Toll-Like Receptor 3 Mediates Aortic Stenosis Through a Conserved Mechanism of Calcification.

BACKGROUND: Calcific aortic valve disease (CAVD) is characterized by a phenotypic switch of valvular interstitial cells to bone-forming cells. Toll-like receptors (TLRs) are evolutionarily conserved pattern recognition receptors at the interface between innate immunity and tissue repair. Type I interferons (IFNs) are not only crucial for an adequate antiviral response but also implicated in bone formation. We hypothesized that the accumulation of endogenous TLR3 ligands in the valvular leaflets may promote the generation of osteoblast-like cells through enhanced type I IFN signaling. METHODS: Human valvular interstitial cells isolated from aortic valves were challenged with mechanical strain or synthetic TLR3 agonists and analyzed for bone formation, gene expression profiles, and IFN signaling pathways. Different inhibitors were used to delineate the engaged signaling pathways. Moreover, we screened a variety of potential lipids and proteoglycans known to accumulate in CAVD lesions as potential TLR3 ligands. Ligand-receptor interactions were characterized by in silico modeling and verified through immunoprecipitation experiments. Biglycan (Bgn), Tlr3, and IFN-α/ß receptor alpha chain (Ifnar1)-deficient mice and a specific zebrafish model were used to study the implication of the biglycan (BGN)-TLR3-IFN axis in both CAVD and bone formation in vivo. Two large-scale cohorts (GERA [Genetic Epidemiology Research on Adult Health and Aging], n=55 192 with 3469 aortic stenosis cases; UK Biobank, n=257 231 with 2213 aortic stenosis cases) were examined for genetic variation at genes implicated in BGN-TLR3-IFN signaling associating with CAVD in humans. RESULTS: Here, we identify TLR3 as a central molecular regulator of calcification in valvular interstitial cells and unravel BGN as a new endogenous agonist of TLR3. Posttranslational BGN maturation by xylosyltransferase 1 (XYLT1) is required for TLR3 activation. Moreover, BGN induces the transdifferentiation of valvular interstitial cells into bone-forming osteoblasts through the TLR3-dependent induction of type I IFNs. It is intriguing that Bgn-/-, Tlr3-/-, and Ifnar1-/- mice are protected against CAVD and display impaired bone formation. Meta-analysis of 2 large-scale cohorts with >300 000 individuals reveals that genetic variation at loci relevant to the XYLT1-BGN-TLR3-interferon-α/ß receptor alpha chain (IFNAR) 1 pathway is associated with CAVD in humans. CONCLUSIONS: This study identifies the BGN-TLR3-IFNAR1 axis as an evolutionarily conserved pathway governing calcification of the aortic valve and reveals a potential therapeutic target to prevent CAVD.

Kinesin family member 12-related hepatopathy: A generally indolent disorder with elevated gamma-glutamyl-transferase activity.

Exome sequencing (ES) has identified biallelic kinesin family member 12 (KIF12) mutations as underlying neonatal cholestatic liver disease. We collected information on onset and progression of this entity. Among consecutively referred pediatric patients at our centers, diagnostic ES identified 4 patients with novel, biallelic KIF12 variants using the human GRCh38 reference sequence, as KIF12 remains incompletely annotated in the older reference sequence GRCh37. A review of these and of 21 reported patients with KIF12 variants found that presentation with elevated serum transaminase activity in the context of trivial respiratory infection, without clinical features of liver disease, was more common (n = 18) than manifest cholestatic disease progressing rapidly to liver transplantation (LT; n = 7). Onset of liver disease was at age <1 year in 15 patients; LT was more common in this group. Serum gamma-glutamyl transpeptidase activity (GGT) was elevated in all patients, and total bilirubin was elevated in 15 patients. Liver fibrosis or cirrhosis was present in 14 of 18 patients who were biopsied. The 16 different pathogenic variants and 11 different KIF12 genotypes found were not correlated with age of onset or progression to LT. Identification of biallelic pathogenic KIF12 variants distinguishes KIF12-related disease from other entities with elevated GGT.

Natural history of Wolcott-Rallison syndrome: A systematic review and follow-up study.

BACKGROUND AND AIMS: To systematically review the literature for reports on Wolcott-Rallison syndrome, focusing on the spectrum and natural history, genotype-phenotype correlations, patient and native liver survival, and long-term outcomes. METHODS: PubMed, Livio, Google Scholar, Scopus and Web of Science databases were searched. Data on genotype, phenotype, therapy, cause of death and follow-up were extracted. Survival and correlation analyses were performed. RESULTS: Sixty-two studies with 159 patients met the inclusion criteria and additional 30 WRS individuals were collected by personal contact. The median age of presentation was 2.5 months (IQR 2) and of death was 36 months (IQR 50.75). The most frequent clinical feature was neonatal diabetes in all patients, followed by liver impairment in 73%, impaired growth in 72%, skeletal abnormalities in 59.8%, the nervous system in 37.6%, the kidney in 35.4%, insufficient haematopoiesis in 34.4%, hypothyroidism in 14.8% and exocrine pancreas insufficiency in 10.6%. Episodes of acute liver failure were frequently reported. Liver transplantation was performed in six, combined liver-pancreas in one and combined liver-pancreas-kidney transplantation in two individuals. Patient survival was significantly better in the transplant cohort (p = .0057). One-, five- and ten-year patient survival rates were 89.4%, 65.5% and 53.1%, respectively. Liver failure was reported as the leading cause of death in 17.9% of cases. Overall survival was better in individuals with missense mutations (p = .013). CONCLUSION: Wolcott-Rallison syndrome has variable clinical courses. Overall survival is better in individuals with missense mutations. Liver- or multi-organ transplantation is a feasible treatment option to improve survival.

Impact of citrulline substitution on clinical outcome after liver transplantation in carbamoyl phosphate synthetase 1 and ornithine transcarbamylase deficiency.

Carbamoyl phosphate synthetase 1 (CPS1) and ornithine transcarbamylase (OTC) deficiencies are rare urea cycle disorders, which can lead to life-threatening hyperammonemia. Liver transplantation (LT) provides a cure and offers an alternative to medical treatment and life-long dietary restrictions with permanent impending risk of hyperammonemia. Nevertheless, in most patients, metabolic aberrations persist after LT, especially low plasma citrulline levels, with questionable clinical impact. So far, little is known about these alterations and there is no consensus, whether l-citrulline substitution after LT improves patients' symptoms and outcomes. In this multicentre, retrospective, observational study of 24 patients who underwent LT for CPS1 (n = 11) or OTC (n = 13) deficiency, 25% did not receive l-citrulline or arginine substitution. Correlation analysis revealed no correlation between substitution dosage and citrulline levels (CPS1, p = 0.8 and OTC, p = 1). Arginine levels after liver transplantation were normal after LT independent of citrulline substitution. Native liver survival had no impact on mental impairment (p = 0.67). Regression analysis showed no correlation between l-citrulline substitution and failure to thrive (p = 0.611) or neurological outcome (p = 0.701). Peak ammonia had a significant effect on mental impairment (p = 0.017). Peak plasma ammonia levels correlate with mental impairment after LT in CPS1 and OTC deficiency. Growth and intellectual impairment after LT are not significantly associated with l-citrulline substitution.

Efficacy of Elimination Diets in Eosinophilic Esophagitis: A Systematic Review and Meta-analysis.

BACKGROUND & AIMS: Eosinophilic esophagitis (EoE) can be treated by proton pump inhibitors, topical corticosteroids, or dietary measures. This study systematically assessed the efficacy of 4 major dietary treatment regimens in EoE, updating the evidence presented in a meta-analysis from 2014. METHODS: Electronic databases such as PubMed, Scopus, and Web of Science, and other sources were searched up to September 2022 to identify studies on dietary treatment of EoE. Based on histologic remission criteria, the efficacy of these treatments was pooled and analyzed with respect to the type of dietary regimen: 6-food elimination diet (SFED), 4-food elimination diet (FFED), 1-food elimination diet (OFED), and a targeted elimination diet (TED). Clinical response rates, food sensitization, and efficacies for a pediatric subpopulation were calculated. Influencing variables on efficacies were estimated via meta-regression analyses. RESULTS: Thirty-four studies with 1762 patients met the inclusion criteria. The overall rate of histologic remission was 53.8% (95% CI, 48.0%-59.6%), and in the individual dietary groups was 61.3% (95% CI, 53.0%-69.3%) for SFED, 49.4% (95% CI, 32.5%-66.3%) for FFED, 51.4% (95% CI, 42.6%-60.1%) for OFED, and 45.7% (95% CI, 32.0%-59.7%) for TED. Dietary regimen and patient age did not significantly affect rates of histologic remission. The overall rate of clinical response was 80.8% (95% CI, 72.3%-88.2%), with response rates of 92.8% (95% CI, 81.2%-99.6%) for SFED, 74.1% (95% CI, 49.8%-92.6%) for FFED, 87.1% (95% CI, 58.4%-99.9%) for OFED, and 69.0% (95% CI, 50.2%85.3%) for TED. CONCLUSIONS: Dietary therapy is an effective treatment for EoE patients of any age. The current results could support a trend toward less-restrictive dietary regimens as a primary treatment option.

Genotypic and phenotypic spectrum of infantile liver failure due to pathogenic TRMU variants.

PURPOSE: This study aimed to define the genotypic and phenotypic spectrum of reversible acute liver failure (ALF) of infancy resulting from biallelic pathogenic TRMU variants and determine the role of cysteine supplementation in its treatment. METHODS: Individuals with biallelic (likely) pathogenic variants in TRMU were studied within an international retrospective collection of de-identified patient data. RESULTS: In 62 individuals, including 30 previously unreported cases, we described 47 (likely) pathogenic TRMU variants, of which 17 were novel, and 1 intragenic deletion. Of these 62 individuals, 42 were alive at a median age of 6.8 (0.6-22) years after a median follow-up of 3.6 (0.1-22) years. The most frequent finding, occurring in all but 2 individuals, was liver involvement. ALF occurred only in the first year of life and was reported in 43 of 62 individuals; 11 of whom received liver transplantation. Loss-of-function TRMU variants were associated with poor survival. Supplementation with at least 1 cysteine source, typically N-acetylcysteine, improved survival significantly. Neurodevelopmental delay was observed in 11 individuals and persisted in 4 of the survivors, but we were unable to determine whether this was a primary or a secondary consequence of TRMU deficiency. CONCLUSION: In most patients, TRMU-associated ALF was a transient, reversible disease and cysteine supplementation improved survival.

Pediatric Malignant Peritoneal Mesothelioma With Meningeal Metastasis.

Malignant peritoneal mesothelioma (MPM) is an extremely rare entity with a poor prognosis. We report on a 16-year-old boy with ascites and abdominal distension. A computed tomography scan showed peritoneal thickening and a mass adjacent to the transverse colon. Neither repeated cytologic testing of ascitic fluid, nor peritoneal tissue biopsy detected malignant cells. After the patient became progressively comatose, a magnetic resonance imaging scan of the brain showed leptomeningeal enhancement. An autopsy showed MPM infiltrating the pleura and the meninges. This is the first report on meningeal metastasis of MPM in a pediatric patient illustrating the enigmatic behavior of the tumor and highlighting the diagnostic pitfalls.

Biogenesis of lysosome-related organelles complex-1 (BORC) regulates late endosomal/lysosomal size through PIKfyve-dependent phosphatidylinositol-3,5-bisphosphate.

Mechanisms that control lysosomal function are essential for cellular homeostasis. Lysosomes adapt in size and number to cellular needs but little is known about the underlying molecular mechanism. We demonstrate that the late endosomal/lysosomal multimeric BLOC-1-related complex (BORC) regulates the size of these organelles via PIKfyve-dependent phosphatidylinositol-3,5-bisphosphate [PI(3,5)P2 ] production. Deletion of the core BORC component Diaskedin led to increased levels of PI(3,5)P2 , suggesting activation of PIKfyve, and resulted in enhanced lysosomal reformation and subsequent reduction in lysosomal size. This process required AMP-activated protein kinase (AMPK), a known PIKfyve activator, and was additionally dependent on the late endosomal/lysosomal adaptor, mitogen-activated protein kinases and mechanistic target of rapamycin activator (LAMTOR/Ragulator) complex. Consistently, in response to glucose limitation, AMPK activated PIKfyve, which induced lysosomal reformation with increased baseline autophagy and was coupled to a decrease in lysosomal size. These adaptations of the late endosomal/lysosomal system reversed under glucose replete growth conditions. In summary, our results demonstrate that BORC regulates lysosomal reformation and size in response to glucose availability.

Influence of early biliary complications on survival rates after pediatric liver transplantation-A positive outlook.

BACKGROUND: Early biliary complications (EBC) constitute a burden after pediatric liver transplantation frequently requiring immediate therapy. We aimed to assess the impact of EBC on short- and long-term patient and graft survival as well as post-transplant morbidity. METHODS: We analyzed 121 pediatric liver transplantations performed between 1984 and 2019 at the Medical University of Innsbruck for the occurrence of early (<90 days) biliary complications and investigated the influence of EBC on patient and graft survival. RESULTS: Early biliary complications occurred in 30 (24.8%) out of the 121 pediatric liver transplant recipients. Patient survival at 15 years (89.2% vs. 84.2%, p = .65) and all-cause (82.5% vs. 74.0%) and death-censored graft survival (82.5% vs. 75.1%, p = .71) at 10 years were similar between the EBC and the non-EBC group. The EBC group had a significantly longer ICU (25 vs. 16 days, p < .001) and initial hospital stay (64 vs. 42 days, p = .002). Livers of patients with EBC were characterized by multiple bile ducts (33.3% vs. 13.2%, p = .027), and patients with EBC had a higher risk to develop late biliary complications (OR 2.821 [95% CI 1.049-7.587], p = .044) and bowel obstruction/perforation (OR 4.388 [95% CI 1.503-12.812], p = .007). CONCLUSION: Early biliary complications after pediatric liver transplantation is frequent. The occurrence of EBC significantly increased post-transplant morbidity without affecting mortality. Multiple bile ducts were the only risk factor for the development of EBC in our cohort.

The haemochromatosis gene Hfe and Kupffer cells control LDL cholesterol homeostasis and impact on atherosclerosis development.

AIMS: Imbalances of iron metabolism have been linked to the development of atherosclerosis. However, subjects with hereditary haemochromatosis have a lower prevalence of cardiovascular disease. The aim of our study was to understand the underlying mechanisms by combining data from genome-wide association study analyses in humans, CRISPR/Cas9 genome editing, and loss-of-function studies in mice. METHODS AND RESULTS: Our analysis of the Global Lipids Genetics Consortium (GLGC) dataset revealed that single nucleotide polymorphisms (SNPs) in the haemochromatosis gene HFE associate with reduced low-density lipoprotein cholesterol (LDL-C) in human plasma. The LDL-C lowering effect could be phenocopied in dyslipidaemic ApoE-/- mice lacking Hfe, which translated into reduced atherosclerosis burden. Mechanistically, we identified HFE as a negative regulator of LDL receptor expression in hepatocytes. Moreover, we uncovered liver-resident Kupffer cells (KCs) as central players in cholesterol homeostasis as they were found to acquire and transfer LDL-derived cholesterol to hepatocytes in an Abca1-dependent fashion, which is controlled by iron availability. CONCLUSION: Our results disentangle novel regulatory interactions between iron metabolism, KC biology and cholesterol homeostasis which are promising targets for treating dyslipidaemia but also provide a mechanistic explanation for reduced cardiovascular morbidity in subjects with haemochromatosis.

Combining high-pressure freezing with pre-embedding immunogold electron microscopy and tomography.

Immunogold labeling of permeabilized whole-mount cells or thin-sectioned material is widely used for the subcellular localization of biomolecules at the high spatial resolution of electron microscopy (EM). Those approaches are well compatible with either 3-dimensional (3D) reconstruction of organelle morphology and antigen distribution or with rapid cryofixation-but not easily with both at once. We describe here a specimen preparation and labeling protocol for animal cell cultures, which represents a novel blend of specifically adapted versions of established techniques. It combines the virtues of reliably preserved organelle ultrastructure, as trapped by rapid freezing within milliseconds followed by freeze-substitution and specimen rehydration, with the advantages of robust labeling of intracellular constituents in 3D through means of pre-embedding NANOGOLD-silver immunocytochemistry. So obtained thin and semi-thick epoxy resin sections are suitable for transmission EM imaging, as well as tomographic reconstruction and modeling of labeling patterns in the 3D cellular context.

AP1S1 missense mutations cause a congenital enteropathy via an epithelial barrier defect.

Congenital diarrheal disorders (CDD) comprise > 50 monogenic entities featuring chronic diarrhea of early-onset, including defects in nutrient and electrolyte absorption, enterocyte polarization, enteroendocrine cell differentiation, and epithelial integrity. Diarrhea is also a predominant symptom in many immunodeficiencies, congenital disorders of glycosylation, and in some defects of the vesicular sorting and transporting machinery. We set out to identify the etiology of an intractable diarrhea in 2 consanguineous families by whole-exome sequencing, and identified two novel AP1S1 mutations, c.269T>C (p.Leu90Pro) and c.346G>A (p.Glu116Lys). AP1S1 encodes the small subunit of the adaptor protein 1 complex (AP-1), which plays roles in clathrin coat-assembly and trafficking between trans-Golgi network, endosomes and the plasma membrane. An AP1S1 knock-out (KO) of a CaCo2 intestinal cell line was generated to characterize intestinal AP1S1 deficiency as well as identified mutations by stable expression in KO background. Morphology and prototype transporter protein distribution were comparable between parental and KO cells. We observed altered localization of tight-junction proteins ZO-1 and claudin 3, decreased transepithelial electrical resistance and an increased dextran permeability of the CaCo2-AP1S1-KO monolayer. In addition, lumen formation in 3D cultures of these cells was abnormal. Re-expression of wild-type AP1S1 in CaCo2-AP1S1-KO cells reverted these abnormalities, while expression of AP1S1 containing either missense mutation did not. Our data indicate that loss of AP1S1 function causes an intestinal epithelial barrier defect, and that AP1S1 mutations can cause a non-syndromic form of congenital diarrhea, whereas 2 reported truncating AP1S1 mutations caused MEDNIK syndrome, characterized by mental retardation, enteropathy, deafness, neuropathy, ichthyosis, and keratodermia.

Stereotactic radiofrequency ablation of a variety of liver masses in children.

BACKGROUND AND AIMS: Surgical resection is currently the cornerstone of liver tumor treatment in children. In adults radiofrequency ablation (RFA) is an established minimally invasive treatment option for small focal liver tumors. Multiprobe stereotactic RFA (SRFA) with intraoperative image fusion to confirm ablation margins allows treatment for large lesions. We describe our experience with SRFA in children with liver masses. METHODS: SRFA was performed in 10 patients with a median age of 14 years (range 0.5-17.0 years) suffering from liver adenoma (n = 3), hepatocellular carcinoma (n = 1), hepatoblastoma (n = 2), myofibroblastic tumor (n = 1), hepatic metastases of extrahepatic tumors (n = 2) and infiltrative hepatic cysts associated with alveolar echinococcosis (n = 1). Overall, 15 lesions with a mean lesion size of 2.6 cm (range 0.7-9.5 cm) were treated in 11 sessions. RESULTS: The technical success rate was 100%, as was the survival rate. No transient adverse effects higher than grade II (Clavien and Dindo) were encountered after interventions. The median hospital stay was 5 d (range 2-33 d). In two patients who subsequently underwent transplant hepatectomy complete ablation was histologically confirmed. Follow-up imaging studies (median 55 months, range 18-129 months) revealed no local or distant recurrence of disease in any patient. CONCLUSIONS: SRFA is an effective minimal-invasive treatment option in pediatric patients with liver tumors of different etiologies.

Abnormal Rab11-Rab8-vesicles cluster in enterocytes of patients with microvillus inclusion disease.

Microvillus inclusion disease (MVID) is a congenital enteropathy characterized by accumulation of vesiculo-tubular endomembranes in the subapical cytoplasm of enterocytes, historically termed "secretory granules." However, neither their identity nor pathophysiological significance is well defined. Using immunoelectron microscopy and tomography, we studied biopsies from MVID patients (3× Myosin 5b mutations and 1× Syntaxin3 mutation) and compared them to controls and genome-edited CaCo2 cell models, harboring relevant mutations. Duodenal biopsies from 2 patients with novel Myosin 5b mutations and typical clinical symptoms showed unusual ultrastructural phenotypes: aberrant subapical vesicles and tubules were prominent in the enterocytes, though other histological hallmarks of MVID were almost absent (ectopic intra-/intercellular microvilli, brush border atrophy). We identified these enigmatic vesiculo-tubular organelles as Rab11-Rab8-positive recycling compartments of altered size, shape and location harboring the apical SNARE Syntaxin3, apical transporters sodium-hydrogen exchanger 3 (NHE3) and cystic fibrosis transmembrane conductance regulator. Our data strongly indicate that in MVID disrupted trafficking between cargo vesicles and the apical plasma membrane is the primary cause of a defect of epithelial polarity and subsequent facultative loss of brush border integrity, leading to malabsorption. Furthermore, they support the notion that mislocalization of transporters, such as NHE3 substantially contributes to the reported sodium loss diarrhea.

An inducible mouse model for microvillus inclusion disease reveals a role for myosin Vb in apical and basolateral trafficking.

Microvillus inclusion disease (MVID) is a rare intestinal enteropathy with an onset within a few days to months after birth, resulting in persistent watery diarrhea. Mutations in the myosin Vb gene (MYO5B) have been identified in the majority of MVID patients. However, the exact pathophysiology of MVID still remains unclear. To address the specific role of MYO5B in the intestine, we generated an intestine-specific conditional Myo5b-deficient (Myo5bfl/fl;Vil-CreERT2) mouse model. We analyzed intestinal tissues and cultured organoids of Myo5bfl/fl;Vil-CreERT2 mice by electron microscopy, immunofluorescence, and immunohistochemistry. Our data showed that Myo5bfl/fl;Vil-CreERT2 mice developed severe diarrhea within 4 d after tamoxifen induction. Periodic Acid Schiff and alkaline phosphatase staining revealed subapical accumulation of intracellular vesicles in villus enterocytes. Analysis by electron microscopy confirmed an almost complete absence of apical microvilli, the appearance of microvillus inclusions, and enlarged intercellular spaces in induced Myo5bfl/fl;Vil-CreERT2 intestines. In addition, we determined that MYO5B is involved not only in apical but also basolateral trafficking of proteins. The analysis of the intestine during the early onset of the disease revealed that subapical accumulation of secretory granules precedes occurrence of microvillus inclusions, indicating involvement of MYO5B in early differentiation of epithelial cells. By comparing our data with a novel MVID patient, we conclude that our mouse model completely recapitulates the intestinal phenotype of human MVID. This includes severe diarrhea, loss of microvilli, occurrence of microvillus inclusions, and subapical secretory granules. Thus, loss of MYO5B disturbs both apical and basolateral trafficking of proteins and causes MVID in mice.

Ultrastructural Morphometry Points to a New Role for LAMTOR2 in Regulating the Endo/Lysosomal System.

The late endosomal adaptor protein LAMTOR2/p14 is essential for tissue homeostasis by controlling MAPK and mTOR signaling, which in turn regulate cell growth and proliferation, migration and spreading. Moreover, LAMTOR2 critically controls architecture and function of the endocytic system, including epidermal growth factor receptor (EGFR) degradation in lysosomes, positioning of late endosomes and defense against intracellular pathogens. Here we describe the multifaceted ultrastructural phenotype of the endo/lysosomal system of LAMTOR2-deficient mouse embryonic fibroblasts. Quantitative (immuno-)electron microscopy of cryo-fixed samples revealed significantly reduced numbers of recycling tubules emanating from maturing multivesicular bodies (MVB). Instead, a distinct halo of vesicles surrounded MVB, tentatively interpreted as detached, jammed recycling tubules. These morphological changes in LAMTOR2-deficient cells correlated with the presence of growth factors (e.g. EGF), but were similarly induced in control cells by inactivating mTOR. Furthermore, proper transferrin receptor trafficking and recycling were apparently dependent on an intact LAMTOR complex. Finally, a severe imbalance in the relative proportions of endo/lysosomes was found in LAMTOR2-deficient cells, resulting from increased amounts of mature MVB and (autophago)lysosomes. These observations suggest that the LAMTOR/Ragulator complex is required not only for maintaining the homeostasis of endo/lysosomal subpopulations but also contributes to the proper formation of MVB-recycling tubules, and regulation of membrane/cargo recycling from MVB.

Microvillus inclusion disease: loss of Myosin vb disrupts intracellular traffic and cell polarity.

Microvillus inclusion disease (MVID) is a congenital enteropathy characterized by loss of apical microvilli and formation of cytoplasmic inclusions lined by microvilli in enterocytes. MVID is caused by mutations in the MYO5B gene, coding for the myosin Vb motor protein. Although myosin Vb is implicated in the organization of intracellular transport and cell surface polarity in epithelial cells, its precise role in the pathogenesis of MVID is unknown. We performed correlative immunohistochemistry analyses of sections from duodenal biopsies of a MVID patient, compound heterozygous for two novel MYO5B mutations, predicting loss of function of myosin Vb in duodenal enterocytes together with a stable MYO5B CaCo2 RNAi cell system. Our findings show that myosin Vb-deficient enterocytes display disruption of cell polarity as reflected by mislocalized apical and basolateral transporter proteins, altered distribution of certain endosomal/lysosomal constituents including Rab GTPases. Together, this severe disturbance of epithelial cell function could shed light on the pathology and symptoms of MVID.