Intratumoral Delivery of Interferon\u3b3-Secreting Mesenchymal Stromal Cells Repolarizes Tumor-Associated Macrophages and Suppresses Neuroblastoma Proliferation In Vivo

Neuroblastoma, the most common extracranial solid tumor in childhood, remains a therapeutic challenge. However, one promising patient treatment strategy is the delivery of anti-tumor therapeutic agents via mesenchymal stromal cell (MSC) therapy. MSCs have been safely used to treat genetic bone diseases such as osteogenesis imperfecta, cardiovascular diseases, autoimmune diseases, and cancer. The pro-inflammatory cytokine interferon-gamma (IFN\u3b3) has been shown to decrease tumor proliferation by altering the tumor microenvironment (TME). Despite this, clinical trials of systemic IFN\u3b3 therapy have failed due to the high blood concentration required and associated systemic toxicities. Here, we developed an intra-adrenal model of neuroblastoma, characterized by liver and lung metastases. We then engineered MSCs to deliver IFN\u3b3 directly to the TME. In vitro, these MSCs polarized murine macrophages to the M1 phenotype. In vivo, we attained a therapeutically active TME concentration of IFN\u3b3 without increased systemic concentration or toxicity. The TME-specific IFN\u3b3 reduced tumor growth rate and increased survival in two models of T cell deficient athymic nude mice. Absence of this benefit in NOD SCID gamma (NSG) immunodeficient mouse model indicates a mechanism dependent on the innate immune system. IL-17 and IL-23p19, both uniquely M1 polarization markers, transiently increased in the tumor interstitial fluid. Finally, the MSC vehicle did not promote tumor growth. These findings reveal that MSCs can deliver effective cytokine therapy directly to the tumor while avoiding systemic toxicity. This method transiently induces inflammatory M1 macrophage polarization, which reduces tumor burden in our novel neuroblastoma murine model

Safety Profile of Good Manufacturing Practice Manufactured Interferon \u3b3-Primed Mesenchymal Stem/Stromal Cells for Clinical Trials

Mesenchymal stem/stromal cells (MSCs) are widely studied by both academia and industry for a broad array of clinical indications. The collective body of data provides compelling evidence of the clinical safety of MSC therapy. However, generally accepted proof of therapeutic efficacy has not yet been reported. In an effort to generate a more effective therapeutic cell product, investigators are focused on modifying MSC processing protocols to enhance the intrinsic biologic activity. Here, we report a Good Manufacturing Practice-compliant two-step MSC manufacturing protocol to generate MSCs or interferon \u3b3 (IFN\u3b3) primed MSCs which allows freshly expanded cells to be infused in patients on a predetermined schedule. This protocol eliminates the need to infuse cryopreserved, just thawed cells which may reduce the immune modulatory activity. Moreover, using (IFN\u3b3) as a prototypic cytokine, we demonstrate the feasibility of priming the cells with any biologic agent. We then characterized MSCs and IFN\u3b3 primed MSCs prepared with our protocol, by karyotype, in vitro potential for malignant transformation, biodistribution, effect on engraftment of transplanted hematopoietic cells, and in vivo toxicity in immune deficient mice including a complete post-mortem examination. We found no evidence of toxicity attributable to the MSC or IFN\u3b3 primed MSCs. Our data suggest that the clinical risk of infusing MSCs or IFN\u3b3 primed MSCs produced by our two-step protocol is not greater than MSCs currently in practice. While actual proof of safety requires phase I clinical trials, our data support the use of either cell product in new clinical studies

Essential metabolic, anti-inflammatory, and anti-tumorigenic functions of miR-122 in liver

miR-122, an abundant liver-specific microRNA (miRNA), regulates cholesterol metabolism and promotes hepatitis C virus (HCV) replication. Reduced miR-122 expression in hepatocellular carcinoma (HCC) correlates with metastasis and poor prognosis. Nevertheless, the consequences of sustained loss of function of miR-122 in vivo have not been determined. Here, we demonstrate that deletion of mouse Mir122 resulted in hepatosteatosis, hepatitis, and the development of tumors resembling HCC. These pathologic manifestations were associated with hyperactivity of oncogenic pathways and hepatic infiltration of inflammatory cells that produce pro-tumorigenic cytokines, including IL-6 and TNF. Moreover, delivery of miR-122 to a MYC-driven mouse model of HCC strongly inhibited tumorigenesis, further supporting the tumor suppressor activity of this miRNA. These findings reveal critical functions for miR-122 in the maintenance of liver homeostasis and have important therapeutic implications, including the potential utility of miR-122 delivery for selected patients with HCC and the need for careful monitoring of patients receiving miR-122 inhibition therapy for HCV.This work was supported, in part, by NIH grants CA122694 (to K. Ghoshal), DK088076 (to K. Ghoshal), CA086978 (to K. Ghoshal and S.T. Jacob), Pelotonia Idea Grant (to J. Yu and K. Ghoshal), CA120185 (to J.T. Mendell), CA134292 (to J.T. Mendell), and the Cancer Prevention and Research Institute of Texas (to J.T. Men- dell). Bo Wang is supported by a Pelotonia graduate fellowship

Elements of Good Training in Anatomic Pathology

The American College of Veterinary Pathologists’ (ACVP’s) 2007–2012 strategic plan recognized the crisis confronting academic training programs and formed a task force to address these concerns. One area of concern identified by the ACVP Training Program Development Task Force was the lack of guidelines to make training more consistent across all programs and provide justification for maintaining or increasing faculty numbers and training resources. Training guidelines for clinical pathology have been outlined in three publications.1,2,4 The current document addresses the need for training guidelines in veterinary anatomic pathology

Targeted Deletion of Kcne2 Causes Gastritis Cystica Profunda and Gastric Neoplasia

Gastric cancer is the second leading cause of cancer death worldwide. Predisposing factors include achlorhydria, Helicobacter pylori infection, oxyntic atrophy and TFF2-expressing metaplasia. In parietal cells, apical potassium channels comprising the KCNQ1 α subunit and the KCNE2 β subunit provide a K+ efflux current to facilitate gastric acid secretion by the apical H+K+ATPase. Accordingly, genetic deletion of murine Kcnq1 or Kcne2 impairs gastric acid secretion. Other evidence has suggested a role for KCNE2 in human gastric cancer cell proliferation, independent of its role in gastric acidification. Here, we demonstrate that 1-year-old Kcne2−/− mice in a pathogen-free environment all exhibit a severe gastric preneoplastic phenotype comprising gastritis cystica profunda, 6-fold increased stomach mass, increased Ki67 and nuclear Cyclin D1 expression, and TFF2- and cytokeratin 7-expressing metaplasia. Some Kcne2−/−mice also exhibited pyloric polypoid adenomas extending into the duodenum, and neoplastic invasion of thin walled vessels in the sub-mucosa. Finally, analysis of human gastric cancer tissue indicated reduced parietal cell KCNE2 expression. Together with previous findings, the results suggest KCNE2 disruption as a possible risk factor for gastric neoplasia

Cranial Ontogeny in Stegoceras validum (Dinosauria: Pachycephalosauria): A Quantitative Model of Pachycephalosaur Dome Growth and Variation

Historically, studies of pachycephalosaurs have recognized plesiomorphically flat-headed taxa and apomorphically domed taxa. More recently, it has been suggested that the expression of the frontoparietal dome is ontogenetic and derived from a flat-headed juvenile morphology. However, strong evidence to support this hypothesis has been lacking. Here we test this hypothesis in a large, stratigraphically constrained sample of specimens assigned to Stegoceras validum, the best known pachycephalosaur, using multiple independent lines of evidence including conserved morphology of ornamentation, landmark-based allometric analyses of frontoparietal shape, and cranial bone histology. New specimens show that the diagnostic ornamentation of the parietosquamosal bar is conserved throughout the size range of the sample, which links flat-headed specimens to domed S. validum. High-resolution CT scans of three frontoparietals reveal that vascularity decreases with size and document a pattern that is consistent with previously proposed histological changes during growth. Furthermore, aspects of dome shape and size are strongly correlated and indicative of ontogenetic growth. These results are complementary and strongly support the hypothesis that the sample represents a growth series of a single taxon. Cranial dome growth is positively allometric, proceeds from a flat-headed to a domed state, and confirms the synonymy of Ornatotholus browni as a juvenile Stegoceras. This dataset serves as the first detailed model of growth and variation in a pachycephalosaur. Flat-headed juveniles possess three characters (externally open cranial sutures, tuberculate dorsal surface texture, and open supratemporal fenestrae) that are reduced or eliminated during ontogeny. These characters also occur in putative flat-headed taxa, suggesting that they may also represent juveniles of domed taxa. However, open cranial sutures and supratemporal fenestrae are plesiomorphic within Ornithischia, and thus should be expected in the adult stage of a primitive pachycephalosaur. Additional lines of evidence will be needed to resolve the taxonomic validity of flat-headed pachycephalosaur taxa

The effect of intravitreal anti-VEGF agents on peripheral wound healing in a rabbit model

John Christoforidis1, Robert Ricketts1, Cedric Pratt1, Jordan Pierce1, Scott Bean1, Michael Wells1, Xiaoli Zhang2, Krista La Perle31College of Medicine, 2Center for Biostatistics, 3College of Veterinary Medicine, The Ohio State University, Columbus, OH, USAPurpose: To investigate the effect of intravitreal pegaptanib, bevacizumab, and ranibizumab on blood-vessel formation during cutaneous wound healing in a rabbit model and to compare this effect to placebo controls.Methods: Forty New Zealand albino rabbits underwent full thickness cutaneous wounds using 6-mm dermatologic punch biopsies. The rabbits were assigned to four groups of ten, each receiving intravitreal injections of pegaptanib, bevacizumab, ranibizumab, or no injection (untreated controls). Five rabbits from each group underwent wound harvesting on day 7 and five from each group on day 14. The skin samples were stained with hematoxylin and eosin (HE), Masson's trichrome (MT), and CD34 for vascular endothelial cells. Semiquantitative evaluation of HE- and MT-stained slides was performed by one pathologist. Quantitative assessment of mean neovascularization (MNV) scores was obtained from five contiguous biopsy margin 400× fields of CD34-stained sections by four independent observers.Results: Week 1 MNV scores in CD-34 stained sections were: untreated controls: 11.51 ± 4.36; bevacizumab: 7.41 ± 2.82 (P = 0.013); ranibizumab: 8.71 ± 4.08 (P = 0.071); and pegaptanib: 10.15 ± 5.59 (P = 0.378). Week 2 MNV data were: untreated controls: 6.14 ± 2.25; bevacizumab: 7.25 ± 2.75 (P = 0.471); ranibizumab: 4.53 ± 3.12 (P = 0.297); and, pegaptanib: 6.35 ± 3.09 (P = 0.892). Interobserver variability using intraclass correlation coefficient was 0.961.Conclusions: At week 1, all three anti-VEGF agents had suppressed MNV scores compared to controls. Although not statistically significant, there was an inhibitory trend, particularly with bevacizumab and ranibizumab. These effects were diminished at 2 weeks, reflecting a transition between the proliferative and remodeling phases of wound healing.Keywords: anti-VEGF, wound healing, intraocular, pegaptanib, bevacizumab, ranibizuma