EpCam is required for maintaining the integrity of the biliary epithelium.

BACKGROUND & AIMS: Tufting enteropathy (TE) is a rare congenital disorder often caused by mutations in the gene encoding epithelial cell adhesion molecule (EpCam). The disease leads to diarrhoea, intestinal failure and dependence on total parenteral nutrition (TPN). These patients often have liver impairments, but the pathology and mechanism of the damage are not well understood. We evaluated liver biopsies from TE patients to understand the pathophysiology. METHODS: We identified three patients with TE who underwent liver biopsy. Two normal controls and 45 patients on TPN secondary to short gut syndrome were selected for comparison (five were age- and TPN duration matched to the TE patients). RESULTS: We found that all TE patients showed a complete loss of EpCam expression in enterocytes and biliary epithelial cells, while the normal and TPN groups show basolateral expression. Histologically TE patients showed ductopenia, which was not seen in control groups. E-cadherin and ß-catenin are normally located along the lateral membrane of biliary epithelial cells. However, they were relocated to the apical membrane in TE patients, indicating a defect in the apical-basal polarity of cholangiocytes. We examined hepatic reparative cells and found near absence of hepatic progenitor cells and intermediate hepatobiliary cells with mild reactive ductular cells in TE patients. CONCLUSION: Our findings show that TE is associated with disrupted polarity of cholangiocyte and ductopenia. We demonstrate for the first time a role of EpCam in the maintenance of integrity of biliary epithelium. We also provided evidence for a disrupted development of hepatic reparative cells.

miR-149 and miR-29c as candidates for bipolar disorder biomarkers.

Bipolar disorder (BD) is a common, recurring psychiatric illness with unknown pathogenesis. Recent studies suggest that microRNA (miRNA) levels in brains of BD patients are significantly altered, and these changes may offer insight into BD pathology or etiology. Previously, we observed significant alterations of miR-29c levels in extracellular vesicles (EVs) extracted from prefrontal cortex (Brodmann area 9, BA9) of BD patients. In this study, we show that EVs extracted from the anterior cingulate cortex (BA24), a crucial area for modulating emotional expression and affect, have increased levels of miR-149 in BD patients compared to controls. Because miR-149 has been shown to inhibit glial proliferation, increased miR-149 expression in BA24-derived EVs is consistent with the previously reported reduced glial cell numbers in BA24 of patients diagnosed with either familial BD or familial major depressive disorder. qPCR analysis of laser-microdissected neuronal and glial cells from BA24 cortical samples of BD patients verified that the glial, but not neuronal, population exhibits significantly increased miR-149 expression. Finally, we report altered expression of both miR-149 and miR-29c in EVs extracted from brains of Flinders Sensitive Line rats, a well-validated animal model exhibiting depressive-like behaviors and glial (astrocytic) dysfunction. These findings warrant future investigations into the potential of using EV miRNA signatures as biomarkers to further enhance the biological definition of BD. © 2017 Wiley Periodicals, Inc.

Liposomal Vincristine as a Bridge Therapy Prior to CAR-T Therapy in Relapsed and Refractory Diffuse Large B-Cell Lymphoma?

We report a case of a 76-year-old male with a history of relapsed and refractory diffuse large B-cell lymphoma (DLBCL).Our patient was initially treated with front line chemotherapy along with central nervous system (CNS) prophylaxis with complete response. He subsequently relapsed, was sensitive to second-line chemotherapy, and underwent autologous stem cell transplantation achieving a complete remission. Only a few months after transplant, the patient suffered his second relapse and was deemed a candidate for Chimeric Antigen Receptor T-Cell Therapy (CAR-T). Given his aggressive disease, combined with the time needed to generate CAR-T cells, a multidisciplinary team recommended to treat our patient with liposomal vincristine in combination with rituximab as a bridge therapy. Durable responses have been seen using liposomal vincristine based on results from a recent phase II trial in heavily pretreated patients with DLBCL1. This therapy was effective in stabilizing and reducing active disease in our patient. This case looks to illustrate the use of liposomal vincristine in combination with immunotherapy in a novel setting bridging highly selected patients with active and refractory lymphoma prior to CAR-T. Moreover, we expanded an additional therapeutic point, highlighting the importance of optimal disease control prior to CAR-T cell harvesting, as recent literature has shown that residual malignant cells in the pheresis product may be inadvertently be transfected with the CAR gene, resulting in resistance and further relapse2.

Systemic clinical tumor regressions and potentiation of PD1 blockade with in situ vaccination.

Indolent non-Hodgkin's lymphomas (iNHLs) are incurable with standard therapy and are poorly responsive to checkpoint blockade. Although lymphoma cells are efficiently killed by primed T cells, in vivo priming of anti-lymphoma T cells has been elusive. Here, we demonstrate that lymphoma cells can directly prime T cells, but in vivo immunity still requires cross-presentation. To address this, we developed an in situ vaccine (ISV), combining Flt3L, radiotherapy, and a TLR3 agonist, which recruited, antigen-loaded and activated intratumoral, cross-presenting dendritic cells (DCs). ISV induced anti-tumor CD8+ T cell responses and systemic (abscopal) cancer remission in patients with advanced stage iNHL in an ongoing trial ( NCT01976585 ). Non-responding patients developed a population of PD1+CD8+ T cells after ISV, and murine tumors became newly responsive to PD1 blockade, prompting a follow-up trial of the combined therapy. Our data substantiate that recruiting and activating intratumoral, cross-priming DCs is achievable and critical to anti-tumor T cell responses and PD1-blockade efficacy.

Abelson phosphorylation of CLASP2 modulates its association with microtubules and actin.

The Abelson (Abl) non-receptor tyrosine kinase regulates the cytoskeleton during multiple stages of neural development, from neurulation, to the articulation of axons and dendrites, to synapse formation and maintenance. We previously showed that Abl is genetically linked to the microtubule (MT) plus end tracking protein (+TIP) CLASP in Drosophila. Here we show in vertebrate cells that Abl binds to CLASP and phosphorylates it in response to serum or PDGF stimulation. In vitro, Abl phosphorylates CLASP with a Km of 1.89 µM, indicating that CLASP is a bona fide substrate. Abl-phosphorylated tyrosine residues that we detect in CLASP by mass spectrometry lie within previously mapped F-actin and MT plus end interaction domains. Using purified proteins, we find that Abl phosphorylation modulates direct binding between purified CLASP2 with both MTs and actin. Consistent with these observations, Abl-induced phosphorylation of CLASP2 modulates its localization as well as the distribution of F-actin structures in spinal cord growth cones. Our data suggest that the functional relationship between Abl and CLASP2 is conserved and provides a means to control the CLASP2 association with the cytoskeleton.

Dystroglycan and mitochondrial ribosomal protein L34 regulate differentiation in the Drosophila eye.

Mutations that diminish the function of the extracellular matrix receptor Dystroglycan (DG) result in muscular dystrophies, with associated neuronal migration defects in the brain and mental retardation e.g. Muscle Eye Brain Disease. To gain insight into the function of DG in the nervous system we initiated a study to examine its contribution to development of the eye of Drosophila melanogaster. Immuno-histochemistry showed that DG is concentrated on the apical surface of photoreceptors (R) cells during specification of cell-fate in the third instar larva and is maintained at this location through early pupal stages. In point mutations that are null for DG we see abortive R cell elongation during differentiation that first appears in the pupa and results in stunted R cells in the adult. Overexpression of DG in R cells results in a small but significant increase in their size. R cell differentiation defects appear at the same stage in a deficiency line Df(2R)Dg(248) that affects Dg and the neighboring mitochondrial ribosomal gene, mRpL34. In the adult, these flies have severely disrupted R cells as well as defects in the lens and ommatidia. Expression of an mRpL34 transgene rescues much of this phenotype. We conclude that DG does not affect neuronal commitment but functions R cell autonomously to regulate neuronal elongation during differentiation in the pupa. We discuss these findings in view of recent work implicating DG as a regulator of cell metabolism and its genetic interaction with mRpL34, a member of a class of mitochondrial genes essential for normal metabolic function.

Evidence that dystroglycan is associated with dynamin and regulates endocytosis.

Disruption of the dystroglycan gene in humans and mice leads to muscular dystrophies and nervous system defects including malformation of the brain and defective synaptic transmission. To identify proteins that interact with dystroglycan in the brain we have used immunoaffinity purification followed by mass spectrometry (LC/MS-MS) and found that the GTPase dynamin 1 is a novel dystroglycan-associated protein. The beta-dystroglycan-dynamin 1 complex also included alpha-dystroglycan and Grb2. Overlay assays indicated that dynamin interacts directly with dystroglycan, and immunodepletion showed that only a pool of dynamin is associated with dystroglycan. Dystroglycan was associated and colocalized immunohistochemically with dynamin 1 in the central nervous system in the outer plexiform layer of retina where photoreceptor terminals are found. Endocytosis in neurons is both constitutive, as in non-neural cells, and regulated by neural activity. To assess the function of dystroglycan in the former, we have assayed transferrin uptake in fibroblastic cells differentiated from embryonic stem cells null for both dystroglycan alleles. In wild-type cells, dystroglycan formed a complex with dynamin and codistributed with cortactin at membrane ruffles, which are organelles implicated in endocytosis. Dystroglycan-null cells had a significantly greater transferrin uptake, a process well known to require dynamin. Expression of dystroglycan in null cells by infection with an adenovirus containing dystroglycan reduced transferrin uptake to levels seen in wild-type embryonic stem cells. These data suggest that dystroglycan regulates endocytosis possibly as a result of its interaction with dynamin.