ZO-1 interactions with F-actin and occludin direct epithelial polarization and single lumen specification in 3D culture.

Epithelia within tubular organs form and expand lumens. Failure of these processes can result in serious developmental anomalies. Although tight junction assembly is crucial to epithelial polarization, the contribution of specific tight junction proteins to lumenogenesis is undefined. Here, we show that ZO-1 (also known as TJP1) is necessary for the formation of single lumens. Epithelia lacking this tight junction scaffolding protein form cysts with multiple lumens and are defective in the earliest phases of polarization, both in two and three dimensions. Expression of ZO-1 domain-deletion mutants demonstrated that the actin-binding region and U5-GuK domain are crucial to single lumen development. For actin-binding region, but not U5-GuK domain, mutants, this could be overcome by strong polarization cues from the extracellular matrix. Analysis of the U5-GuK binding partners shroom2, α-catenin and occludin showed that only occludin deletion led to multi-lumen cysts. Like ZO-1-deficiency, occludin deletion led to mitotic spindle orientation defects. Single lumen formation required the occludin OCEL domain, which binds to ZO-1. We conclude that ZO-1-occludin interactions regulate multiple phases of epithelial polarization by providing cell-intrinsic signals that are required for single lumen formation.

Insufficient autoantigen presentation and failure of tolerance in a mouse model of rheumatoid arthritis.

OBJECTIVE: In the K/BxN mouse model of rheumatoid arthritis, T cells reactive for the self antigen glucose-6-phosphate isomerase (GPI) escape negative selection even though GPI expression is ubiquitous. We sought to determine whether insufficient GPI presentation could account for the failure of negative selection and for the development of arthritis. METHODS: To increase the antigen presentation of GPI, we generated transgenic mice expressing a membrane-bound form of GPI (mGPI) and crossed them with K/BxN mice. A monoclonal antibody specific for the α-chain of the KRN T cell receptor was generated to examine the fate of GPI-specific T cells. RESULTS: The mGPI-transgenic mice presented GPI more efficiently and showed a dramatic increase in negative selection and an inhibition of arthritis. Interestingly, thymic negative selection remained incomplete in these mice, and the escaped autoreactive T cells were anergic in the peripheral lymphoid organs, suggesting that enhanced antigen presentation also induces peripheral tolerance. Despite this apparent tolerance induction toward GPI, these mice developed a chronic wasting disease, characterized by colonic inflammation with epithelial dysplasia, as well as a dramatic reduction in Treg cells. CONCLUSION: These data indicate that insufficient autoantigen expression or presentation results in defects of both central and peripheral tolerance in the K/BxN mice. Our findings also support the idea that insufficient autoantigen levels may underlie the development of autoimmunity.

Robotic Distal Pancreatectomy and Splenectomy for an Intrapancreatic Hepatocellular Carcinoma: A Case Report and Review of the Literature.

Background: Liver parenchyma that resides outside of the normal hepatic confines is defined as accessory liver if in communication with the native biliary tree, or ectopic liver (EL) if it is not. EL can develop in a variety of tissues, including but not limited to the gallbladder, the hepatic ligaments, the pancreas, and retroperitoneum. EL has an increased propensity for malignant degeneration resulting in hepatocellular carcinoma (HCC). Presentation: A 67-year-old Korean male presented with epigastric discomfort and was found to have an elevation in his transaminases. Cross-sectional imaging demonstrated a 1.3 cm solid mass in the body of the pancreas with features concerning for either a pancreatic ductal adenocarcinoma or pancreatic neuroendocrine tumor. Subsequent endoscopic ultrasound and fine needle aspiration demonstrated cells of epithelial origin with hepatocellular differentiation. A robotic-assisted distal pancreatectomy and splenectomy was performed with final pathology demonstrating a well-differentiated HCC. Conclusions: EL with malignant degeneration resulting in HCC requires surgical excision. The majority of patients reported with EL resulting in HCC in the pancreas have had the tumors located in the body and tail. Therefore, definitive treatment requires distal pancreatectomy and splenectomy. Herein, we describe the presentation, workup, and definitive treatment of HCC arising in the pancreas.

Pathologic Response to Primary Systemic Therapy With FOLFIRINOX in Patients With Resectable Pancreatic Cancer.

BACKGROUND: Primary systemic therapy in resectable pancreatic cancer is currently under investigation. FOLFIRINOX has been shown to be effective in both the adjuvant and metastatic settings and is increasingly being used on and off study in the neoadjuvant setting. The objective pathologic response elicited by this regimen in truly resectable disease has not as yet been widely reported. METHODS: This analysis focuses on 14 patients with resectable pancreatic cancer who were treated in a pilot study of primary systemic therapy, using 4 cycles of neoadjuvant FOLFIRINOX before surgery. A dedicated pancreatic pathologist reviewed all of the subsequent surgical specimens to assess the degree of tumor regression elicited by this approach, according to the scoring system proposed by Evans. RESULTS: Four patients (28.6%) had Evans grade I, 4 (28.6%) Evans grade IIa, 2 (14.2%) Evans grade IIb, and 4 (28.6%) Evans grade III response to the primary systemic therapy. There were no Evans grade IV responses. CONCLUSIONS: The results are intriguing with 28% of the specimens showing destruction of <10% of tumor cells, and only 28% achieving >90% destruction of tumor cells. The significant variation in response once again confirms the known heterogeneity in the biology of this cancer and clearly FOLFIRINOX is not equally effective in all patients. Future studies evaluating primary systemic therapy in pancreatic cancer should examine the optimal duration of therapy before surgery and should include a standardized pathologic grading scheme to better enable comparison of results.

A Case of Severe, Nilotinib-Induced Liver Injury.

Idiosyncratic hepatotoxicity is a leading reason for the discontinuation or dose modification of Food and Drug Administration (FDA)-approved medications in the United States. We report the case of a 53-year-old woman with chronic myeloid leukemia who developed acute cholestatic hepatitis in response to the tyrosine kinase inhibitor nilotinib. Nilotinib was discontinued, and the patient's liver function tests normalized over the next 3 months. We conclude that nilotinib may cause life-threatening hepatotoxicity and recommend that patients on the medication undergo regular monitoring of their liver tests.

Graft-versus-host disease propagation depends on increased intestinal epithelial tight junction permeability.

Graft-versus-host disease (GVHD) is a complication of hematopoietic stem cell transplantation (HSCT) that affects multiple organs. GVHD-associated intestinal damage can be separated into two distinct phases, initiation and propagation, which correspond to conditioning-induced damage and effector T cell activation and infiltration, respectively. Substantial evidence indicates that intestinal damage induced by pretransplant conditioning is a key driver of GVHD initiation. Here, we aimed to determine the impact of dysregulated intestinal permeability on the subsequent GVHD propagation phase. The initiation phase of GVHD was unchanged in mice lacking long MLCK (MLCK210), an established regulator of epithelial tight junction permeability. However, MLCK210-deficient mice were protected from sustained barrier loss and exhibited limited GVHD propagation, as indicated by reduced histopathology, fewer CD8+ effector T cells in the gut, and improved overall survival. Consistent with these findings, intestinal epithelial MLCK210 expression and enzymatic activity were similarly increased in human and mouse GVHD biopsies. Intestinal epithelial barrier loss mediated by MLCK210 is therefore a key driver of the GVHD propagation. These data suggest that inhibition of MLCK210-dependent barrier regulation may be an effective approach to limiting GVHD progression.

Protein Kinase C theta (PKCtheta) expression and constitutive activation in gastrointestinal stromal tumors (GISTs).

KIT expression is a key diagnostic feature of gastrointestinal stromal tumors (GISTs), and virtually all of the GISTs express oncogenic forms of the KIT or PDGFRA receptor tyrosine kinase proteins, which serve as therapeutic targets of imatinib mesylate (Gleevec; Novartis, Basel, Switzerland). However, KIT expression can be low in PDGFRA-mutant GISTs, increasing the likelihood of misdiagnosis as other types of sarcoma. We report that the signaling intermediate protein kinase C theta (PKCtheta) is a diagnostic marker in GISTs, including those that lack KIT expression and/or contain PDGFRA mutations. PKCtheta is strongly activated in most GISTs and hence may serve, along with KIT/PDGFRA, as a novel therapeutic target.

Mechanisms of oncogenic KIT signal transduction in primary gastrointestinal stromal tumors (GISTs).

Most gastrointestinal stromal tumors (GISTs) express constitutively activated forms of the KIT receptor tyrosine kinase protein, resulting from oncogenic mutations in the extracellular, juxtamembrane, or kinase domains. KIT oncoproteins are detected early in GIST tumorigenesis, and most GIST patients respond well to treatment with the KIT kinase inhibitor imatinib mesylate (STI571, Gleevec). However, GISTs can develop resistance to imatinib, and additional therapeutic strategies are needed. Little is known about oncogenic KIT signal transduction in GISTs, and whether the type of KIT mutation accounts for selective activation of downstream signaling intermediates. We therefore evaluated KIT downstream signaling profiles in 15 primary GISTs with mutations in KIT exons 9, 11, 13, and 17, and in two human GIST cell lines. All GISTs showed constitutive phosphorylation at KIT tyrosine residues Y703 and Y721. Additionally, most GISTs showed activation of MAPK p42/44, AKT, S6K, STAT1, and STAT3. STAT5 and JNK were not demonstrably activated in any GIST. Using GIST in vitro models, we showed that activation of MAPK p42/44, AKT, and S6K was KIT dependent, whereas STAT1 and STAT3 phosphorylation was only partially dependent on KIT activation. Correlation of activated signaling pathways with the type of KIT mutation revealed low levels of AKT phosphorylation in exon 9 mutant GISTs in contrast to a subset of GISTs with exon 11 mutations. However, additional factors are likely to modify the engagement of signaling pathways in GISTs as suggested by the fact that four GISTs with identical KIT exon 9 mutations had differential activation of MAPK p42/44 and STAT proteins. In summary, in this first report on KIT signal transduction in primary GISTs and GIST cell lines, we identified pathways that are constitutively activated in a KIT-dependent manner and therefore warrant further study as molecular targets in GISTs.

Recipient NK cell inactivation and intestinal barrier loss are required for MHC-matched graft-versus-host disease.

Previous studies have shown a correlation between pretransplant conditioning intensity, intestinal barrier loss, and graft-versus-host disease (GVHD) severity. However, because irradiation and other forms of pretransplant conditioning have pleiotropic effects, the precise role of intestinal barrier loss in GVHD pathogenesis remains unclear. We developed GVHD models that allowed us to isolate the specific contributions of distinct pretransplant variables. Intestinal damage was required for the induction of minor mismatch [major histocompatibility complex (MHC)-matched] GVHD, but was not necessary for major mismatch GVHD, demonstrating fundamental pathogenic distinctions between these forms of disease. Moreover, recipient natural killer (NK) cells prevented minor mismatch GVHD by limiting expansion and target organ infiltration of alloreactive T cells via a perforin-dependent mechanism, revealing an immunoregulatory function of MHC-matched recipient NK cells in GVHD. Minor mismatch GVHD required MyD88-mediated Toll-like receptor 4 (TLR4) signaling on donor cells, and intestinal damage could be bypassed by parenteral lipopolysaccharide (LPS) administration, indicating a critical role for the influx of bacterial components triggered by intestinal barrier loss. In all, the data demonstrate that pretransplant conditioning plays a dual role in promoting minor mismatch GVHD by both depleting recipient NK cells and inducing intestinal barrier loss.