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Objective The glands of the stomach body and antral mucosa contain a complex compendium of cell lineages. In lower mammals, the distribution of oxyntic glands and antral glands define the anatomical regions within the stomach. We examined in detail the distribution of the full range of cell lineages within the human stomach. Design We determined the distribution of gastric gland cell lineages with specific immunocytochemical markers in entire stomach specimens from three non-obese organ donors. Results The anatomical body and antrum of the human stomach were defined by the presence of ghrelin and gastrin cells, respectively. Concentrations of somatostatin cells were observed in the proximal stomach. Parietal cells were seen in all glands of the body of the stomach as well as in over 50% of antral glands. MIST1 expressing chief cells were predominantly observed in the body although individual glands of the antrum also showed MIST1 expressing chief cells. While classically described antral glands were observed with gastrin cells and deep antral mucous cells without any parietal cells, we also observed a substantial population of mixed type glands containing both parietal cells and G cells throughout the antrum. Conclusions Enteroendocrine cells show distinct patterns of localisation in the human stomach. The existence of antral glands with mixed cell lineages indicates that human antral glands may be functionally chimeric with glands assembled from multiple distinct stem cell populations.

The Rab GTPases are the largest family of proteins regulating membrane traffic. Rab proteins form a nidus for the assembly of multiprotein complexes on distinct vesicle membranes to regulate particular membrane trafficking pathways. Recent investigations have demonstrated that Myosin Vb (Myo5B) is an effector for Rab8a, Rab10, and Rab11a, all of which are implicated in regulating different pathways for recycling of proteins to the plasma membrane. It remains unclear how specific interactions of Myo5B with individual Rab proteins can lead to specificity in the regulation of alternate trafficking pathways. We examined the relative contributions of Rab/Myo5B interactions with specific pathways using Myo5B mutants lacking binding to either Rab11a or Rab8a. Myo5B Q1300L and Y1307C mutations abolished Rab8a association, whereas Myo5B Y1714E and Q1748R mutations uncoupled association with Rab11a. Expression of Myo5B tails containing these mutants demonstrated that Rab11a, but not Rab8a, was required for recycling of transferrin in nonpolarized cells. In contrast, in polarized epithelial cyst cultures, Myo5B was required for apical membrane trafficking and de novo lumen formation, dependent on association with both Rab8a and Rab11a. These data demonstrate that different combinations of Rab GTPase association with Myo5B control distinct membrane trafficking pathways.

Crohn’s disease (CD) is a complex chronic inflammatory disorder with both gastrointestinal and extra-intestinal manifestations associated immune dysregulation. Analyzing 202,359 cells from 170 specimens across 83 patients, we identify a distinct epithelial cell type in both terminal ileum and ascending colon (hereon as ‘LND’) with high expression of LCN2 , NOS2 , and DUOX2 and genes related to antimicrobial response and immunoregulation. LND cells, confirmed by in-situ RNA and protein imaging, are rare in non-IBD controls but expand in active CD, and actively interact with immune cells and specifically express IBD/CD susceptibility genes, suggesting a possible function in CD immunopathogenesis. Furthermore, we discover early and late LND subpopulations with different origins and developmental potential. A higher ratio of late-to-early LND cells correlates with better response to anti-TNF treatment. Our findings thus suggest a potential pathogenic role for LND cells in both Crohn’s ileitis and colitis. Crohn’s disease (CD) is a complex disease associated with immune dysregulation. Here the authors use multimodal data to identify and characterize an epithelial cell population, termed ‘LND’ cells, in both terminal ileum and ascending colon, with LND interacting locally with immune cells and potentially contributing to CD pathology.

Microvillus inclusion disease (MVID), caused by loss-of-function mutations in the motor protein myosin Vb (MYO5B), is a severe infantile disease characterized by diarrhea, malabsorption, and acid/base instability, requiring intensive parenteral support for nutritional and fluid management. Human patient-derived enteroids represent a model for investigation of monogenic epithelial disorders but are a rare resource from MVID patients. We developed human enteroids with different loss-of function MYO5B variants and showed that they recapitulated the structural changes found in native MVID enterocytes. Multiplex immunofluorescence imaging of patient duodenal tissues revealed patient-specific changes in localization of brush border transporters. Functional analysis of electrolyte transport revealed profound loss of Na+/H+ exchange (NHE) activity in MVID patient enteroids with near-normal chloride secretion. The chloride channel-blocking antidiarrheal drug crofelemer dose-dependently inhibited agonist-mediated fluid secretion. MVID enteroids exhibited altered differentiation and maturation versus healthy enteroids. γ-Secretase inhibition with DAPT recovered apical brush border structure and functional Na+/H+ exchange activity in MVID enteroids. Transcriptomic analysis revealed potential pathways involved in the rescue of MVID cells including serum/glucocorticoid-regulated kinase 2 (SGK2) and NHE regulatory factor 3 (NHERF3). These results demonstrate the utility of patient-derived enteroids for developing therapeutic approaches to MVID.

Background While immune checkpoint blockade (ICB) is the current first-line treatment for metastatic melanoma, it is effective for ~ 52% of patients and has dangerous side effects. The objective here was to identify the feasibility and mechanism of RAS/RAF/PI3K pathway inhibition in melanoma to sensitize tumors to ICB therapy. Methods Rigosertib (RGS) is a non-ATP-competitive small molecule RAS mimetic. RGS monotherapy or in combination therapy with ICB were investigated using immunocompetent mouse models of BRAF wt and BRAF mut melanoma and analyzed in reference to patient data. Results RGS treatment (300 mg/kg) was well tolerated in mice and resulted in ~ 50% inhibition of tumor growth as monotherapy and ~ 70% inhibition in combination with αPD1 + αCTLA4. RGS-induced tumor growth inhibition depends on CD40 upregulation in melanoma cells followed by immunogenic cell death, leading to enriched dendritic cells and activated T cells in the tumor microenvironment. The RGS-initiated tumor suppression was partially reversed by either knockdown of CD40 expression in melanoma cells or depletion of CD8 + cytotoxic T cells. Treatment with either dabrafenib and trametinib or with RGS, increased CD40 + SOX10 + melanoma cells in the tumors of melanoma patients and patient-derived xenografts. High CD40 expression level correlates with beneficial T-cell responses and better survival in a TCGA dataset from melanoma patients. Expression of CD40 by melanoma cells is associated with therapeutic response to RAF/MEK inhibition and ICB. Conclusions Our data support the therapeutic use of RGS + αPD1 + αCTLA4 in RAS/RAF/PI3K pathway-activated melanomas and point to the need for clinical trials of RGS + ICB for melanoma patients who do not respond to ICB alone. Trial registration NCT01205815 (Sept 17, 2010). Graphical abstract

Intestinal tuft cells have recently been the focus of many studies due to their function in chemosensation and type 2 immunity in human gastrointestinal diseases. This study investigated the impact of acute tuft cell loss on intestinal physiological function. Tuft cell deletion was induced in DCLK1‐IRES‐GFP‐CreERT2/+;Rosa‐DTA (DCLK1‐DTA) mice by a single tamoxifen injection, concomitant with littermate controls. Transient deletion of intestinal and biliary tuft cells was maximal on day 4 and recovered by day 7 post tamoxifen. DCLK1‐DTA mice presented with significantly shortened small intestinal length and greater body weight loss by day 4. The activity of Na+‐dependent glucose transporter 1 (SGLT1) and cystic fibrosis transmembrane regulator (CFTR) was reduced. Correlated with tuft cell reduction, the frequency of cholecystokinin (CCK)+ enteroendocrine and intermediate secretory cells, which co‐express Paneth and goblet cell markers, was increased. In the lamina propria, fewer mast cells and leukocytes were found in the Day 4 DCLK1‐DTA mice compared to controls. Ablation of tuft cells may induce nutrient malabsorption through alterations in epithelial cell proliferation and differentiation, along with changes in the mucosal defense response. These observations identify a new role for tuft cells in regulating intestinal absorption and mucosal regeneration.

Functional loss of myosin Vb (MYO5B) induces a variety of deficits in intestinal epithelial cell function and causes a congenital diarrheal disorder, microvillus inclusion disease (MVID). The impact of MYO5B loss on differentiated cell lineage choice has not been investigated. We quantified the populations of differentiated epithelial cells in tamoxifen-induced, epithelial cell-specific MYO5B-knockout (VilCreERT2 Myo5bfl/fl) mice utilizing digital image analysis. Consistent with our RNA-sequencing data, MYO5B loss induced a reduction in tuft cells in vivo and in organoid cultures. Paneth cells were significantly increased by MYO5B deficiency along with expansion of the progenitor cell zone. We further investigated the effect of lysophosphatidic acid (LPA) signaling on epithelial cell differentiation. Intraperitoneal LPA significantly increased tuft cell populations in both control and MYO5B-knockout mice. Transcripts for Wnt ligands were significantly downregulated by MYO5B loss in intestinal epithelial cells, whereas Notch signaling molecules were unchanged. Additionally, treatment with the Notch inhibitor dibenzazepine (DBZ) restored the populations of secretory cells, suggesting that the Notch pathway is maintained in MYO5B-deficient intestine. MYO5B loss likely impairs progenitor cell differentiation in the small intestine in vivo and in vitro, partially mediated by Wnt/Notch imbalance. Notch inhibition and/or LPA treatment may represent an effective therapeutic approach for treatment of MVID.

Urinary concentration is an energy-dependent process that minimizes body water loss by increasing aquaporin 2 (AQP2) expression in collecting duct (CD) principal cells. To investigate the role of mitochondrial (mt) ATP production in renal water clearance, we disrupted mt electron transport in CD cells by targeting ubiquinone (Q) binding protein QPC (UQCRQ), a subunit of mt complex III essential for oxidative phosphorylation. QPC-deficient mice produced less concentrated urine than controls, both at baseline and after type 2 vasopressin receptor stimulation with desmopressin. Impaired urinary concentration in QPC-deficient mice was associated with reduced total AQP2 protein levels in CD tubules, while AQP2 phosphorylation and membrane trafficking remained unaffected. In cultured inner medullary CD cells treated with mt complex III inhibitor antimycin A, the reduction in AQP2 abundance was associated with activation of 5' adenosine monophosphate-activated protein kinase (AMPK) and was reversed by treatment with AMPK inhibitor SBI-0206965. In summary, our studies demonstrated that the physiological regulation of AQP2 abundance in principal CD cells was dependent on mt electron transport. Furthermore, our data suggested that oxidative phosphorylation in CD cells was dispensable for maintaining water homeostasis under baseline conditions, but necessary for maximal stimulation of AQP2 expression and urinary concentration.

BackgroundImmune checkpoint blockade (ICB) therapies are an important treatment for patients with advanced cancers; however, only a subset of patients with certain types of cancer achieve durable remission. Cancer vaccines are an attractive strategy to boost patient immune responses, but less is known about whether and how immunization can induce long-term tumor immune reprogramming and arrest cancer progression. We developed a clinically relevant genetic cancer mouse model in which hepatocytes sporadically undergo oncogenic transformation. We compared how tumor-specific CD8 T cells (TST) differentiated in mice with early sporadic lesions as compared with late lesions and tested how immunotherapeutic strategies, including vaccination and ICB, impact TST function and liver cancer progression.MethodsMice with a germline floxed allele of the SV40 large T antigen (TAG) undergo spontaneous recombination and activation of the TAG oncogene, leading to rare early cancerous TAG-expressing lesions that inevitably progress to established liver cancer. We assessed the immunophenotype (CD44, PD1, TCF1, and TOX expression) and function (TNFα and IFNγ cytokine production) of tumor/TAG-specific CD8 T cells in mice with early and late liver lesions by flow cytometry. We vaccinated mice, either alone or in combination with ICB, to test whether these immunotherapeutic interventions could stop liver cancer progression and improve survival.ResultsIn mice with early lesions, a subset of TST were PD1+ TCF1+ TOX− and could produce IFNγ while TST present in mice with late liver cancers were PD1+ TCF1lo/− TOX+ and unable to make effector cytokines. Strikingly, vaccination with attenuated TAG epitope-expressing Listeria monocytogenes (LMTAG) blocked liver cancer development and led to a population of TST that were PD1-heterogeneous, TCF1+ TOX− and polyfunctional cytokine producers. Vaccine-elicited TCF1+TST could self-renew and differentiate, establishing them as progenitor TST. In contrast, ICB administration did not slow cancer progression or improve LMTAG vaccine efficacy.ConclusionVaccination, but not ICB, generated a population of functional progenitor TST and halted cancer progression in a clinically relevant model of sporadic liver cancer. In patients with early cancers or at high risk of cancer recurrence, immunization may be the most effective strategy.

Acquired resistance to BRAF/MEK-targeted therapy occurs in the majority of melanoma patients that harbor BRAF mutated tumors, leading to relapse or progression and the underlying mechanism is unclear in many cases. Using multiplex immunohistochemistry and spatial imaging analysis of paired tumor sections obtained from 11 melanoma patients prior to BRAF/MEK-targeted therapy and when the disease progressed on therapy, we observed a significant increase of tumor cellularity in the progressed tumors and the close association of SOX10 + melanoma cells with CD8 + T cells negatively correlated with patient’s progression-free survival (PFS). In the TCGA-melanoma dataset ( n  = 445), tumor cellularity exhibited additive prognostic value in the immune score signature to predict overall survival in patients with early-stage melanoma. Moreover, tumor cellularity prognoses OS independent of immune score in patients with late-stage melanoma.