Incidence, management and outcomes in hepatic artery complications after paediatric liver transplantation: protocol of the retrospective, international, multicentre HEPATIC Registry.

INTRODUCTION: Hepatic artery complications (HACs), such as a thrombosis or stenosis, are serious causes of morbidity and mortality after paediatric liver transplantation (LT). This study will investigate the incidence, current management practices and outcomes in paediatric patients with HAC after LT, including early and late complications. METHODS AND ANALYSIS: The HEPatic Artery stenosis and Thrombosis after liver transplantation In Children (HEPATIC) Registry is an international, retrospective, multicentre, observational study. Any paediatric patient diagnosed with HAC and treated for HAC (at age <18 years) after paediatric LT within a 20-year time period will be included. The primary outcomes are graft and patient survivals. The secondary outcomes are technical success of the intervention, primary and secondary patency after HAC intervention, intraprocedural and postprocedural complications, description of current management practices, and incidence of HAC. ETHICS AND DISSEMINATION: All participating sites will obtain local ethical approval and (waiver of) informed consent following the regulations on the conduct of observational clinical studies. The results will be disseminated through scientific presentations at conferences and through publication in peer-reviewed journals. TRIAL REGISTRATION NUMBER: The HEPATIC registry is registered at the ClinicalTrials.gov website; Registry Identifier: NCT05818644.

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.

Prevalence, management and efficacy of treatment in portal vein obstruction after paediatric liver transplantation: protocol of the retrospective international multicentre PORTAL registry.

INTRODUCTION: Portal vein obstruction (PVO) consists of anastomotic stenosis and thrombosis, which occurs due to a progression of the former. The aim of this large-scale international study is to assess the prevalence, current management practices and efficacy of treatment in patients with PVO. METHODS AND ANALYSIS: The Portal vein Obstruction Revascularisation Therapy After Liver transplantation registry will facilitate an international, retrospective, multicentre, observational study, with 25 centres around the world already actively involved. Paediatric patients (aged <18 years) with a diagnosed PVO between 1 January 2001 and 1 January 2021 after liver transplantation will be eligible for inclusion. The primary endpoints are the prevalence of PVO, primary and secondary patency after PVO intervention and current management practices. Secondary endpoints are patient and graft survival, severe complications of PVO and technical success of revascularisation techniques. ETHICS AND DISSEMINATION: Medical Ethics Review Board of the University Medical Center Groningen has approved the study (METc 2021/072). The results of this study will be disseminated via peer-reviewed publications and scientific presentations at national and international conferences. TRIAL REGISTRATION NUMBER: Netherlands Trial Register (NL9261).

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.

A CRISPR screen in intestinal epithelial cells identifies novel factors for polarity and apical transport.

Epithelial polarization and polarized cargo transport are highly coordinated and interdependent processes. In our search for novel regulators of epithelial polarization and protein secretion, we used a genome-wide CRISPR/Cas9 screen and combined it with an assay based on fluorescence-activated cell sorting (FACS) to measure the secretion of the apical brush-border hydrolase dipeptidyl peptidase 4 (DPP4). In this way, we performed the first CRISPR screen to date in human polarized epithelial cells. Using high-resolution microscopy, we detected polarization defects and mislocalization of DPP4 to late endosomes/lysosomes after knockout of TM9SF4, anoctamin 8, and ARHGAP33, confirming the identification of novel factors for epithelial polarization and apical cargo secretion. Thus, we provide a powerful tool suitable for studying polarization and cargo secretion in epithelial cells. In addition, we provide a dataset that serves as a resource for the study of novel mechanisms for epithelial polarization and polarized transport and facilitates the investigation of novel congenital diseases associated with these processes.

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.

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.

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.

LAMTOR/Ragulator is a negative regulator of Arl8b- and BORC-dependent late endosomal positioning.

Signaling from lysosomes controls cellular clearance and energy metabolism. Lysosomal malfunction has been implicated in several pathologies, including neurodegeneration, cancer, infection, immunodeficiency, and obesity. Interestingly, many functions are dependent on the organelle position. Lysosomal motility requires the integration of extracellular and intracellular signals that converge on a competition between motor proteins that ultimately control lysosomal movement on microtubules. Here, we identify a novel upstream control mechanism of Arl8b-dependent lysosomal movement toward the periphery of the cell. We show that the C-terminal domain of lyspersin, a subunit of BLOC-1-related complex (BORC), is essential and sufficient for BORC-dependent recruitment of Arl8b to lysosomes. In addition, we establish lyspersin as the linker between BORC and late endosomal/lysosomal adaptor and mitogen activated protein kinase and mechanistic target of rapamycin activator (LAMTOR) complexes and show that epidermal growth factor stimulation decreases LAMTOR/BORC association, thereby promoting BORC- and Arl8b-dependent lysosomal centrifugal transport.

Recruitment dynamics of ESCRT-III and Vps4 to endosomes and implications for reverse membrane budding.

The ESCRT machinery mediates reverse membrane scission. By quantitative fluorescence lattice light-sheet microscopy, we have shown that ESCRT-III subunits polymerize rapidly on yeast endosomes, together with the recruitment of at least two Vps4 hexamers. During their 3-45 s lifetimes, the ESCRT-III assemblies accumulated 75-200 Snf7 and 15-50 Vps24 molecules. Productive budding events required at least two additional Vps4 hexamers. Membrane budding was associated with continuous, stochastic exchange of Vps4 and ESCRT-III components, rather than steady growth of fixed assemblies, and depended on Vps4 ATPase activity. An all-or-none step led to final release of ESCRT-III and Vps4. Tomographic electron microscopy demonstrated that acute disruption of Vps4 recruitment stalled membrane budding. We propose a model in which multiple Vps4 hexamers (four or more) draw together several ESCRT-III filaments. This process induces cargo crowding and inward membrane buckling, followed by constriction of the nascent bud neck and ultimately ILV generation by vesicle fission.

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.

Cargo-selective apical exocytosis in epithelial cells is conducted by Myo5B, Slp4a, Vamp7, and Syntaxin 3.

Mutations in the motor protein Myosin Vb (Myo5B) or the soluble NSF attachment protein receptor Syntaxin 3 (Stx3) disturb epithelial polarity and cause microvillus inclusion disease (MVID), a lethal hereditary enteropathy affecting neonates. To understand the molecular mechanism of Myo5B and Stx3 interplay, we used genome editing to introduce a defined Myo5B patient mutation in a human epithelial cell line. Our results demonstrate a selective role of Myo5B and Stx3 for apical cargo exocytosis in polarized epithelial cells. Apical exocytosis of NHE3, CFTR (cystic fibrosis transmembrane conductance regulator), and GLUT5 required an interaction cascade of Rab11, Myo5B, Slp4a, Munc18-2, and Vamp7 with Stx3, which cooperate in the final steps of this selective apical traffic pathway. The brush border enzymes DPPIV and sucrase-isomaltase still correctly localize at the apical plasma membrane independent of this pathway. Hence, our work demonstrates how Myo5B, Stx3, Slp4a, Vamp7, Munc18-2, and Rab8/11 cooperate during selective apical cargo trafficking and exocytosis in epithelial cells and thereby provides further insight into MVID pathophysiology.

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.

Interleukin-like epithelial-to-mesenchymal transition inducer activity is controlled by proteolytic processing and plasminogen-urokinase plasminogen activator receptor system-regulated secretion during breast cancer progression.

INTRODUCTION: Interleukin-like epithelial-to-mesenchymal transition inducer (ILEI) is an essential cytokine in tumor progression that is upregulated in several cancers, and its altered subcellular localization is a predictor of poor survival in human breast cancer. However, the regulation of ILEI activity and the molecular meaning of its altered localization remain elusive. METHODS: The influence of serum withdrawal, broad-specificity protease inhibitors, different serine proteases and plasminogen depletion on the size and amount of the secreted ILEI protein was investigated by Western blot analysis of EpRas cells. Proteases with ILEI-processing capacity were identified by carrying out an in vitro cleavage assay. Murine mammary tumor and metastasis models of EpC40 and 4T1 cells overexpressing different mutant forms of ILEI were used-extended with in vivo aprotinin treatment for the inhibition of ILEI-processing proteases-to test the in vivo relevance of proteolytic cleavage. Stable knockdown of urokinase plasminogen activator receptor (uPAR) in EpRas cells was performed to investigate the involvement of uPAR in ILEI secretion. The subcellular localization of the ILEI protein in tumor cell lines was analyzed by immunofluorescence. Immunohistochemistry for ILEI localization and uPAR expression was performed on two human breast cancer arrays, and ILEI and uPAR scores were correlated with the metastasis-free survival of patients. RESULTS: We demonstrate that secreted ILEI requires site-specific proteolytic maturation into its short form for its tumor-promoting function, which is executed by serine proteases, most efficiently by plasmin. Noncleaved ILEI is tethered to fibronectin-containing fibers of the extracellular matrix through a propeptide-dependent interaction. In addition to ILEI processing, plasmin rapidly increases ILEI secretion by mobilizing its intracellular protein pool in a uPAR-dependent manner. Elevated ILEI secretion correlates with an altered subcellular localization of the protein, most likely representing a shift into secretory vesicles. Moreover, altered subcellular ILEI localization strongly correlates with high tumor cell-associated uPAR protein expression, as well as with poor survival, in human breast cancer. CONCLUSIONS: Our findings point out extracellular serine proteases, in particular plasmin, and uPAR as valuable therapeutic targets against ILEI-driven tumor progression and emphasize the prognostic relevance of ILEI localization and a combined ILEI-uPAR marker analysis in human breast cancer.

Loss of syntaxin 3 causes variant microvillus inclusion disease.

Microvillus inclusion disease (MVID) is a disorder of intestinal epithelial differentiation characterized by life-threatening intractable diarrhea. MVID can be diagnosed based on loss of microvilli, microvillus inclusions, and accumulation of subapical vesicles. Most patients with MVID have mutations in myosin Vb that cause defects in recycling of apical vesicles. Whole-exome sequencing of DNA from patients with variant MVID showed homozygous truncating mutations in syntaxin 3 (STX3). STX3 is an apical receptor involved in membrane fusion of apical vesicles in enterocytes. Patient-derived organoid cultures and overexpression of truncated STX3 in Caco-2 cells recapitulated most characteristics of variant MVID. We conclude that loss of STX3 function causes variant MVID.

Coordinated binding of Vps4 to ESCRT-III drives membrane neck constriction during MVB vesicle formation.

Five endosomal sorting complexes required for transport (ESCRTs) mediate the degradation of ubiquitinated membrane proteins via multivesicular bodies (MVBs) in lysosomes. ESCRT-0, -I, and -II interact with cargo on endosomes. ESCRT-II also initiates the assembly of a ringlike ESCRT-III filament consisting of Vps20, Snf7, Vps24, and Vps2. The AAA-adenosine triphosphatase Vps4 disassembles and recycles the ESCRT-III complex, thereby terminating the ESCRT pathway. A mechanistic role for Vps4 in intraluminal vesicle (ILV) formation has been unclear. By combining yeast genetics, biochemistry, and electron tomography, we find that ESCRT-III assembly on endosomes is required to induce or stabilize the necks of growing MVB ILVs. Yet, ESCRT-III alone is not sufficient to complete ILV biogenesis. Rather, binding of Vps4 to ESCRT-III, coordinated by interactions with Vps2 and Snf7, is coupled to membrane neck constriction during ILV formation. Thus, Vps4 not only recycles ESCRT-III subunits but also cooperates with ESCRT-III to drive distinct membrane-remodeling steps, which lead to efficient membrane scission at the end of ILV biogenesis in vivo.

Development of an innovative 3D cell culture system to study tumour--stroma interactions in non-small cell lung cancer cells.

INTRODUCTION: We describe a novel 3D co-culture model using non-small cell lung cancer (NSCLC) cell lines in combination with lung fibroblasts. This model allows the investigation of tumour-stroma interactions and addresses the importance of having a more in vivo like cell culture model. METHODS: Automation-compatible multi-well hanging drop microtiter plates were used for the production of 3D mono- and co-cultures. In these hanging drops the two NSCLC cell lines A549 and Colo699 were cultivated either alone or co-cultured with lung fibroblasts. The viability of tumour spheroids was confirmed after five and ten days by using Annexin V/Propidium Iodide staining for flow-cytometry. Tumour fibroblast spheroid formation was characterized by scanning electron microscope (SEM), semi-thin sections, fluorescence microscope and immunohistochemistry (IHC). In addition to conventional histology, protein expression of E-Cadherin, vimentin, Ki67, fibronectin, cytokeratin 7 and α-smooth muscle actin (α-SMA) was investigated by IHC. RESULTS: Lower viability was observed in A549 monocultures compared to co-cultures, whereas Colo699 monocultures showed better viability compared to co-cultures. Ki67 expression varied significantly between mono- and co-cultures in both tumour cell lines. An increase of vimentin and decreased E-Cadherin expression could be detected during the course of the cultivation suggesting a transition to a more mesenchymal phenotype. Furthermore, the fibroblast cell line showed an expression of α-SMA only in co-culture with the cancer cell line A549, thereby indicating a mesenchymal to mesenchymal shift to an even more myofibroblast phenotype. CONCLUSION: We demonstrate that our method is a promising tool for the generation of tumour spheroid co-cultures. Furthermore, these spheroids allow the investigation of tumour-stroma interactions and a better reflection of in vivo conditions of cancer cells in their microenvironment. Our method holds potential to contribute to the development of anti-cancer agents and support the search for biomarkers.

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.