Cytokine-modified VSV is attenuated for neural pathology, but is both highly immunogenic and oncolytic

Vesicular stomatitis virus (VSV), an enveloped, nonsegmented, negative-stranded RNA virus, is being tested by several laboratories as an antitumor agent. Unfortunately, viral infection of the central nervous system (CNS) has been observed by many groups following administration to tumor-bearing animals. In rodents, VSV encephalitis is characterized by weight-loss, paralysis, and high mortality. In order to provide protection from VSV infection of the CNS after therapeutic administration, we have attenuated VSV by the introduction of the gene encoding the proinflammatory cytokine interleukin (IL)-23, and designated the new virus VSV23. We hypothesize that while VSV23 is replicating within tumors, resulting in tumor destruction, the expression of IL-23 will enhance host antitumor and antiviral immune responses. In the event that the virus escapes from the tumor, the host’s immune system will be activated and the virus will be rapidly cleared from healthy tissue. Experimental VSV23 infection of the CNS is characterized by decreased viral replication, morbidity, and mortality. VSV23 is capable of stimulating the enhanced production of nitric oxide in the CNS, which is critical for elimination of VSV from infected neurons. Intraperitoneal administration of VSV23 stimulates both nonspecific natural killer cell, virus-specific cytolytic T lymphocyte and memory virus-specific proliferative T cell responses against wild-type VSV in splenocytes. Furthermore, VSV23 is able to replicate in, and induce apoptosis of tumor cells in vitro. These data indicate that VSV23 is immunogenic, attenuated and suitable for testing as an efficacious and safe oncolytic agent

Foxl1-Expressing Mesenchymal Cells Constitute the Intestinal Stem Cell NicheSummary

Background & Aims: Intestinal epithelial stem cells that express leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5) and/or B cell specific Moloney murine leukemia virus integration site 1 (Bmi1) continuously replicate and generate differentiated cells throughout life. Previously, Paneth cells were suggested to constitute an epithelium-intrinsic niche that regulates the behavior of these stem cells. However, ablating Paneth cells has no effect on the maintenance of functional stem cells. Here, we show definitively that a small subset of mesenchymal subepithelial cells expressing the winged-helix transcription factor forkhead box l1 (Foxl1) are a critical component of the intestinal stem cell niche. Methods: We genetically ablated Foxl1+ mesenchymal cells in adult mice using 2 separate models by expressing either the human or simian diphtheria toxin receptor under Foxl1 promoter control. Conclusions: Killing Foxl1+ cells by diphtheria toxin administration led to an abrupt cessation of proliferation of both epithelial stem- and transit-amplifying progenitor cell populations that was associated with a loss of active Wnt signaling to the intestinal epithelium. Therefore, Foxl1-expressing mesenchymal cells constitute the fundamental niche for intestinal stem cells. Keywords: Intestinal Stem Cell Niche, Wnt, Mesenchym

A Confocal and Electron Microscopic Comparison of Interferon β–Induced Changes in Vesicular Stomatitis Virus Infection of Neuroblastoma and Nonneuronal Cells

Vesicular stomatitis virus (VSV) replication is highly sensitive to interferon (IFN)-induced antiviral responses. Pretreatment of sensitive cultured cells with IFNβ results in a 104-fold reduction in the release of infectious VSV particles. However, differences exist between the mechanisms of reduced infectious particle titers in cell lines of neuroblastoma and nonneuronal lineage. In L929-fibroblast-derived cells, using immunofluorescence confocal microscopy, infection under control conditions reveals the accumulation of VSV matrix, phosphoprotein (P), and nucleocapsid (N) proteins over time, with induced cellular morphological changes indicative of cytopathic effects (CPEs). Upon observing L929 cells that had been pretreated with IFNβ, neither detectable VSV proteins nor CPEs were seen, consistent with type I IFN antiviral protection. When using the same techniques to observe VSV infections of NB41A3 cells, a neuroblastoma cell line, aside from similar viral progression in the untreated control cells, IFNβ-treated cells illustrated a severely attenuated VSV infection. Attenuated VSV progression was observed through detection of VSV matrix, P, and N proteins in isolated cells during the first 8 h of infection. However, by 18–24 h postinfection all neuroblastomas had succumbed to the viral infection. Finally, upon closer inspection of IFNβ-treated NB41A3 cells, no detectable changes in VSV protein localization were identified compared with untreated, virally infected neuroblastomas. Next, to extend our study to test our hypothesis that virion assembly is compromised within type I IFN-treated neuroblastoma cells, we employed electron microscopy to examine our experimental conditions at the ultrastructural level. Using VSV-specific antibodies in conjunction with immuno-gold reagents, we observed several similarities between the two cell lines, such as identification of viroplasmic regions containing VSV N and P proteins and signs of stress-induced CPEs of VSV-infected cells, which had either been mock-treated or pretreated with interferon-β (IFNβ). One difference we observed between nonneuronal and neuroblastoma cells was more numerous actively budding VSV virions across untreated L929 plasma membranes compared with untreated NB41A3 cells. Additionally, IFNβ-treated, VSV-infected L929 cells exhibited neither cytoplasmic viroplasm nor viral protein expression. In contrast, IFNβ-treated, VSV-infected NB41A3 cells showed evidence of VSV infection at a very low frequency as well as small-scale viroplasmic regions that colocalized with viral N and P proteins. Finally, we observed that VSV viral particles harvested from untreated VSV-infected L929 and NB41A3 cells were statistically similar in size and shape. A portion of VSV virions from IFNβ-treated, virally infected NB41A3 cells were similar in size and shape to virus from both untreated cell types. However, among the sampling of virions, pleomorphic viral particles that were identified from IFNβ-treated, VSV-infected NB41A3 cells were different enough to suggest a misassembly mechanism as part of the IFNβ antiviral state in neuroblastoma cells

IFN-β-Induced Alteration of VSV Protein Phosphorylation in Neuronal Cells

Vesicular stomatitis virus (VSV) replication is highly sensitive to interferon (IFN)-induced antiviral responses. VSV infection of well-known cell lines pretreated with IFN-β results in a 104-fold reduction in the release of infectious particles, with a concomitant abrogation in viral transcript and/or protein levels. However, in cell lines of neuronal lineage only a threefold reduction in viral transcript and protein levels was observed, despite the same 104-fold reduction in released infectious virions, suggesting an assembly defect. Examination of VSV matrix (M) protein ubiquitination yielded no differences between mock- and IFN-β-treated neuronal cells. Further analysis of potential post-translational modification events, by scintillation and two-dimensional electrophoretic methods, revealed IFN-β-induced alterations in M protein and phosphoprotein (P) phosphorylation. Hypophosphorylated P protein was demonstrated by reduced 32P counts, normalized by 35S-cysteine/methionine incorporation, and by a shift in isoelectric focusing. Hypophosphorylation of VSV P protein was found to occur in neuronal cell lysates, but not within budded virions from the same IFN-β-treated cells. In contrast, hyperphosphorylation of VSV M protein was observed in both cell lysates and viral particles from IFN-β-treated neuronal cells. Hyperphosphorylated M protein was demonstrated by increased 32P counts relative to 35S-cysteine/methionine normalization, and by altered isoelectric focusing in protein populations from cell and viral lysates. Hyperphosphorylated VSV M protein was found to inhibit its association with VSV nucleocapsid, suggesting a possible mechanism for type I IFN-mediated misassembly through disruption of the interactions between ribonucleoprotein cores, and hyperphosphorylated M protein bound to the plasma membrane inner leaflet

Novel variants in the stem cell niche factor WNT2B define the disease phenotype as a congenital enteropathy with ocular dysgenesis

WNT2B is a member of the Wnt family, a group of signal transduction proteins involved in embryologic development and stem cell renewal and maintenance. We recently reported homozygous nonsense variants in WNT2B in three individuals with severe, neonatal-onset diarrhea, and intestinal failure. Here we present a fourth case, from a separate family, with neonatal diarrhea associated with novel compound heterozygous WNT2B variants. One of the two variants was a frameshift variant (c.423del [p.Phe141fs]), while the other was a missense change (c.722 G > A [p.G241D]) that we predict through homology modeling to be deleterious, disrupting post-translational acylation. This patient presented as a neonate with severe diet-induced (osmotic) diarrhea and growth failure resulting in dependence on parenteral nutrition. Her gastrointestinal histology revealed abnormal cellular architecture particularly in the stomach and colon, including oxyntic atrophy, abnormal distribution of enteroendocrine cells, and a paucity of colonic crypt glands. In addition to her gastrointestinal findings, she had bilateral corneal clouding and atypical genital development later identified as a testicular 46,XX difference/disorder of sexual development. Upon review of the previously reported cases, two others also had anterior segment ocular anomalies though none had atypical genital development. This growing case series suggests that variants in WNT2B are associated with an oculo-intestinal (and possibly gonadal) syndrome, due to the protein’s putative involvement in multiple developmental and stem cell maintenance pathways