Matriptase drives early-onset intestinal failure in a mouse model of congenital tufting enteropathy.

Syndromic congenital tufting enteropathy (CTE) is a life-threatening recessive human genetic disorder that is caused by mutations in SPINT2, encoding the protease inhibitor HAI-2, and is characterized by severe intestinal dysfunction. We recently reported the generation of a Spint2-deficient mouse model of CTE. Here, we show that the CTE-associated early-onset intestinal failure and lethality of Spint2-deficient mice is caused by unchecked activity of the serine protease matriptase. Macroscopic and histological defects observed in the absence of HAI-2, including villous atrophy, luminal bleeding, loss of mucin-producing goblet cells, loss of defined crypt architecture and the resulting acute inflammatory response in the large intestine, were all prevented by intestinal-specific inactivation of the St14 gene encoding matriptase. The CTE-associated loss of the cell junctional proteins EpCAM and claudin 7 was also prevented. As a result, inactivation of intestinal matriptase allowed Spint2-deficient mice to gain weight after birth and dramatically increased their lifespan. These data implicate matriptase as a causative agent in the development of CTE and may provide a new target for the treatment of CTE in individuals carrying SPINT2 mutations.This article has an associated 'The people behind the papers' interview.

Malabsorption Spectrums in India.

Among the causes of malabsorption, tropical sprue is one of the leading cause.Several reports indicating that celiac disease, now being recognised more frequently. MATERIAL: 94 patients, aged more than 12 years, presenting with Chronic diarrhoea and malabsorption syndrome were analyzed by clinical presentation, endoscopic and histopathological examination.The spectrum of disease in these patients and features differentiating celiac disease and tropical sprue are reported here. OBSERVATION: Most common cause was Celiac Disease (65%), followed by Tropical Sprue (21%), common variable immunodeficiency (2%), lymphangiectasia (1%), idiopathic (3%). Patients with celiac disease were younger,having anemia, scalloping of folds,moderate or severe villous atrophy, crypt hyperplasia, diffuse epithelial damage. Patients with tropical sprue were older and more often normal duodenal epithelium. CONCLUSION: Malabsorption, a disease which is often missed and not recognised by clinicians. A meticulous search for diagnosis is required.

A proton-coupled folate transporter mutation causing hereditary folate malabsorption locks the protein in an inward-open conformation.

The proton-coupled folate transporter (PCFT, SLC46A1) is required for folate intestinal absorption and transport across the choroid plexus. Recent work has identified a F392V mutation causing hereditary folate malabsorption. However, the residue properties responsible for this loss of function remains unknown. Using site-directed mutagenesis, we observed complete loss of function with charged (Lys, Asp, and Glu) and polar (Thr, Ser, and Gln) Phe-392 substitutions and minimal function with some neutral substitutions; however, F392M retained full function. Using the substituted-cysteine accessibility method (with N-biotinyl aminoethyl methanethiosulfonate labeling), Phe-392 mutations causing loss of function, although preserving membrane expression and trafficking, also resulted in loss of accessibility of the substituted cysteine in P314C-PCFT located within the aqueous translocation pathway. F392V function and accessibility of the P314C cysteine were restored by insertion of a G305L (suppressor) mutation. A S196L mutation localized in proximity to Gly-305 by homology modeling was inactive. However, when inserted into the inactive F392V scaffold, function was restored (mutually compensatory mutations), as was accessibility of the P314C cysteine residue. Reduced function, documented with F392H PCFT, was due to a 15-fold decrease in methotrexate influx Vmax, accompanied by a decreased influx Kt (4.5-fold) and Ki (3-fold). The data indicate that Phe-392 is required for rapid oscillation of the carrier among its conformational states and suggest that this is achieved by dampening affinity of the protein for its folate substrates. F392V and other inactivating Phe-392 PCFT mutations lock the protein in its inward-open conformation. Reach (length) and hydrophobicity of Phe-392 appear to be features required for full activity.

Pathogenic STX3 variants affecting the retinal and intestinal transcripts cause an early-onset severe retinal dystrophy in microvillus inclusion disease subjects.

Biallelic STX3 variants were previously reported in five individuals with the severe congenital enteropathy, microvillus inclusion disease (MVID). Here, we provide a significant extension of the phenotypic spectrum caused by STX3 variants. We report ten individuals of diverse geographic origin with biallelic STX3 loss-of-function variants, identified through exome sequencing, single-nucleotide polymorphism array-based homozygosity mapping, and international collaboration. The evaluated individuals all presented with MVID. Eight individuals also displayed early-onset severe retinal dystrophy, i.e., syndromic-intestinal and retinal-disease. These individuals harbored STX3 variants that affected both the retinal and intestinal STX3 transcripts, whereas STX3 variants affected only the intestinal transcript in individuals with solitary MVID. That STX3 is essential for retinal photoreceptor survival was confirmed by the creation of a rod photoreceptor-specific STX3 knockout mouse model which revealed a time-dependent reduction in the number of rod photoreceptors, thinning of the outer nuclear layer, and the eventual loss of both rod and cone photoreceptors. Together, our results provide a link between STX3 loss-of-function variants and a human retinal dystrophy. Depending on the genomic site of a human loss-of-function STX3 variant, it can cause MVID, the novel intestinal-retinal syndrome reported here or, hypothetically, an isolated retinal dystrophy.

Lysophosphatidic Acid Increases Maturation of Brush Borders and SGLT1 Activity in MYO5B-deficient Mice, a Model of Microvillus Inclusion Disease.

BACKGROUND & AIM: Myosin VB (MYO5B) is an essential trafficking protein for membrane recycling in gastrointestinal epithelial cells. The inactivating mutations of MYO5B cause the congenital diarrheal disease, microvillus inclusion disease (MVID). MYO5B deficiency in mice causes mislocalization of SGLT1 and NHE3, but retained apical function of CFTR, resulting in malabsorption and secretory diarrhea. Activation of lysophosphatidic acid (LPA) receptors can improve diarrhea, but the effect of LPA on MVID symptoms is unclear. We investigated whether LPA administration can reduce the epithelial deficits in MYO5B-knockout mice. METHODS: Studies were conducted with tamoxifen-induced, intestine-specific knockout of MYO5B (VilCreERT2;Myo5bflox/flox) and littermate controls. Mice were given LPA, an LPAR2 agonist (GRI977143), or vehicle for 4 days after a single injection of tamoxifen. Apical SGLT1 and CFTR activities were measured in Üssing chambers. Intestinal tissues were collected, and localization of membrane transporters was evaluated by immunofluorescence analysis in tissue sections and enteroids. RNA sequencing and enrichment analysis were performed with isolated jejunal epithelial cells. RESULTS: Daily administration of LPA reduced villus blunting, frequency of multivesicular bodies, and levels of cathepsins in intestinal tissues of MYO5B-knockout mice compared with vehicle administration. LPA partially restored the brush border height and the localization of SGLT1 and NHE3 in small intestine of MYO5B-knockout mice and enteroids. The SGLT1-dependent short-circuit current was increased and abnormal CFTR activities were decreased in jejunum from MYO5B-knockout mice given LPA compared with vehicle. CONCLUSIONS: LPA may regulate a MYO5B-independent trafficking mechanism and brush border maturation, and therefore be developed for treatment of MVID.

Editing Myosin VB Gene to Create Porcine Model of Microvillus Inclusion Disease, With Microvillus-Lined Inclusions and Alterations in Sodium Transporters.

BACKGROUND & AIMS: Microvillus inclusion disease (MVID) is caused by inactivating mutations in the myosin VB gene (MYO5B). MVID is a complex disorder characterized by chronic, watery, life-threatening diarrhea that usually begins in the first hours to days of life. We developed a large animal model of MVID to better understand its pathophysiology. METHODS: Pigs were cloned by transfer of chromatin from swine primary fetal fibroblasts, which were edited with TALENs and single-strand oligonucleotide to introduce a P663-L663 substitution in the endogenous swine MYO5B (corresponding to the P660L mutation in human MYO5B, associated with MVID) to fertilized oocytes. We analyzed duodenal tissues from patients with MVID (with the MYO5B P660L mutation) and without (controls), and from pigs using immunohistochemistry. Enteroids were generated from pigs with MYO5B(P663L) and without the substitution (control pigs). RESULTS: Duodenal tissues from patients with MVID lacked MYO5B at the base of the apical membrane of intestinal cells; instead MYO5B was intracellular. Intestinal tissues and derived enteroids from MYO5B(P663L) piglets had reduced apical levels and diffuse subapical levels of sodium hydrogen exchanger 3 and SGLT1, which regulate transport of sodium, glucose, and water, compared with tissues from control piglets. However, intestinal tissues and derived enteroids from MYO5B(P663L) piglets maintained CFTR on apical membranes, like tissues from control pigs. Liver tissues from MYO5B(P663L) piglets had alterations in bile salt export pump, a transporter that facilitates bile flow, which is normally expressed in the bile canaliculi in the liver. CONCLUSIONS: We developed a large animal model of MVID that has many features of the human disease. Studies of this model could provide information about the functions of MYO5B and MVID pathogenesis, and might lead to new treatments.

The role of histopathology in the diagnosis and management of coeliac disease and other malabsorptive conditions.

Most absorption of nutrients takes place in the proximal small intestine, and the most common disorders leading to malabsorption are associated with a morphological abnormality in the duodenal mucosa that is appreciable in histological sections of biopsy specimens. Coeliac disease is the most well-known example, causing intraepithelial lymphocytosis, inflammation and villous atrophy in the duodenum. Remarkably similar inflammatory changes can be induced by other processes, including medications, e.g. angiotensin II receptor blockers and immune checkpoint inhibitors, immune dysregulation disorders, e.g. common variable immunodeficiency and autoimmune enteropathy, infections, collagenous sprue, and tropical sprue. However, there are often subtle histological differences from coeliac disease in the type of inflammatory infiltrate, the presence of crypt apoptosis, and the extent and type of inflammation beyond the duodenum. The clinical setting and serological investigation usually allow diagnostic separation, but some cases remain challenging. Histopathology is also important in assessing the response to treatment, such as the change in villous architecture caused by a gluten-free diet, or the response to cessation of a potentially causative medication. This review examines the practical role that histopathology of duodenal biopsy specimens plays in the assessment and management of inflammatory malabsorptive processes of the proximal small intestine, with a particular emphasis on coeliac disease.

Expanding the clinical spectrum in trichohepatoenteric syndrome.

Trichohepatoenteric syndrome (THES) is a very rare autosomal recessive genetic disorder, which is characterized by intractable diarrhea during infancy, dysmorphic features, immunodeficiency, and a failure to thrive. There are still significant difficulties for patients and clinicians in terms of the management of THES, even though its molecular basis has been uncovered in the last decade. In this article, we have presented two cases relating to siblings that have been diagnosed with the condition. Concerning one of the patients, we described a novel variation (c.2114 + 5G > A) in the TTC37 gene and a mild clinical course; meanwhile, the other one was clinically diagnosed with THES at 17 years of age, but they had seizures and died suddenly. These cases expand the spectrum of clinical findings in relation to THES.

Gremlin 1 depletion in vivo causes severe enteropathy and bone marrow failure.

The intestinal epithelium is perpetually renewed from a stem cell niche in the base of crypts to maintain a healthy bowel mucosa. Exit from this niche and maturation of epithelial cells requires tightly controlled gradients in BMP signalling, progressing from low BMP signalling at the crypt base to high signalling at the luminal surface. The BMP antagonist gremlin 1 (Grem1) is highly expressed by subepithelial myofibroblasts adjacent to the intestinal crypts but its role in regulating the stem cell niche and epithelial renewal in vivo has not been explored. To explore the effects of Grem1 loss in adulthood following normal growth and development, we bred mice (ROSA26CreER-Grem1 flx/flx ) in which Grem1 could be deleted by tamoxifen administration. While Grem1 remained intact, these mice were healthy, grew normally, and reproduced successfully. Following Grem1 depletion, the mice became unwell and were euthanised (at 7-13 days). Post-mortem examination revealed extensive mucosal abnormalities throughout the small and large intestines with failure of epithelial cell replication and maturation, villous atrophy, and features of malabsorption. Bone marrow hypoplasia was also observed with associated early haematopoietic failure. These results demonstrate an essential homeostatic role for gremlin 1 in maintaining normal bowel epithelial function in adulthood, suggesting that abnormalities in gremlin 1 expression can contribute to enteropathies. We also identified a previously unsuspected requirement for gremlin 1 in normal haematopoiesis. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Fermentable fibers induce rapid macro- and micronutrient depletion in Toll-like receptor 5-deficient mice.

Functional fermentable fibers are considered essential for a healthy diet. Recently, we demonstrated that gut microbiota dysbiotic mice fed an inulin-containing diet (ICD) developed hepatocellular carcinoma (HCC) within 6 mo. In particular, a subset of Toll-like receptor 5-deficient (T5KO) mice prone to HCC exhibited rapid onset of hyperbilirubinemia (HB) and cholemia; these symptoms provide rationale that ICD induces cholestasis. Our objective in the present study was to determine whether inulin-fed T5KO-HB mice exhibit other known consequences of cholestasis, including essential fatty acid and fat-soluble vitamin deficiencies. Here, we measured hepatic fatty acids and serum vitamin A and D levels from wild-type (WT), T5KO low bilirubin (LB) and T5KO-HB mice fed ICD for 4 wk. Additionally, hepatic RNAseq and proteomics were performed to ascertain other metabolic alterations. Compared with WT and T5KO-LB, T5KO-HB mice exhibited steatorrhea, i.e., ~50% increase in fecal lipids. This could contribute to the significant reduction of linoleate in hepatic neutral lipids in T5KO-HB mice. Additionally, serum vitamins A and D were ~50% reduced in T5KO-HB mice, which was associated with metabolic compromises. Overall, our study highlights that fermentable fiber-induced cholestasis is further characterized by depletion of macro-and micronutrients.NEW & NOTEWORTHY Feeding a dietary, fermentable fiber diet to a subset of Toll-like receptor 5 deficient (T5KO) mice induces early onset hyperbilirubinemia and cholemia that later manifests to hepatocellular carcinoma (HCC). Our study highlights that fermentable fiber-induced cholestasis is characterized with modest macro- and micronutrient deficiencies that may further contribute to hepatic biliary disease. Compared with chemical induction, immunization, surgery, or genetic manipulation, these findings provide a novel approach to study the cholestatic subtype of HCC.

The Endosomal Recycling Pathway-At the Crossroads of the Cell.

The endosomal recycling pathway lies at the heart of the membrane trafficking machinery in the cell. It plays a central role in determining the composition of the plasma membrane and is thus critical for normal cellular homeostasis. However, defective endosomal recycling has been linked to a wide range of diseases, including cancer and some of the most common neurological disorders. It is also frequently subverted by many diverse human pathogens in order to successfully infect cells. Despite its importance, endosomal recycling remains relatively understudied in comparison to the endocytic and secretory transport pathways. A greater understanding of the molecular mechanisms that support transport through the endosomal recycling pathway will provide deeper insights into the pathophysiology of disease and will likely identify new approaches for their detection and treatment. This review will provide an overview of the normal physiological role of the endosomal recycling pathway, describe the consequences when it malfunctions, and discuss potential strategies for modulating its activity.

Chylomicron retention disease: genetics, biochemistry, and clinical spectrum.

PURPOSE OF REVIEW: Chylomicron retention disease (CRD) is an autosomic recessive disorder, in which intestinal fat malabsorption is the main cause of diverse severe manifestations. The specific molecular defect was identified in 2003 and consists of mutations in the SAR1B or SARA2 gene encoding for intracellular SAR1B GTPase protein. The aim of this review is first to provide an update of the recent biochemical, genetic and clinical findings, and second to discuss novel mechanisms related to hallmark symptoms. RECENT FINDINGS: CRD patients present with SAR1B mutations, which disable the formation of coat protein complex II and thus blocks the transport of chylomicron cargo from the endoplasmic reticulum to the Golgi. Consequently, there is a total absence of chylomicron and apolipoprotein B-48 in the blood circulation following a fat meal, accompanied by a deficiency in liposoluble vitamins and essential fatty acids. The recent discovery of Transport and Golgi organization and Transport and Golgi organization-like proteins may explain the intriguing export of large chylomicron, exceeding coat protein complex II size. Hypocholesterolemia could be accounted for by a decrease in HDL cholesterol, likely a reflection of limited production of intestinal HDL in view of reduced ATP-binding cassette family A protein 1 and apolipoprotein A-I protein. In experimental studies, the paralog SAR1A compensates for the lack of the SAR1B GTPase protein. SUMMARY: Molecular testing for CRD is recommended to distinguish the disease from other congenital fat malabsorptions, and to early define molecular aberrations, accelerate treatment, and prevent complications.

EPCAM mutation update: Variants associated with congenital tufting enteropathy and Lynch syndrome.

The epithelial cell adhesion molecule gene (EPCAM, previously known as TACSTD1 or TROP1) encodes a membrane-bound protein that is localized to the basolateral membrane of epithelial cells and is overexpressed in some tumors. Biallelic mutations in EPCAM cause congenital tufting enteropathy (CTE), which is a rare chronic diarrheal disorder presenting in infancy. Monoallelic deletions of the 3' end of EPCAM that silence the downstream gene, MSH2, cause a form of Lynch syndrome, which is a cancer predisposition syndrome associated with loss of DNA mismatch repair. Here, we report 13 novel EPCAM mutations from 17 CTE patients from two separate centers, review EPCAM mutations associated with CTE and Lynch syndrome, and structurally model pathogenic missense mutations. Statistical analyses indicate that the c.499dupC (previously reported as c.498insC) frameshift mutation was associated with more severe treatment regimens and greater mortality in CTE, whereas the c.556-14A>G and c.491+1G>A splice site mutations were not correlated with treatments or outcomes significantly different than random simulation. These findings suggest that genotype-phenotype correlations may be useful in contributing to management decisions of CTE patients. Depending on the type and nature of EPCAM mutation, one of two unrelated diseases may occur, CTE or Lynch syndrome.

Enteroids expressing a disease-associated mutant of EpCAM are a model for congenital tufting enteropathy.

Congenital tufting enteropathy (CTE) is an autosomal recessive disease characterized by severe intestinal failure in infancy and mutations in the epithelial cell adhesion molecule (EPCAM) gene. Previous studies of CTE in mice expressing mutant EpCAM show neonatal lethality. Hence, to study the cellular, molecular, and physiological alterations that result from EpCAM mutation, a tamoxifen-inducible mutant EpCAM enteroid model has been generated. The presence of mutant EpCAM in the model was confirmed at both mRNA and protein levels. Immunofluorescence microscopy demonstrated the reduced expression of mutant EpCAM. Mutant enteroids had reduced budding potential as well as significantly decreased mRNA expression for epithelial lineage markers (Mucin 2, lysozyme, sucrase-isomaltase), proliferation marker Ki67, and secretory pathway transcription factors (Atoh1, Hnf1b). Significantly decreased numbers of Paneth and goblet cells were confirmed by staining. These findings were correlated with intestinal tissue from CTE patients and the mutant mice model that had significantly fewer Paneth and goblet cells than in healthy counterparts. FITC-dextran studies demonstrated significantly impaired barrier function in monolayers derived from mutant enteroids compared with control monolayers. In conclusion, we have established an ex vivo CTE model. The role of EpCAM in the budding potential, differentiation, and barrier function of enteroids is noted. Our study establishes new facets of EpCAM biology that will aid in understanding the pathophysiology of CTE and role of EpCAM in health and disease.NEW & NOTEWORTHY Here, we develop a novel ex vivo enteroid model for congenital tufting enteropathy (CTE) based on epithelial cell adhesion molecule (EPCAM) gene mutations found in patients. With this model we demonstrate the role of EpCAM in maintaining the functional homeostasis of the intestinal epithelium, including differentiation, proliferation, and barrier integrity. This study further establishes a new direction in EpCAM biology that will help in understanding the detailed pathophysiology of CTE and role of EpCAM.

Loss of MYO5B Leads to Reductions in Na+ Absorption With Maintenance of CFTR-Dependent Cl- Secretion in Enterocytes.

BACKGROUND & AIMS: Inactivating mutations in MYO5B cause microvillus inclusion disease (MVID), but the physiological cause of the diarrhea associated with this disease is unclear. We investigated whether loss of MYO5B results in aberrant expression of apical enterocyte transporters. METHODS: We studied alterations in apical membrane transporters in MYO5B-knockout mice, as well as mice with tamoxifen-inducible, intestine-specific disruption of Myo5b (VilCreERT2;Myo5bflox/flox mice) or those not given tamoxifen (controls). Intestinal tissues were collected from mice and analyzed by immunostaining, immunoelectron microscopy, or cultured enteroids were derived. Functions of brush border transporters in intestinal mucosa were measured in Ussing chambers. We obtained duodenal biopsy specimens from individuals with MVID and individuals without MVID (controls) and compared transporter distribution by immunocytochemistry. RESULTS: Compared to intestinal tissues from littermate controls, intestinal tissues from MYO5B-knockout mice had decreased apical localization of SLC9A3 (also called NHE3), SLC5A1 (also called SGLT1), aquaporin (AQP) 7, and sucrase isomaltase, and subapical localization of intestinal alkaline phosphatase and CDC42. However, CFTR was present on apical membranes of enterocytes from MYO5B knockout and control mice. Intestinal biopsies from patients with MVID had subapical localization of NHE3, SGLT1, and AQP7, but maintained apical CFTR. After tamoxifen administration, VilCreERT2;Myo5bflox/flox mice lost apical NHE3, SGLT1, DRA, and AQP7, similar to germline MYO5B knockout mice. Intestinal tissues from VilCreERT2;Myo5bflox/flox mice had increased CFTR in crypts and CFTR localized to the apical membranes of enterocytes. Intestinal mucosa from VilCreERT2;Myo5bflox/flox mice given tamoxifen did not have an intestinal barrier defect, based on Ussing chamber analysis, but did have decreased SGLT1 activity and increased CFTR activity. CONCLUSIONS: Although trafficking of many apical transporters is regulated by MYO5B, trafficking of CFTR is largely independent of MYO5B. Decreased apical localization of NHE3, SGLT1, DRA, and AQP7 might be responsible for dysfunctional water absorption in enterocytes of patients with MVID. Maintenance of apical CFTR might exacerbate water loss by active secretion of chloride into the intestinal lumen.

The Differentiation of Intestinal-Failure-Associated Liver Disease from Nonalcoholic Fatty Liver and Nonalcoholic Steatohepatitis.

Intestinal failure-associated liver disease (IFALD), formerly known as parenteral nutrition-associated liver disease has often been listed in textbooks as an example of nonalcoholic fatty liver disease (NAFLD). However, the etiology, pathophysiology, epidemiology, histology, and progression differ substantially between the conditions defined as NAFLD and the disease, IFALD. Therefore, IFALD should not be defined or considered as a type or a cause of nonalcoholic fatty liver or nonalcoholic steatohepatitis, but rather as a distinct disease.

The localisation of the apical Par/Cdc42 polarity module is specifically affected in microvillus inclusion disease.

BACKGROUND INFORMATION: Microvillus inclusion disease (MVID) is a genetic disorder affecting intestinal absorption. It is caused by mutations in MYO5B or syntaxin 3 (STX3) affecting apical membrane trafficking. Morphologically, MVID is characterised by a depletion of apical microvilli and the formation of microvillus inclusions inside the cells, suggesting a loss of polarity. To investigate this hypothesis, we examined the location of essential apical polarity determinants in five MVID patients. RESULTS: We found that the polarity determinants Cdc42, Par6B, PKCζ/ι and the structural proteins ezrin and phospho-ezrin were lost from the apical membrane and accumulated either in the cytoplasm or on the basal side of enterocytes in patients, which suggests an inversion of cell polarity. Moreover, microvilli-like structures were observed at the basal side as per electron microscopy analysis. We next performed Myo5B depletion in three dimensional grown human Caco2 cells forming cysts and found a direct link between the loss of Myo5B and the mislocalisation of the same apical proteins; furthermore, we observed that a majority of cysts displayed an inverted polarity phenotype as seen in some patients. Finally, we found that this loss of polarity was specific for MVID: tissue samples of patients with Myo5B-independent absorption disorders showed normal polarity but we identified Cdc42 as a potentially essential biomarker for trichohepatoenteric syndrome. CONCLUSION: Our findings indicate that the loss of Myo5B induces a strong loss of enterocyte polarity, potentially leading to polarity inversion. SIGNIFICANCE: Our results show that polarity determinants could be useful markers to help establishing a diagnosis in patients. Furthermore, they could be used to characterise other rare intestinal absorption diseases.

Identification of intestinal ion transport defects in microvillus inclusion disease.

Loss of function mutations in the actin motor myosin Vb (Myo5b) lead to microvillus inclusion disease (MVID) and death in newborns and children. MVID results in secretory diarrhea, brush border (BB) defects, villus atrophy, and microvillus inclusions (MVIs) in enterocytes. How loss of Myo5b results in increased stool loss of chloride (Cl(-)) and sodium (Na(+)) is unknown. The present study used Myo5b loss-of-function human MVID intestine, polarized intestinal cell models of secretory crypt (T84) and villus resembling (CaCo2BBe, C2BBe) enterocytes lacking Myo5b in conjunction with immunofluorescence confocal stimulated emission depletion (gSTED) imaging, immunohistochemical staining, transmission electron microscopy, shRNA silencing, immunoblots, and electrophysiological approaches to examine the distribution, expression, and function of the major BB ion transporters NHE3 (Na(+)), CFTR (Cl(-)), and SLC26A3 (DRA) (Cl(-)/HCO3 (-)) that control intestinal fluid transport. We hypothesized that enterocyte maturation defects lead villus atrophy with immature secretory cryptlike enterocytes in the MVID epithelium. We investigated the role of Myo5b in enterocyte maturation. NHE3 and DRA localization and function were markedly reduced on the BB membrane of human MVID enterocytes and Myo5bKD C2BBe cells, while CFTR localization was preserved. Forskolin-stimulated CFTR ion transport in Myo5bKD T84 cells resembled that of control. Loss of Myo5b led to YAP1 nuclear retention, retarded enterocyte maturation, and a cryptlike phenotype. We conclude that preservation of functional CFTR in immature enterocytes, reduced functional expression of NHE3, and DRA contribute to Cl(-) and Na(+) stool loss in MVID diarrhea.

Multilabel immunofluorescence and antigen reprobing on formalin-fixed paraffin-embedded sections: novel applications for precision pathology diagnosis.

We report new methods for multilabel immunofluorescence (MIF) and reprobing of antigen epitopes on the same formalin-fixed paraffin-embedded (FFPE) sections. The MIF method includes an antigen-retrieval step followed by multilabel immunostaining and examination by confocal microscopy. As examples, we illustrate epitopes localized to the apical and basolateral membranes, and the cytoplasm of enterocytes of normal small intestine and in cases of congenital enteropathies (microvillous inclusion disease and congenital tufting enteropathy). We also demonstrate localization of the bile salt excretion pump protein (BSEP) in bile canalicular membrane of normal hepatocytes and in cases of primary sclerosing cholangitis. To demonstrate colocalization of cytoplasmic and nuclear epitopes we analyzed normal control and hyperplastic pulmonary neuroendocrine cells (PNEC) and neuroepithelial bodies (NEBs), presumed airway sensors in the lungs of infants with bronchopulmonary dysplasia (BPD). As cytoplasmic markers we used anti-bombesin or anti-synaptic vesicle protein 2 (SV2) antibody, respectively, and for nuclear localization, antibodies against neurogenic genes mammalian achaete-scute homolog (Mash1) and prospero homeobox 1 (Prox1), essential for NEB cells differentiation and maturation, hypoxia-inducible factor 1α (HIF1α) a downstream modulator of hypoxia response and a proliferation marker Ki67. The reprobing method consisted of removal of the previously immunolabeled target and immunostaining with different antibodies, facilitating colocalization of enterocyte brush border epitopes as well as HIF1α, Mash1 and Prox1 in PNEC/NEB PNEC and NEBs. As these methods are suitable for routine FFPE pathology samples from various tissues, allowing visualization of multiple epitopes in the same cells/sections with superior contrast and resolution, they are suitable for a wide range of applications in diagnostic pathology and may be particularly well suited for precision medicine diagnostics.