Novel Murine Pancreatic Tumor Model Demonstrates Immunotherapeutic Control of Tumor Progression by a Toxoplasma gondii Protein.

Pancreatic ductal adenocarcinoma is the fourth leading cause of cancer-related death in the United States, with few effective treatments available and only 10% of those diagnosed surviving 5 years. Although immunotherapeutics is a growing field of study in cancer biology, there has been little progress in its use for the treatment of pancreatic cancer. Pancreatic cancer is considered a nonimmunogenic tumor because the tumor microenvironment does not easily allow for the immune system, even when stimulated, to attack the cancer. Infection with the protozoan parasite Toxoplasma gondii has been shown to enhance the immune response to clear cancer tumors. A subset of T. gondii proteins called soluble Toxoplasma antigen (STAg) contains an immunodominant protein called profilin. Both STAg and profilin have been shown to stimulate an immune response that reduces viral, bacterial, and parasitic burdens. Here, we use STAg and profilin to treat pancreatic cancer in a KPC mouse-derived allograft murine model. These mice exhibit pancreatic cancer with both Kras and P53 mutations as subcutaneous tumors. Pancreatic cancer tumors in C57BL/6J mice with a wild-type background showed a significant response to treatment with either profilin or STAg, exhibiting a decrease in tumor volume accompanied by an influx of CD4+ and CD8+ T cells into the tumors. Both IFN-γ-/- mice and Batf3-/- mice, which lack conventional dendritic cells, failed to show significant decreases in tumor volumes when treated. These results indicate that gamma interferon (IFN-γ) and dendritic cells may play critical roles in the immune response necessary to treat pancreatic cancer.

ICTV Virus Taxonomy Profile: Bornaviridae.

Members of the family Bornaviridae produce enveloped virions containing a linear negative-sense non-segmented RNA genome of about 9 kb. Bornaviruses are found in mammals, birds, reptiles and fish. The most-studied viruses with public health and veterinary impact are Borna disease virus 1 and variegated squirrel bornavirus 1, both of which cause fatal encephalitis in humans. Several orthobornaviruses cause neurological and intestinal disorders in birds, mostly parrots. Endogenous bornavirus-like sequences occur in the genomes of various animals. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Bornaviridae, which is available at ictv.global/report/bornaviridae.

Patient-Derived Cancer Organoid Cultures to Predict Sensitivity to Chemotherapy and Radiation.

PURPOSE: Cancer treatment is limited by inaccurate predictors of patient-specific therapeutic response. Therefore, some patients are exposed to unnecessary side effects and delays in starting effective therapy. A clinical tool that predicts treatment sensitivity for individual patients is needed. EXPERIMENTAL DESIGN: Patient-derived cancer organoids were derived across multiple histologies. The histologic characteristics, mutation profile, clonal structure, and response to chemotherapy and radiation were assessed using bright-field and optical metabolic imaging on spheroid and single-cell levels, respectively. RESULTS: We demonstrate that patient-derived cancer organoids represent the cancers from which they were derived, including key histologic and molecular features. These cultures were generated from numerous cancers, various biopsy sample types, and in different clinical settings. Next-generation sequencing reveals the presence of subclonal populations within the organoid cultures. These cultures allow for the detection of clonal heterogeneity with a greater sensitivity than bulk tumor sequencing. Optical metabolic imaging of these organoids provides cell-level quantification of treatment response and tumor heterogeneity allowing for resolution of therapeutic differences between patient samples. Using this technology, we prospectively predict treatment response for a patient with metastatic colorectal cancer. CONCLUSIONS: These studies add to the literature demonstrating feasibility to grow clinical patient-derived organotypic cultures for treatment effectiveness testing. Together, these culture methods and response assessment techniques hold great promise to predict treatment sensitivity for patients with cancer undergoing chemotherapy and/or radiation.

Taxonomy of the order Mononegavirales: second update 2018.

In October 2018, the order Mononegavirales was amended by the establishment of three new families and three new genera, abolishment of two genera, and creation of 28 novel species. This article presents the updated taxonomy of the order Mononegavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV).

Strengthening the Interaction of the Virology Community with the International Committee on Taxonomy of Viruses (ICTV) by Linking Virus Names and Their Abbreviations to Virus Species.

The International Committee on Taxonomy of Viruses (ICTV) is tasked with classifying viruses into taxa (phyla to species) and devising taxon names. Virus names and virus name abbreviations are currently not within the ICTV's official remit and are not regulated by an official entity. Many scientists, medical/veterinary professionals, and regulatory agencies do not address evolutionary questions nor are they concerned with the hierarchical organization of the viral world, and therefore, have limited use for ICTV-devised taxa. Instead, these professionals look to the ICTV as an expert point source that provides the most current taxonomic affiliations of viruses of interests to facilitate document writing. These needs are currently unmet as an ICTV-supported, easily searchable database that includes all published virus names and abbreviations linked to their taxa is not available. In addition, in stark contrast to other biological taxonomic frameworks, virus taxonomy currently permits individual species to have several members. Consequently, confusion emerges among those who are not aware of the difference between taxa and viruses, and because certain well-known viruses cannot be located in ICTV publications or be linked to their species. In addition, the number of duplicate names and abbreviations has increased dramatically in the literature. To solve this conundrum, the ICTV could mandate listing all viruses of established species and all reported unclassified viruses in forthcoming online ICTV Reports and create a searchable webpage using this information. The International Union of Microbiology Societies could also consider changing the mandate of the ICTV to include the nomenclature of all viruses in addition to taxon considerations. With such a mandate expansion, official virus names and virus name abbreviations could be catalogued and virus nomenclature could be standardized. As a result, the ICTV would become an even more useful resource for all stakeholders in virology.

Activating Mutations in Pik3ca Contribute to Anal Carcinogenesis in the Presence or Absence of HPV-16 Oncogenes.

PURPOSE: Over 95% of human anal cancers are etiologically associated with high-risk HPVs, with HPV type 16 (HPV16) the genotype most commonly found. Activating mutations in the catalytic subunit of Phosphatidylinositol (3,4,5)-trisphosphate kinase (PI3K), encoded by the Pik3ca gene, are detected in approximately 20% of human anal cancers.Experimental Design: We asked if common activating mutations in Pik3ca contribute to anal carcinogenesis using an established mouse model for anal carcinogenesis in which mice are topically treated with the chemical carcinogen 7,12-Dimethylbenz(a)anthracene (DMBA). Mice expressing in their anal epithelium one of two activating mutations in Pik3ca genes, Pik3caH1047R or Pik3caE545K , were monitored for anal carcinogenesis in the presence or absence of transgenes expressing the HPV16 E6 and E7 oncogenes. RESULTS: Both mutant forms of Pik3ca increased susceptibility to anal carcinogenesis in the absence of HPV16 oncogenes, and cooperated with HPV16 oncogenes to induce the highest level and earliest onset of anal cancers. The combination of HPV16 oncogenes and Pik3ca mutations led to anal cancers even in the absence of treatment with DMBA. We further observed that the investigational mTOR1/2 dual inhibitor, TAK-228, significantly reduced the size of anal cancer-derived tumor spheroids in vitro and reduced the growth rates of anal cancer-derived tumor grafts in vivo. CONCLUSIONS: These data demonstrate that activating mutations in Pik3ca drive anal carcinogenesis together with HPV16 oncogenes, and that the PI3K/mTOR pathway is a relevant target for therapeutic intervention.

MTORC1/2 Inhibition as a Therapeutic Strategy for PIK3CA Mutant Cancers.

PIK3CA mutations are common in clinical molecular profiling, yet an effective means to target these cancers has yet to be developed. MTORC1 inhibitors are often used off-label for patients with PIK3CA mutant cancers with only limited data to support this approach. Here we describe a cohort of patients treated with cancers possessing mutations activating the PI3K signaling cascade with minimal benefit to treatment with the MTORC1 inhibitor everolimus. Previously, we demonstrated that dual PI3K/mTOR inhibition could decrease proliferation, induce differentiation, and result in a treatment response in APC and PIK3CA mutant colorectal cancer. However, reactivation of AKT was identified, indicating that the majority of the benefit may be secondary to MTORC1/2 inhibition. TAK-228, an MTORC1/2 inhibitor, was compared with dual PI3K/mTOR inhibition using BEZ235 in murine colorectal cancer spheroids. A reduction in spheroid size was observed with TAK-228 and BEZ235 (-13% and -14%, respectively) compared with an increase of >200% in control (P < 0.001). These spheroids were resistant to MTORC1 inhibition. In transgenic mice possessing Pik3ca and Apc mutations, BEZ235 and TAK-228 resulted in a median reduction in colon tumor size of 19% and 20%, respectively, with control tumors having a median increase of 18% (P = 0.02 and 0.004, respectively). This response correlated with a decrease in the phosphorylation of 4EBP1 and RPS6. MTORC1/2 inhibition is sufficient to overcome resistance to everolimus and induce a treatment response in PIK3CA mutant colorectal cancers and deserves investigation in clinical trials and in future combination regimens.

Avian Vaccination: Current Options and Strategies.

Researchers have generated an array of potential avian antiviral vaccines. However, vaccine and viral complexity, small profit margins, the cost of development and manufacturing, and the small population at risk relegate most avian vaccine use to commercial species. Some vaccines designed for use in nonavian species are used to prevent or ameliorate disease in exotic and companion birds. This article highlights newly developed vaccines that may be used in exotic and pet birds. Information pertinent to vaccine choice and strategy is provided, including disease lethality, species affected, and previous knowledge regarding vaccine safety and efficacy. Other avian species of concern are also included.

Studies on immunity and immunopathogenesis of parrot bornaviral disease in cockatiels.

We have demonstrated that vaccination of cockatiels (Nymphicus hollandicus) with killed parrot bornavirus (PaBV) plus recombinant PaBV-4 nucleoprotein (N) in alum was protective against disease in birds challenged with a virulent bornavirus isolate (PaBV-2). Unvaccinated birds, as well as birds vaccinated after challenge, developed gross and histologic lesions typical of proventricular dilatation disease (PDD). There was no evidence that vaccination either before or after challenge made the infection more severe. Birds vaccinated prior to challenge largely remained free of disease, despite the persistence of the virus in many organs. Similar results were obtained when recombinant N, in alum, was used for vaccination. In some rodent models, Borna disease is immune mediated thus we did an additional study whereby cyclosporine A was administered to unvaccinated birds starting 1day prior to challenge. This treatment also conferred complete protection from disease, but not infection.

Versican-Derived Matrikines Regulate Batf3-Dendritic Cell Differentiation and Promote T Cell Infiltration in Colorectal Cancer.

Colorectal cancer originates within immunologically complex microenvironments. To date, the benefits of immunotherapy have been modest, except in neoantigen-laden mismatch repair-deficient tumors. Approaches to enhance tumor-infiltrating lymphocytes in the tumor bed may substantially augment clinical immunotherapy responses. In this article, we report that proteolysis of the tolerogenic matrix proteoglycan versican (VCAN) strongly correlated with CD8+ T cell infiltration in colorectal cancer, regardless of mismatch repair status. Tumors displaying active VCAN proteolysis and low total VCAN were associated with robust (10-fold) CD8+ T cell infiltration. Tumor-intrinsic WNT pathway activation was associated with CD8+ T cell exclusion and VCAN accumulation. In addition to regulating VCAN levels at the tumor site, VCAN proteolysis results in the generation of bioactive fragments with novel functions (VCAN-derived matrikines). Versikine, a VCAN-derived matrikine, enhanced the generation of CD103+CD11chiMHCIIhi conventional dendritic cells (cDCs) from Flt3L-mobilized primary bone marrow-derived progenitors, suggesting that VCAN proteolysis may promote differentiation of tumor-seeding DC precursors toward IRF8- and BATF3-expressing cDCs. Intratumoral BATF3-dependent DCs are critical determinants for T cell antitumor immunity, effector T cell trafficking to the tumor site, and response to immunotherapies. Our findings provide a rationale for testing VCAN proteolysis as a predictive and/or prognostic immune biomarker and VCAN-derived matrikines as novel immunotherapy agents.

Aquatic Bird Bornavirus-Associated Disease in Free-Living Canada Geese ( Branta canadensis ) in the Northeastern USA.

During the winter of 2013-14, 22 Canada geese ( Branta canadensis ) were admitted to the Wildlife Clinic at the Cummings School of Veterinary Medicine at Tufts University with nonspecific neurologic abnormalities and emaciation. Five of these geese, along with three geese that were submitted dead, were evaluated via histopathology, immunohistochemistry, and reverse transcription PCR (RT-PCR) for bornaviruses. Histopathologically, six of the eight birds had lymphoplasmacytic encephalitis. One bird, which also had encephalitis, had a dilated esophagus. Lead poisoning, West Nile virus, avian influenza, and avian paramyxovirus infection were excluded from the diagnosis. Brain tissue from all eight geese was positive for bornaviral N-antigen on immunohistochemistry. Frozen brain tissue from five birds was available for bornavirus RT-PCR. Three of the five birds were positive for the bornavirus M gene. Formalin-fixed paraffin-embedded brain tissue was evaluated on the remaining three geese via RT-PCR, with one of these geese testing positive. A bornavirus was subsequently cultured in duck embryo fibroblasts from the brain of one Canada Goose. This virus genome was sequenced, and the virus was identified as aquatic bird bornavirus 1. We were unable to identify any unusual features of this genome that would account for its apparent pathogenicity, given that subclinical infection with bornavirus in waterfowl is common in North America.

Taxonomy of the order Mononegavirales: update 2017.

In 2017, the order Mononegavirales was expanded by the inclusion of a total of 69 novel species. Five new rhabdovirus genera and one new nyamivirus genus were established to harbor 41 of these species, whereas the remaining new species were assigned to already established genera. Furthermore, non-Latinized binomial species names replaced all paramyxovirus and pneumovirus species names, thereby accomplishing application of binomial species names throughout the entire order. This article presents the updated taxonomy of the order Mononegavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV).

Dual PI3K/mTOR Inhibition in Colorectal Cancers with APC and PIK3CA Mutations.

Therapeutic targeting of the PI3K pathway is an active area of research in multiple cancer types, including breast and endometrial cancers. This pathway is commonly altered in cancer and plays an integral role in numerous vital cellular functions. Mutations in the PIK3CA gene, resulting in a constitutively active form of PI3K, often occur in colorectal cancer, though the population of patients who would benefit from targeting this pathway has yet to be identified. In human colorectal cancers, PIK3CA mutations most commonly occur concomitantly with loss of adenomatous polyposis coli (APC). Here, treatment strategies are investigated that target the PI3K pathway in colon cancers with mutations in APC and PIK3CA Colorectal cancer spheroids with Apc and Pik3ca mutations were generated and characterized confirming that these cultures represent the tumors from which they were derived. Pan and alpha isomer-specific PI3K inhibitors did not induce a significant treatment response, whereas the dual PI3K/mTOR inhibitors BEZ235 and LY3023414 induced a dramatic treatment response through decreased cellular proliferation and increased differentiation. The significant treatment responses were confirmed in mice with Apc and Pik3ca-mutant colon cancers as measured using endoscopy with a reduction in median lumen occlusion of 53% with BEZ235 and a 24% reduction with LY3023414 compared with an increase of 53% in controls (P < 0.001 and P = 0.03, respectively). This response was also confirmed with 18F-FDG microPET/CT imaging.Implications: Spheroid models and transgenic mice suggest that dual PI3K/mTOR inhibition is a potential treatment strategy for APC and PIK3CA-mutant colorectal cancers. Thus, further clinical studies of dual PI3K/mTOR inhibitors are warranted in colorectal cancers with these mutations. Mol Cancer Res; 15(3); 1-11. ©2016 AACR.

Possibility and Challenges of Conversion of Current Virus Species Names to Linnaean Binomials.

Botanical, mycological, zoological, and prokaryotic species names follow the Linnaean format, consisting of an italicized Latinized binomen with a capitalized genus name and a lower case species epithet (e.g., Homo sapiens). Virus species names, however, do not follow a uniform format, and, even when binomial, are not Linnaean in style. In this thought exercise, we attempted to convert all currently official names of species included in the virus family Arenaviridae and the virus order Mononegavirales to Linnaean binomials, and to identify and address associated challenges and concerns. Surprisingly, this endeavor was not as complicated or time-consuming as even the authors of this article expected when conceiving the experiment. [Arenaviridae; binomials; ICTV; International Committee on Taxonomy of Viruses; Mononegavirales; virus nomenclature; virus taxonomy.].

Apparent resolution of parrot bornavirus infection in cockatiels (Nymphicus hollandicus).

Parrot bornavirus (PaBV), the etiologic agent of proventricular dilatation disease (PDD), is a major cause of concern in the avian health community. Within an infected flock, some birds will develop PDD and succumb to disease, while others remain healthy. Until now, there has been no study describing the results of long-term infection in apparently healthy carriers. For the last 5 years, the Schubot Exotic Bird Health Center at Texas A&M University has monitored individual PaBV shedding data in a flock of 66 naturally infected cockatiels. Of these birds, 53 were detected shedding PaBV4 in their droppings by reverse transcriptase polymerase chain reaction on at least one occasion. However, the prevalence of shedding declined over time, with the last positive cloacal swab being in October 2013. To determine whether the decline and eventual lack of shedding was an indication of virus elimination, seven previously shedding birds were euthanized and necropsied in 2016. Neither any gross lesion of PDD was observed nor was there any evidence of PDD or bornaviral encephalitis detected by histopathology. All tissues tested were negative for the presence of PaBV by reverse transcriptase polymerase chain reaction and immunohistochemistry. Thus, there was no evidence of an ongoing, productive infection in these birds. There are two possible explanations for these results. One possibility is that the birds were previously infected and have subsequently eliminated the virus. Alternatively, there may have been as few as three truly infected birds in the flock and the transient detection of PaBV in the droppings of other birds may simply be a "pass-through" phenomenon.

The pathogenesis of bornaviral diseases in mammals.

Natural bornavirus infections and their resulting diseases are largely restricted to horses and sheep in Central Europe. The disease also occurs naturally in cats, and can be induced experimentally in laboratory rodents and numerous other mammals. Borna disease virus-1 (BoDV-1), the cause of most cases of mammalian Borna disease, is a negative-stranded RNA virus that replicates within the nucleus of target cells. It causes severe, often lethal, encephalitis in susceptible species. Recent events, especially the discovery of numerous new species of bornaviruses in birds and a report of an acute, lethal bornaviral encephalitis in humans, apparently acquired from squirrels, have revived interest in this remarkable family of viruses. The clinical manifestations of the bornaviral diseases are highly variable. Thus, in addition to acute lethal encephalitis, they can cause persistent neurologic disease associated with diverse behavioral changes. They also cause a severe retinitis resulting in blindness. In this review, we discuss both the pathological lesions observed in mammalian bornaviral disease and the complex pathogenesis of the neurologic disease. Thus infected neurons may be destroyed by T-cell-mediated cytotoxicity. They may die as a result of excessive inflammatory cytokine release from microglia. They may also die as a result of a 'glutaminergic storm' due to a failure of infected astrocytes to regulate brain glutamate levels.

Colon Cancer Tumorigenesis Initiated by the H1047R Mutant PI3K.

The phosphoinositide 3-kinase (PI3K) signaling pathway is critical for multiple important cellular functions, and is one of the most commonly altered pathways in human cancers. We previously developed a mouse model in which colon cancers were initiated by a dominant active PI3K p110-p85 fusion protein. In that model, well-differentiated mucinous adenocarcinomas developed within the colon and initiated through a non-canonical mechanism that is not dependent on WNT signaling. To assess the potential relevance of PI3K mutations in human cancers, we sought to determine if one of the common mutations in the human disease could also initiate similar colon cancers. Mice were generated expressing the Pik3caH1047R mutation, the analog of one of three human hotspot mutations in this gene. Mice expressing a constitutively active PI3K, as a result of this mutation, develop invasive adenocarcinomas strikingly similar to invasive adenocarcinomas found in human colon cancers. These tumors form without a polypoid intermediary and also lack nuclear CTNNB1 (ß-catenin), indicating a non-canonical mechanism of tumor initiation mediated by the PI3K pathway. These cancers are sensitive to dual PI3K/mTOR inhibition indicating dependence on the PI3K pathway. The tumor tissue remaining after treatment demonstrated reduction in cellular proliferation and inhibition of PI3K signaling.

Horses naturally infected with EIAV harbor 2 distinct SU populations but are monophyletic with respect to IN.

Equine infectious anemia virus (EIAV) causes lifelong infections ranging from acutely fatal, to chronic, to asymptomatic. Within infected animals, EIAV is found as a quasispecies. Many experimental studies on EIAV, carried out in the U.S. over the past 70 years, have used either the highly virulent Wyoming (EIAVWYO) field strain or various derivatives of that strain. These infections have provided insights into the variety of genetic changes that accumulate in the env gene and LTR in experimentally infected horses. In the current study, we obtained EIAV sequences from blood samples collected from naturally infected Texas horses between 2000 and 2002. We found surface (SU) and long terminal repeat (LTR) sequences clearly related to EIAVWYO and its cell culture-adapted derivatives. Some blood samples yielded SU or LTR sequences belonging to 2 discrete clusters. In these cases, SU and LTR variation between animals was no greater than sequence variation within animals. In contrast, a portion of integrase (IN) was more homogeneous within animals than between animals. These results suggest that specific selective pressures are applied to SU and LTR sequences, potentially driving generation of two distinct sequence clusters within a horse. We speculate that viruses in one cluster may be more highly expressed and easily transmitted while those in the second cluster support long-term inapparent infection. The presence of homogeneous IN sequences within a horse supports the hypothesis that SU and LTR sequences diverged after the initial infection.

The pathogenesis of proventricular dilatation disease.

Bornaviruses cause neurologic diseases in several species of birds, especially parrots, waterfowl and finches. The characteristic lesions observed in these birds include encephalitis and gross dilatation of the anterior stomach - the proventriculus. The disease is thus known as proventricular dilatation disease (PDD). PDD is characterized by extreme proventricular dilatation, blockage of the passage of digesta and consequent death by starvation. There are few clinical resemblances between this and the bornaviral encephalitides observed in mammals. Nevertheless, there are common virus-induced pathogenic pathways shared across this disease spectrum that are explored in this review. Additionally, a review of the literature relating to gastroparesis in humans and the control of gastric mobility in mammals and birds points to several plausible mechanisms by which bornaviral infection may result in extreme proventricular dilatation.

Colon Tumors with the Simultaneous Induction of Driver Mutations in APC, KRAS, and PIK3CA Still Progress through the Adenoma-to-carcinoma Sequence.

Human colorectal cancers often possess multiple mutations, including three to six driver mutations per tumor. The timing of when these mutations occur during tumor development and progression continues to be debated. More advanced lesions carry a greater number of driver mutations, indicating that colon tumors might progress from adenomas to carcinomas through the stepwise accumulation of mutations following tumor initiation. However, mutations that have been implicated in tumor progression have been identified in normal-appearing epithelial cells of the colon, leaving the possibility that these mutations might be present before the initiation of tumorigenesis. We utilized mouse models of colon cancer to investigate whether tumorigenesis still occurs through the adenoma-to-carcinoma sequence when multiple mutations are present at the time of tumor initiation. To create a model in which tumors could concomitantly possess mutations in Apc, Kras, and Pik3ca, we developed a novel minimally invasive technique to administer an adenovirus expressing Cre recombinase to a focal region of the colon. Here, we demonstrate that the presence of these additional driver mutations at the time of tumor initiation results in increased tumor multiplicity and an increased rate of progression to invasive adenocarcinomas. These cancers can even metastasize to retroperitoneal lymph nodes or the liver. However, despite having as many as three concomitant driver mutations at the time of initiation, these tumors still proceed through the adenoma-to-carcinoma sequence.