytokines. These effects were mitigated upon exposure to B. vulgatus 20220303-A2 CFS, which had matched phenotype gene product mutations. These protective effects were mediated by epigenetic reprogramming of the organoids treated with B. vulgatus CFS. CONCLUSIONS: We identified a unique strain of B. vulgatus that may exert a beneficial role by protecting CeD epithelium against a gluten-induced break of epithelial tolerance through miRNA reprogramming. IMPACT: Gut dysbiosis precedes the onset of celiac disease in genetically at-risk infants. This dysbiosis is characterized by the loss of protective bacterial strains in those children who will go on to develop celiac disease. The paper reports the mechanism by which one of these protective strains, B. vulgatus, ameliorates the gluten-induced break of gut epithelial homeostasis by epigenetically re-programming the target intestinal epithelium involving pathways controlling permeability, immune response, and cell turnover.

Zonulin as a Biomarker for the Development of Celiac Disease.

OBJECTIVES: Increased intestinal permeability seems to be a key factor in the pathogenesis of autoimmune diseases, including celiac disease (CeD). However, it is unknown whether increased permeability precedes CeD onset. This study's objective was to determine whether intestinal permeability is altered before celiac disease autoimmunity (CDA) in at-risk children. We also examined whether environmental factors impacted zonulin, a widely used marker of gut permeability. METHODS: We evaluated 102 children in the CDGEMM study from 2014-2022. We included 51 CDA cases and matched controls, who were enrolled for 12 months or more and consumed gluten. We measured serum zonulin from age 12 months to time of CDA onset, and the corresponding time point in controls, and examined clinical factors of interest. We ran a mixed-effects longitudinal model with dependent variable zonulin. RESULTS: Children who developed CDA had a significant increase in zonulin in the 18.3 months (range 6-78) preceding CDA compared to those without CDA (slope differential = ß = 0.1277, 95% CI: 0.001, 0.255). Among metadata considered, zonulin trajectory was only influenced by increasing number of antibiotic courses, which increased the slope of trajectory of zonulin over time in CDA subjects (P = .04). CONCLUSIONS: Zonulin levels significantly rise in the months that precede CDA diagnosis. Exposure to a greater number of antibiotic courses was associated with an increase in zonulin levels in CDA subjects. This suggests zonulin may be used as a biomarker for preclinical CeD screening in at-risk children, and multiple antibiotic courses may increase their risk of CDA by increasing zonulin levels.

Cohort profile: Celiac disease genomic, environmental, microbiome and metabolome study; a prospective longitudinal birth cohort study of children at-risk for celiac disease.

The Celiac Disease Genomic, Environmental, Microbiome and Metabolomic (CDGEMM) study is an international prospective birth cohort in children at-risk of developing celiac disease (CD). The CDGEMM study has been designed to take a multi-omic approach to predicting CD onset in at-risk individuals. Participants are required to have a first-degree family member with biopsy diagnosed CD and must be enrolled prior to the introduction of solid food. Participation involves providing blood and stool samples longitudinally over a period of five years as well as answering questionnaires related to the participant, their family, and environment. Recruitment and data collection have been ongoing since 2014. As of 2022 we have a total of 554 participants and the average age of the cohort is 56.4 months. A total of 54 participants have developed positive antibodies for CD and 31 have confirmed CD. Approximately 80% of the 54 participants with CD have developed it by 3 years of age. To date we have identified several microbial strains, pathways, and metabolites occurring in increased abundance and detected before CD onset, which have previously been linked to autoimmune and inflammatory conditions while others occurred in decreased abundance before CD onset and are known to have anti-inflammatory effects. Our ongoing analysis includes expanding our metagenomic and metabolomic analyses, evaluating environmental risk factors linked to CD onset, and mechanistic studies investigating how alterations in the microbiome and metabolites may protect against or contribute to CD development.

Early Antibody Dynamics in a Prospective Cohort of Children At Risk of Celiac Disease.

INTRODUCTION: The purpose of this study was to identify possible serum biomarkers predicting celiac disease (CD) onset in children at risk. METHODS: A subgroup from an ongoing, international prospective study of children at risk of CD was classified according to an early trajectory of deamidated gliadin peptides (DGPs) immunoglobulin (Ig) G and clinical outcomes (CD, potential CD, and CD autoimmunity). RESULTS: Thirty-eight of 325 children developed anti-tissue transglutaminase IgA antibody (anti-tTG IgA) seroconversion. Twenty-eight of 38 children (73.6%) showed an increase in anti-DGPs IgG before their first anti-tTG IgA seroconversion. DISCUSSION: Anti-DGPs IgG can represent an early preclinical biomarker predicting CD onset in children at risk.

Budesonide and the Gluten Containing Elimination Diet as Treatments for Non-responsive Celiac Disease in Children.

OBJECTIVES: Non-responsive celiac disease (NRCD) is defined as patients having persistent symptoms and enteropathy (Marsh 3 histology) suggestive of active celiac disease (CeD), after following a gluten-free diet (GFD) for at least 12 months. NRCD is suggested to affect 15% of children with CeD but data are limited and there is no research to date describing treatment of children with this condition. The aim of this study was to describe our center's approach to identifying and treating NRCD with budesonide and the Gluten Containing Elimination Diet (GCED). METHODS: We performed a retrospective, single center analysis over a 5-year period of patients with CD less than 18 years of age (inclusive) who underwent treatment for persistent symptoms and enteropathy despite following a GFD. RESULTS: We identified 22 patients with NRCD. Thirteen were treated with the GCED for 3 months with 46% achieving both histological and symptomatic resolution. Nine patients were treated with budesonide (6-9 mg), with 89% achieving both symptomatic and histologic resolution after a median 3-month treatment course. Further, 67% of patients who responded to the GCED and 100% of patients who responded to budesonide remained in remission for at least 6 months following treatment transition back to exclusive GFD. CONCLUSIONS: The GCED and budesonide can provide benefit for NRCD. Most patients with NRCD can return to a GFD after 3 months of treatment.

Estimating typhoid incidence from community-based serosurveys: a multicohort study.

BACKGROUND: The incidence of enteric fever, an invasive bacterial infection caused by typhoidal Salmonellae (Salmonella enterica serovars Typhi and Paratyphi), is largely unknown in regions without blood culture surveillance. The aim of this study was to evaluate whether new diagnostic serological markers for typhoidal Salmonella can reliably estimate population-level incidence. METHODS: We collected longitudinal blood samples from patients with blood culture-confirmed enteric fever enrolled from surveillance studies in Bangladesh, Nepal, Pakistan, and Ghana between 2016 and 2021 and conducted cross-sectional serosurveys in the catchment areas of each surveillance site. We used ELISAs to measure quantitative IgA and IgG antibody responses to hemolysin E and S Typhi lipopolysaccharide. We used Bayesian hierarchical models to fit two-phase power-function decay models to the longitudinal antibody responses among enteric fever cases and used the joint distributions of the peak antibody titres and decay rate to estimate population-level incidence rates from cross-sectional serosurveys. FINDINGS: The longitudinal antibody kinetics for all antigen-isotypes were similar across countries and did not vary by clinical severity. The seroincidence of typhoidal Salmonella infection among children younger than 5 years ranged between 58·5 per 100 person-years (95% CI 42·1-81·4) in Dhaka, Bangladesh, to 6·6 per 100 person-years (4·3-9·9) in Kavrepalanchok, Nepal, and followed the same rank order as clinical incidence estimates. INTERPRETATION: The approach described here has the potential to expand the geographical scope of typhoidal Salmonella surveillance and generate incidence estimates that are comparable across geographical regions and time. FUNDING: Bill & Melinda Gates Foundation. TRANSLATIONS: For the Nepali, Bengali and Urdu translations of the abstract see Supplementary Materials section.

Zonulin Antagonist, Larazotide (AT1001), As an Adjuvant Treatment for Multisystem Inflammatory Syndrome in Children: A Case Series.

OBJECTIVES: A recent study suggests that Multisystem Inflammatory Syndrome in Children (MIS-C) is triggered by gastrointestinal breach of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral particles from the gut lumen into systemic circulation. The virus remains in the gut weeks to months after respiratory infection, causing zonulin release from the intestinal epithelial cells. Zonulin loosens tight junctions, permitting trafficking of highly inflammatory viral particles into circulation. Current MIS-C treatments target the subsequent immune hyperactivation, not the causative loss of mucosal barrier integrity. Larazotide, a zonulin inhibitor, prevents breakdown of tight junctions, limiting antigen trafficking. DESIGN: Children with MIS-C were treated with larazotide as an adjuvant to steroid/intravenous immunoglobulin therapy. Clinical outcomes, SARS-CoV-2 antigenemia, and cytokine profiles are reported. Outcomes were compared with children with MIS-C receiving steroids and/or IVIG therapy alone. PATIENTS: Four children with MIS-C, ages 3-17 years, were enrolled. INTERVENTIONS: Patients were treated with open label larazotide 10 mcg/kg (maximum 500 mcg/dose) orally four times daily for 21 days. MEASUREMENTS AND MAIN RESULTS: All four patients tolerated larazotide without adverse effects and displayed reduction in Spike antigenemia to undetectable levels. When compared with 22 children with MIS-C receiving steroids and/or intravenous immunoglobulin therapy alone, larazotide-treated patients reported significantly improved time to resolution of gastrointestinal symptoms (p = 0.03), and time to clearance of Spike antigenemia (p = 0.04), plus a trend towards shorter length of stay. CONCLUSIONS: Larazotide appears safe and well-tolerated and may offer potential benefit as an adjuvant to immune-targeted therapies. Expansion of clinical trials is urgently needed to ascertain the clinical impact of larazotide on MIS-C.

Characterization of the blood microbiota in children with Celiac disease.

Celiac Disease (CD) is an autoimmune disorder triggered by gluten ingestion that can develop in genetically predisposed individuals. Alterations in the gut microbiota have been suggested to contribute to development of autoimmune conditions including CD. Recent work suggests the existence of a blood microbiota. Evidence that alterations in the blood microbiota potentially influence the development of chronic immune based diseases is increasing. However, there is no published literature regarding the blood microbiota in children, including those with CD. This study aimed to characterize the diversity and taxonomic composition of the blood microbiota of children with CD compared to controls. Whole blood samples were collected from children with active CD, CD in remission, and control subjects and 16S rRNA sequencing was utilized to analyze the blood microbiota. We found 16s rRNA present throughout all pediatric blood samples, providing evidence for the presence of a pediatric blood microbiota. We found significant differences in beta diversity and in abundance of certain taxa (Campylobacterales order, Odoribacteraceae and Helicobacteraceae families, Odoribacter genus and species, and Bacteroides acidifaciens species) between subjects with active CD and controls. These taxa have been previously reported to be associated with immune response and gut-inflammatory diseases. We did not find significant differences between subjects with active and remission CD or between remission CD and controls. Conclusions: We provide evidence for a pediatric blood microbiota and identified higher beta diversity and alterations in the composition of blood microbiota in subjects with active CD compared to controls.

Corrigendum: Immunological Impact of a Gluten-Free Dairy-Free Diet in Children With Kidney Disease: A Feasibility Study.

[This corrects the article DOI: 10.3389/fimmu.2021.624821.].

Microbiota and Metabolomic Patterns in the Breast Milk of Subjects with Celiac Disease on a Gluten-Free Diet.

The intestinal microbiome may trigger celiac disease (CD) in individuals with a genetic disposition when exposed to dietary gluten. Research demonstrates that nutrition during infancy is crucial to the intestinal microbiome engraftment. Very few studies to date have focused on the breast milk composition of subjects with a history of CD on a gluten-free diet. Here, we utilize a multi-omics approach with shotgun metagenomics to analyze the breast milk microbiome integrated with metabolome profiling of 36 subjects, 20 with CD on a gluten-free diet and 16 healthy controls. These analyses identified significant differences in bacterial and viral species/strains and functional pathways but no difference in metabolite abundance. Specifically, three bacterial strains with increased abundance were identified in subjects with CD on a gluten-free diet of which one (Rothia mucilaginosa) has been previously linked to autoimmune conditions. We also identified five pathways with increased abundance in subjects with CD on a gluten-free diet. We additionally found four bacterial and two viral species/strains with increased abundance in healthy controls. Overall, the differences observed in bacterial and viral species/strains and in functional pathways observed in our analysis may influence microbiome engraftment in neonates, which may impact their future clinical outcomes.

Microbiome signatures of progression toward celiac disease onset in at-risk children in a longitudinal prospective cohort study.

Other than exposure to gluten and genetic compatibility, the gut microbiome has been suggested to be involved in celiac disease (CD) pathogenesis by mediating interactions between gluten/environmental factors and the host immune system. However, to establish disease progression markers, it is essential to assess alterations in the gut microbiota before disease onset. Here, a prospective metagenomic analysis of the gut microbiota of infants at risk of CD was done to track shifts in the microbiota before CD development. We performed cross-sectional and longitudinal analyses of gut microbiota, functional pathways, and metabolites, starting from 18 mo before CD onset, in 10 infants who developed CD and 10 matched nonaffected infants. Cross-sectional analysis at CD onset identified altered abundance of six microbial strains and several metabolites between cases and controls but no change in microbial species or pathway abundance. Conversely, results of longitudinal analysis revealed several microbial species/strains/pathways/metabolites occurring in increased abundance and detected before CD onset. These had previously been linked to autoimmune and inflammatory conditions (e.g., Dialister invisus, Parabacteroides sp., Lachnospiraceae, tryptophan metabolism, and metabolites serine and threonine). Others occurred in decreased abundance before CD onset and are known to have anti-inflammatory effects (e.g., Streptococcus thermophilus, Faecalibacterium prausnitzii, and Clostridium clostridioforme). Additionally, we uncovered previously unreported microbes/pathways/metabolites (e.g., Porphyromonas sp., high mannose-type N-glycan biosynthesis, and serine) that point to CD-specific biomarkers. Our study establishes a road map for prospective longitudinal study designs to better understand the role of gut microbiota in disease pathogenesis and therapeutic targets to reestablish tolerance and/or prevent autoimmunity.

Immunological Impact of a Gluten-Free Dairy-Free Diet in Children With Kidney Disease: A Feasibility Study.

Kidney disease affects 10% of the world population and is associated with increased mortality. Steroid-resistant nephrotic syndrome (SRNS) is a leading cause of end-stage kidney disease in children, often failing standard immunosuppression. Here, we report the results of a prospective study to investigate the immunological impact and safety of a gluten-free and dairy-free (GF/DF) diet in children with SRNS. The study was organized as a four-week summer camp implementing a strict GF/DF diet with prospective collection of blood, urine and stool in addition to whole exome sequencing WES of DNA of participants. Using flow cytometry, proteomic assays and microbiome metagenomics, we show that GF/DF diet had a major anti-inflammatory effect in all participants both at the protein and cellular level with 4-fold increase in T regulatory/T helper 17 cells ratio and the promotion of a favorable regulatory gut microbiota. Overall, GF/DF can have a significant anti-inflammatory effect in children with SRNS and further trials are warranted to investigate this potential dietary intervention in children with SRNS.

An updated overview on celiac disease: from immuno-pathogenesis and immuno-genetics to therapeutic implications.

INTRODUCTION: Celiac disease (CD) is an autoimmune enteropathy triggered by ingestion of gluten. While presenting many similarities with other autoimmune diseases, celiac disease is unique in that the external trigger, gluten, and the genetic background necessary for disease development (HLA DQ2/DQ8) are well described. The prevalence of celiac disease is dramatically increasing over the years and new epidemiologic data show changes regarding age of onset and symptoms. A better understanding of CD-pathogenesis is fundamental to highlight the reasons of this rise of celiac diagnoses. AREAS COVERED: In this review we describe CD-pathogenesis by dissecting all the components necessary to lose tolerance to gluten (ingestion of gluten, genetic predisposition, loss of barrier function and immune response). Additionally, we also highlight the role that microbiome plays in celiac disease as well as new proposed therapies and experimental tools. EXPERT OPINION: Prevalence of autoimmune diseases is increasing around the world. As a result, modern society is strongly impacted by a social and economic burden. Given the unique characteristics of celiac disease, a better understanding of its pathogenesis and the factors that contribute to it may shed light on other autoimmune diseases for which external trigger and genetic background are not known.

Multi-omics analysis reveals the influence of genetic and environmental risk factors on developing gut microbiota in infants at risk of celiac disease.

BACKGROUND: Celiac disease (CD) is an autoimmune digestive disorder that occurs in genetically susceptible individuals in response to ingesting gluten, a protein found in wheat, rye, and barley. Research shows that genetic predisposition and exposure to gluten are necessary but not sufficient to trigger the development of CD. This suggests that exposure to other environmental stimuli early in life, e.g., cesarean section delivery and exposure to antibiotics or formula feeding, may also play a key role in CD pathogenesis through yet unknown mechanisms. Here, we use multi-omics analysis to investigate how genetic and early environmental risk factors alter the development of the gut microbiota in infants at risk of CD. RESULTS: Toward this end, we selected 31 infants from a large-scale prospective birth cohort study of infants with a first-degree relative with CD. We then performed rigorous multivariate association, cross-sectional, and longitudinal analyses using metagenomic and metabolomic data collected at birth, 3 months and 6 months of age to explore the impact of genetic predisposition and environmental risk factors on the gut microbiota composition, function, and metabolome prior to the introduction of trigger (gluten). These analyses revealed several microbial species, functional pathways, and metabolites that are associated with each genetic and environmental risk factor or that are differentially abundant between environmentally exposed and non-exposed infants or between time points. Among our significant findings, we found that cesarean section delivery is associated with a decreased abundance of Bacteroides vulgatus and Bacteroides dorei and of folate biosynthesis pathway and with an increased abundance of hydroxyphenylacetic acid, alterations that are implicated in immune system dysfunction and inflammatory conditions. Additionally, longitudinal analysis revealed that, in infants not exposed to any environmental risk factor, the abundances of Bacteroides uniformis and of metabolite 3-3-hydroxyphenylproprionic acid increase over time, while those for lipoic acid and methane metabolism pathways decrease, patterns that are linked to beneficial immunomodulatory and anti-inflammatory effects. CONCLUSIONS: Overall, our study provides unprecedented insights into major taxonomic and functional shifts in the developing gut microbiota of infants at risk of CD linking genetic and environmental risk factors to detrimental immunomodulatory and inflammatory effects. Video Abstract.

Traumatic injury and access to care in rural areas: leveraging linked data and geographic information systems for planning and advocacy.

INTRODUCTION: The purpose of this ecological study was to apply Geographic Information System (GIS) methods to patterns of traumatic injury and access to trauma care to facilitate system planning and advocacy. METHODS: Four US state (Colorado) and national data sources were linked to examine county-level disparities. Average ambulance drive times to trauma centers for populated places in each county were estimated and mapped. RESULTS: Independent samples t-tests demonstrated Colorado's rural counties had significantly higher injury hospitalization rates (mean (M)=685.4 v M=566.3; p=0.005)) and fatality rates (M=93.8 v M=71.6, p<0.001), indicating residents with the least access to care are the most impacted by the burden of injury; this finding was supported by GIS analyses of drive times to level I and II trauma centers and underlying injury rates, which are visually displayed. CONCLUSIONS: These methods are useful tools for rural public health professionals to conduct system optimization, identify training and resource needs, assess prevention priorities, and advocate for trauma system support.

Targeted genotyping for the prediction of celiac disease autoimmunity development in patients with type 1 diabetes and their family members.

BACKGROUND: Patients with type 1 diabetes (T1D) and their first-degree relatives (FDRs) have an increased risk of developing celiac disease (CD) compared to the general population. This is largely explained by the shared association with major histocompatibility class II human leukocyte antigen (HLA) DQ2 and/or DQ8 between the two disease states. AIM: To describe the frequency of CD autoimmunity (CDA) and the distribution of HLA and haptoglobin genotypes in patients with T1D and their FDRs. Additionally, we aimed at identifying predictors associated with an increased risk of developing CDA in patients with T1D and their family members. METHODS: We obtained clinical information and blood samples from 1027 participants (302 with T1D and 725 FDRs) over a five-year period. Samples were tested for autoantibodies associated with CD, HLA-DQ alleles, and haptoglobin genotype. We fit univariate and multiple logistic regression models for CDA separately for subjects with T1D and for FDRs of subjects with T1D. RESULTS: Implementation of a screening program increased the frequency of CDA by 2-fold in participants with T1D and 2.8-fold in their FDRs. Multivariate analysis found that, in participants with T1D, having both DR7-DQ2 and DR4-DQ8 was associated with an increased frequency of CDA. In FDRs of T1D patients, reported CD in the family was associated with an increased frequency of CDA during screening. Haptoglobin 2 genotype was not associated with developing CDA in the multivariate analysis. CONCLUSION: Patients with T1D and their FDRs have a high frequency of CDA. Carrying both DR7-DQ2 and DR4-DQ8 was associated with development of CDA in patients with T1D.

Pediatric Nonceliac Gluten Sensitivity: A Gluten-related Disorder Treatment Center Experience.

OBJECTIVE: The aim of the study was to identify the prevalence and clinical characteristics of children with nonceliac gluten sensitivity (NCGS) presenting to a tertiary care center specialized for evaluation of gluten-related disorders. METHODS: The medical records of all patients aged 0 to 18 years who presented to our center over a 4-year period (July 2013-June 2018) and consented to participate in our research registry were reviewed. Patients meeting the clinical criteria for NCGS were reviewed in detail. RESULTS: Among 500 pediatric patients who volunteered to participate in the registry during the study period, we identified 26 (5.2%) with NCGS. Both gastrointestinal and extraintestinal symptoms associated with gluten ingestion were common with abdominal pain (57.7%), bloating (53.9%), rash (53.9%), diarrhea/loose stool (42.3%), and emotional/behavioral issues (42.3%) emerging as the predominant complaints. In addition, children with NCGS demonstrated a high personal history (61.5%) and family history (61.5%) of concomitant allergic/atopic disease. CONCLUSIONS: Even within our highly specialized population of patients with a suspected gluten-related disorder, pediatric NCGS is relatively uncommon. The estimated prevalence and clinical features mirror those previously reported in a similarly highly selective population of adults. In the absence of celiac disease, clinical suspicion for NCGS should arise in a child with gastrointestinal and/or extraintestinal complaints alleviated with gluten removal and considered in symptomatic patients with associated allergic/atopic disease. Proper and adequate exclusion of celiac disease and other potential causes of the clinical complaints is essential to justify adoption of the gluten-free diet according to an appropriate stringency and with dietitian supervision to avoid nutritional deficiencies.

Effect of Combined Gluten-Free, Dairy-Free Diet in Children With Steroid-Resistant Nephrotic Syndrome: An Open Pilot Trial.

INTRODUCTION: Steroid-resistant nephrotic syndrome (SRNS) affects both children and adults and has a high rate of progression to end-stage renal disease. Although a subset of patients have well-characterized genetic mutation(s), in the majority of cases, the etiology is unknown. Over the past 50 years, a number of case reports have suggested the potential impact of dietary changes in controlling primary nephrotic syndrome, especially gluten and dairy restrictions. METHODS: We have designed a prospective, open-label, nonrandomized, pilot clinical trial, to study the effect of a gluten-free and dairy-free (GF/DF) diet in children with SRNS. The study will be organized as a 4-week summer camp to implement a GF/DF diet in a tightly controlled and monitored setting. Blood, urine, and stool samples will be collected at different time points during the study. RESULTS: The primary end point is a reduction of more than 50% in the urine protein:creatinine ratio. The secondary end points include changes in urine protein, kidney function, and serum albumin, as well as effects in immune activation, kidney injury biomarkers, and gut microbiome composition and function (metagenomic/metatranscriptomic). CONCLUSION: This study will advance the field by testing the effect of dietary changes in patients with SRNS in a highly controlled camp environment. In addition, we hope the results will help to identify a responder profile that may guide the design of a larger trial for further investigation.