Independent Blinded Validation of a Tissue Systems Pathology Test to Predict Progression in Patients With Barrett's Esophagus.

INTRODUCTION: A risk prediction test was previously validated to predict progression to high-grade dysplasia (HGD) and esophageal adenocarcinoma (EAC) in patients with Barrett's esophagus (BE). The aim of our study was to independently validate this test to predict the risk of progression to HGD/EAC in BE patients with nondysplastic (ND), indefinite for dysplasia and low-grade dysplasia (LGD). METHODS: A single-blinded, case-control study was conducted to stratify patients with BE as low, intermediate, or high risk for progression to HGD/EAC within 5 years using a previously described risk prediction test. Patients with BE who progressed to HGD/EAC after at least 1 year (n = 58) were matched to patients undergoing surveillance without progression (n = 210, median surveillance 7 years). Baseline biopsies with subspecialist diagnoses of ND, indefinite for dysplasia, or LGD were tested in a blinded manner, and the predictive performance of the test was assessed. RESULTS: This risk prediction test stratified patients with BE based on progression risk with the high-risk group at 4.7-fold increased risk for HGD/EAC compared with the low-risk group (95% confidence interval 2.5-8.8, P < 0.0001). Prevalence-adjusted positive predictive value at 5 years was 23%. The high-risk class and male sex provided predictive power that was independent of pathologic diagnosis, age, segment length, and hiatal hernia. Patients with ND BE who scored high risk progressed at a higher rate (26%) than patients with subspecialist-confirmed LGD (21.8%) at 5 years. DISCUSSION: A risk prediction test identifies patients with ND BE who are at high risk for progression to HGD/EAC and may benefit from early endoscopic therapy or increased surveillance.

Cellular heterogeneity in the mouse esophagus implicates the presence of a nonquiescent epithelial stem cell population.

Because the esophageal epithelium lacks a defined stem cell niche, it is unclear whether all basal epithelial cells in the adult esophagus are functionally equivalent. In this study, we showed that basal cells in the mouse esophagus contained a heterogeneous population of epithelial cells, similar to other rapidly cycling tissues such as the intestine or skin. Using a combination of cell-surface markers, we separated primary esophageal tissue into distinct cell populations that harbored differences in stem cell potential. We also used an in vitro 3D organoid assay to demonstrate that Sox2, Wnt, and bone morphogenetic protein signaling regulate esophageal self-renewal. Finally, we labeled proliferating basal epithelial cells in vivo to show differing cell-cycle profiles and proliferation kinetics. Based on our results, we propose that a nonquiescent stem cell population resides in the basal epithelium of the mouse esophagus.

Ectopic transplantation sites for cell-based therapy.

PURPOSE OF REVIEW: This review outlines the concept of cell-based therapy to restore tissue function, and addresses four key points to consider in cell transplantation: source, surveillance, safety, and site. Whereas each point is essential, additional attention should be given to transplantation sites if cell therapy is going to be successful in the clinic. Various ectopic locations are discussed, and the strengths and weaknesses of each are compared as suitable candidates for cell therapy. RECENT FINDINGS: Studies in rodents often demonstrate cell transplantation and engraftment in ectopic sites, with little evidence to suggest why it may also work in humans. For example, transplantation to the subcapsular space of the kidney is often performed in rodents, but has not been a good predictor of clinical success. Recent work has shown that the lymph node may be a good site for transplantation of multiple tissue types, and several reasons are highlighted as to why it should be considered for future studies. SUMMARY: The use of cell-based therapy in the clinic has been hampered by the lack of appropriate sites for transplantation. The lymph node is a promising alternative for cell transplantation, and offers hope for clinical application.

Potential barriers to human hepatocyte transplantation in MUP-uPAtg(⁺/⁺)Rag2⁻/⁻γC⁻/⁻ mice.

Primary human fetal and adult hepatocytes have been considered feasible donor cell sources for cell transplantation. We compared the engraftment efficiencies between adult human, fetal human, and adult porcine hepatocytes after transplantation into MUP-uPA(tg(+/+))Rag2(-/-)γC(-/-)mice. Transplantation of adult human hepatocytes yielded a 1,000-fold higher serum albumin level compared to transplantation of fetal human hepatocytes, while transplantation of adult porcine hepatocytes resulted in a 100-fold higher serum albumin level than adult human hepatocytes. These results suggest that adult liver cells are superior to fetal liver cells for transplantation, and caution should be applied if porcine hepatocytes are used for preclinical studies as a proof of concept for human hepatocytes.

The role of formins in human disease.

Formins are a conserved family of proteins that play key roles in cytoskeletal remodeling. They nucleate and processively elongate non-branched actin filaments and also modulate microtubule dynamics. Despite their significant contributions to cell biology and development, few studies have directly implicated formins in disease pathogenesis. This review highlights the roles of formins in cell division, migration, immunity, and microvesicle formation in the context of human disease. In addition, we discuss the importance of controlling formin activity and protein expression to maintain cell homeostasis.

Loss of RhoB expression enhances the myelodysplastic phenotype of mammalian diaphanous-related Formin mDia1 knockout mice.

Myelodysplastic syndrome (MDS) is characterized by ineffective hematopoiesis and hyperplastic bone marrow. Complete loss or interstitial deletions of the long arm of chromosome 5 occur frequently in MDS. One candidate tumor suppressor on 5q is the mammalian Diaphanous (mDia)-related formin mDia1, encoded by DIAPH1 (5q31.3). mDia-family formins act as effectors for Rho-family small GTP-binding proteins including RhoB, which has also been shown to possess tumor suppressor activity. Mice lacking the Drf1 gene that encodes mDia1 develop age-dependent myelodysplastic features. We crossed mDia1 and RhoB knockout mice to test whether the additional loss of RhoB expression would compound the myelodysplastic phenotype. Drf1(-/-)RhoB(-/-) mice are fertile and develop normally. Relative to age-matched Drf1(-/-)RhoB(+/-) mice, the age of myelodysplasia onset was earlier in Drf1(-/-)RhoB(-/-) animals--including abnormally shaped erythrocytes, splenomegaly, and extramedullary hematopoiesis. In addition, we observed a statistically significant increase in the number of activated monocytes/macrophages in both the spleen and bone marrow of Drf1(-/-)RhoB(-/-) mice relative to Drf1(-/-)RhoB(+/-) mice. These data suggest a role for RhoB-regulated mDia1 in the regulation of hematopoietic progenitor cells.

Ubiquitin-mediated degradation of the formin mDia2 upon completion of cell division.

Formins assemble non-branched actin filaments and modulate microtubule dynamics during cell migration and cell division. At the end of mitosis formins contribute to the generation of actin filaments that form the contractile ring. Rho small GTP-binding proteins activate mammalian diaphanous-related (mDia) formins by directly binding and disrupting an intramolecular autoinhibitory mechanism. Although the Rho-regulated activation mechanism is well characterized, little is known about how formins are switched off. Here we reveal a novel mechanism of formin regulation during cytokinesis based on the following observations; 1) mDia2 is degraded at the end of mitosis, 2) mDia2 is targeted for disposal by post-translational ubiquitin modification, 3) forced expression of activated mDia2 yields binucleate cells due to failed cytokinesis, and 4) the cytokinesis block is dependent upon mDia2-mediated actin assembly as versions of mDia2 incapable of nucleating actin but that still stabilize microtubules have no effect on cytokinesis. We propose that the tight control of mDia2 expression and ubiquitin-mediated degradation is essential for the completion of cell division. Because of the many roles for formins in cell morphology, we discuss the relevance of mDia protein turnover in other processes where ubiquitin-mediated proteolysis is an essential component.

Microtubule stabilization: formins assert their independence.

Mammalian Diaphanous-related (mDia) formins are well known for their actin nucleation and filament elongation activities. They have since emerged as microtubule-binding proteins, and a recent study shows that mDia2 stabilizes microtubules independently of its actin nucleation activity.

RhoB and the mammalian Diaphanous-related formin mDia2 in endosome trafficking.

Rho GTPases and the dynamic assembly and disassembly of actin filaments have been shown to have critical roles in both the internalization and trafficking of growth factor receptors. While all three mammalian Diaphanous-related (mDia1/2/3) formin GTPase effector proteins have been localized on endosomes, a role for their actin nucleation, filament elongation, and/or bundling remains poorly understood in the context of intracellular trafficking. In a study of a functional relationship between RhoB, a GTPase known to associate with both early- and late-endosomes, and the formin mDia2, we show that 1) RhoB and mDia2 interact on endosomes; 2) GTPase activity-the ability to hydrolyze GTP to GDP-is required for the ability of RhoB to govern endosome dynamics; and 3) the actin dynamics controlled by RhoB and mDia2 is necessary for vesicle trafficking. These studies further suggest that Rho GTPases significantly influence the activity of mDia family formins in driving cellular membrane remodeling through the regulation of actin dynamics.