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Background Although gut fungi have been implicated in the immunopathogenesis of inflammatory bowel disease, the fungal microbiome has not been deeply explored across endohistologic activity and treatment exposure in ulcerative colitis. Methods We analyzed data from the SPARC IBD (Study of a Prospective Adult Research Cohort with Inflammatory Bowel Disease) registry. We evaluated the fungal composition of fecal samples from 98 patients with ulcerative colitis across endoscopic activity (n = 43), endohistologic activity (n = 41), and biologic exposure (n = 82). Across all subgroups, we assessed fungal diversity and differential abundance of taxonomic groups. Results We identified 500 unique fungal amplicon sequence variants across the cohort of 82 patients, dominated by phylum Ascomycota. Compared with endoscopic remission, patients with endoscopic activity had increased Saccharomyces (log2 fold change = 4.54; adjusted P Conclusions Endoscopic inflammation in ulcerative colitis is associated with an expansion of Saccharomyces and Candida compared with remission. The role of these fungal taxa as potential biomarkers and targets for personalized approaches to therapeutics in ulcerative colitis should be evaluated.

Supplementary Video 1 MPG file - 708K, 3-dimensional tumor model of 10 cm HCC tumor depicted in Fig. 4a (baseline; prior to JX-594 treatment). Beige = perfused; green = hypoperfused; red = necrotic

Supplementary Video 2 MPG file - 1158K, 3-dimensional tumor model of 10 cm HCC tumor depicted in Fig. 4b (baseline; 5 days post JX-594 treatment). Beige = perfused; green = hypoperfused; red = necrotic

Supplementary Figure 4 PDF file - 69K, Day 5 enhancement waterfall plot

Supplementary Figure 3 PDF file - 83K, Normal fibroblasts (GM38 cells) do not express VEGFR2 and are not sensitized to JX-594 infection upon incubation with VEGF

Supplementary Figure 1 PDF file - 74K, VEGF- and FGF-2 stimulate vaccinia replication

Supplementary Methods PDF file - 44K, GLP toxicology study, In vivo imaging and mouse immunohistochemical analyses

Supplementary Table 1 PDF file - 30K, Rabbit GLP toxicology study of intravenous JX-594

Purpose To review safety and efficacy of combined plasma rich in growth factors (PRGF) eye drops and scleral contact lens (SCL) therapy in patients with ocular surface disease. Methods Patients with ocular surface disease of various etiologies were screened for at least 3 months of concurrent treatment with PRGF and SCL. Retrospective pre- and post-treatment measurements were collected, including patient satisfaction, severity and frequency of dry eye symptoms measured by a modified Symptom Assessment in Dry Eye (SANDE) questionnaire, visual acuity, and number of concurrent treatments. Results 26 patients with ocular surface disease were included in the study with 20 patients answering the questionnaire (77% response rate). There were no adverse events reported. Most patients thought the combined therapy was better than previous treatments and would recommend to others (80%, 90% respectively). SANDE scores significantly decreased after use of concurrent therapy. There was a small but significant decrease in the number of other concurrent treatments. Visual acuity was unchanged. Conclusions This retrospective cohort study found PRGF used in combination with SCL is safe and significantly decreases symptoms in patients with recalcitrant ocular surface disease.

BACKGROUND The fungal mycobiome has been increasingly implicated in the pathogenesis of inflammatory bowel disease (IBD). Circulating antibodies to Saccharomyces antibodies are detected in 60-70% of Crohn’s disease and in 10-15% of patients with ulcerative colitis (UC). Specific fungal species, including Candida albicans, are increased in active IBD compared to healthy controls. Recently, UC patients with high fecal Candida prior to fecal microbial transplantation (FMT) were more likely to demonstrate robust treatment responses to transplant, with low Candida levels post-FMT predicting ameliorated inflammation in the recipient. Despite the accumulation of findings supporting the role of fungi in IBD, the mycobiome remains poorly characterized in UC. Few studies have examined changes in the UC mycobiome with respect to validated endoscopic indices or histologic parameters; furthermore, the effects of treatment exposure, including biologic medications, on fungal communities in UC are unknown. AIMS In this study, we aim to characterize the fungal mycobiome in UC patients with varying levels of endoscopic activity, endo-histologic activity, and treatment exposure. METHODS We performed a secondary analysis using the data extracted from the Crohn’s and Colitis Foundation’s Study of a Prospective Adult Research Cohort with IBD, which contains clinical, endoscopic, histologic, and metagenomic data. Using Internal Transcribed Spacer based deep sequencing of fungal rDNA from fecal samples, we classified sequences utilizing the UNITE fungal database and a fitted classifier. The R package DESeq2 was used to calculate the log-2 fold change of differential abundance. RESULTS We identified 504 unique fungal amplicon sequence variants across the cohort of 98 patients, belonging to phylum Ascomycota (71.5%), Basidiomycota (11%), Mucoromycota (0.16%), or unidentified (17.2%). Compared to endoscopic remission, patients with endoscopic activity (n=25) were enriched with Saccharomyces (4.31, p< 5 x 10-5) and Candida (2.53, p< 1 x 10-3). Similarly, compared to endo-histologic remission, patients with endo-histologic activity (n=19) were also enriched for Saccharomyces (2.71, p CONCLUSION These data demonstrate that endoscopic and histologic inflammation in UC is associated with expansion of Saccharomyces and Candida compared to remission, even after controlling for exposure to biologic exposure. These findings are recapitulated during endo-histologic activity. The role of these fungal taxa as potential biomarkers and targets for personalized approaches to therapeutics in UC should be evaluated.

Background Contemporary outcomes of open repair of thoracoabdominal aortic aneurysms (TAAAs) and descending thoracic aortic aneurysms (DTAs) have not been analyzed in an inclusive meta-analysis. Methods After a systematic literature search, studies from 2008 to 2018 reporting outcomes of open repair of DTAs or TAAAs were pooled in a single-arm meta-analysis performed using the generic inverse variance method. Primary outcome was operative mortality. Secondary outcomes were late mortality, postoperative stroke, permanent and temporary spinal cord injury, renal failure, respiratory failure, and myocardial infarction. Results Fifty-four studies with 12,245 patients were included. Pooled operative mortality for open repair was 10.4% (95% confidence interval [CI], 8.3-12.8): 6.6% (95% CI, 3.7-11.6) for DTA and 10.5% (95% CI, 7.5-14.5) for TAAA. Pooled incidence rate of late mortality was 0.6% (95% CI, 0.5-0.8) per person-year. Pooled rates for postoperative outcomes were 4.9% (95% CI, 3.9-6.1) for stroke; 5.7% (95% CI, 4.3-7.5) and 3.0% (95% CI, 2.1-4.2) for permanent and temporary spinal cord injury, respectively; 13.2% (95% CI, 9.9-17.3) for renal failure; 23.3% (95% CI, 17.5-30.4) for respiratory failure; and 2.7% (95% CI, 1.8-4.1) for myocardial infarction. At metaregression, year of publication, use of the clamp-and-sew technique, and use of the cerebrospinal fluid drain were associated with lower operative mortality. Ruptured aneurysms were associated with higher operative mortality. Conclusions Despite improvement, open repair of DTAs and TAAAs continues to be associated with a considerable risk for operative death and perioperative complications. Use of the cerebrospinal fluid drain is associated with better outcomes.

OTTO is an open-source automated liquid handler that can be fabricated at a cost of $1,500 using off-the-shelf and 3D-printable parts as an alternative to commercial devices. Open-source approaches have been applied to build syringe pumps, centrifuges, and other laboratory equipment. These devices are affordable but generally rely on a single motor to perform simple operations and thus do not fully utilize the potential of the Maker Movement. Open-source linear actuators and microcontrollers enable the fabrication of more complex laboratory instruments that rely on 3D positioning and accurate dispensing of fluids, such as automated liquid handlers. These instruments can be built rapidly and affordably, thereby providing access to highly reproducible sample preparation for common biological assays such as qPCR. We applied the design principles of speed and accuracy, unattended automation, and open-source components to build an automated liquid handler that controls micropipetting of liquids in 3D space at speeds and positional resolutions required for qPCR. In benchmarking studies, OTTO showed accuracy and sample preparation times comparable to manual qPCR. The ability to control linear motion and liquid dispensing using affordable off-the-shelf and 3D-printable parts can facilitate the adoption of open-source automated liquid handlers for qPCR, bioplotting, and other bioinstrumentation applications.

Aims Immune checkpoint blockade (ICB) is the mainstay of treatment for metastatic melanoma and lung adenocarcinoma, and their use is growing in cancer. Immune checkpoint blockade induced colitis (ICB colitis) presents a management challenge and its mechanisms remain poorly elucidated. Methods We performed next generation single-cell RNA sequencing of immune cells from patients given ICB. We validated findings using confocal microscopy, drawing comparisons bioinformatically with ulcerative colitis. In parallel, we conducted a review of 1,074 patients given checkpoint inhibitors across two tertiary centres between 2011–2018 to discover patterns in incidence and predictors of clinical outcome. Results Using single-cell RNA sequencing, we discovered excessive local CD8 T cell proliferation was a key feature of ICB colitis, and we were able to visualise higher numbers of replicating CD8 T cells in gut tissue sections of patients with colitis than those without ICB colitis. The degree of replication was greater than seen in ulcerative colitis by bioinformatic analysis. From our clinical review, age, gender and smoking status did not alter the risk of developing colitis, whereas type of immunotherapy did (incidence 9% in PD-1 Monotherapy vs 32% Combination Therapy). Having prior IBD did not guarantee the development of ICB Colitis. Systemic markers of inflammation (C-Reactive Protein, Albumin) did not predict outcome, whereas local markers of inflammation (endoscopic UCEIS scoring, histological Nancy Index) did. Conclusions We putatively link novel insights from bench science to clinical trends. ICB colitis may be driven more by a gut localised inflammation response in comparison to ulcerative colitis. As we also demonstrate, this may explain why endoscopic and histological scoring may have better prognostic value than systemic measures of inflammation.

Most NLP datasets are manually labeled, so suffer from inconsistent labeling or limited size. We propose methods for automatically improving datasets by viewing them as graphs with expected semantic properties. We construct a paraphrase graph from the provided sentence pair labels, and create an augmented dataset by directly inferring labels from the original sentence pairs using a transitivity property. We use structural balance theory to identify likely mislabelings in the graph, and flip their labels. We evaluate our methods on paraphrase models trained using these datasets starting from a pretrained BERT model, and find that the automatically-enhanced training sets result in more accurate models., Comment: EMNLP 2020 (Findings)

The prevailing approach for training and evaluating paraphrase identification models is constructed as a binary classification problem: the model is given a pair of sentences, and is judged by how accurately it classifies pairs as either paraphrases or non-paraphrases. This pointwise-based evaluation method does not match well the objective of most real world applications, so the goal of our work is to understand how models which perform well under pointwise evaluation may fail in practice and find better methods for evaluating paraphrase identification models. As a first step towards that goal, we show that although the standard way of fine-tuning BERT for paraphrase identification by pairing two sentences as one sequence results in a model with state-of-the-art performance, that model may perform poorly on simple tasks like identifying pairs with two identical sentences. Moreover, we show that these models may even predict a pair of randomly-selected sentences with higher paraphrase score than a pair of identical ones., Comment: ACL 2020 Student Research Workshop

Inflammatory bowel disease is a chronic, relapsing condition with two subtypes, Crohn’s disease (CD) and ulcerative colitis (UC). Genome-wide association studies (GWASs) in UC implicate a FCGR2A variant that alters the binding affinity of the antibody receptor it encodes, FcγRIIA, for immunoglobulin G (IgG). Here, we aimed to understand the mechanisms whereby changes in FcγRIIA affinity would affect inflammation in an IgA-dominated organ. We found a profound induction of anti-commensal IgG and a concomitant increase in activating FcγR signaling in the colonic mucosa of UC patients. Commensal-IgG immune complexes engaged gut-resident FcγR-expressing macrophages, inducing NLRP3- and reactive-oxygen-species-dependent production of interleukin-1β (IL-1β) and neutrophil-recruiting chemokines. These responses were modulated by the FCGR2A genotype. In vivo manipulation of macrophage FcγR signal strength in a mouse model of UC determined the magnitude of intestinal inflammation and IL-1β-dependent type 17 immunity. The identification of an important contribution of IgG-FcγR-dependent inflammation to UC has therapeutic implications.

With the rise of technology, modern healthcare and biomedical sciences have largely shifted their content to cyberspace. This raises the challenges of accessibility and security of such data, and the solution may lie in a recent technology called blockchain. Blockchain is a public transaction ledger that offers two unique capabilities, immutability and decentralization of data, that make it stand out as an unparalleled technology in the spheres of healthcare and biomedicine.

Background IL10 receptor (IL10R) deficiency causes severe infantile-onset IBD. Intact IL10R-dependent signals have been shown to be important for innate and adaptive immune cell function in mice. We have previously reported a key role of IL10 is the generation and function of human anti-inflammatory macrophages. Independent of innate immune cell defects, the aim of the current study was to determine the role of IL10R signaling in regulating human CD4+ T cell function. Methods Peripheral blood mononuclear cells and intestinal biopsies cells were collected from IL10/IL10R-deficient patients and controls. Frequencies of CD4+ T cell subsets, naïve T cell proliferation, regulatory T cell (Treg)-mediated suppression and Treg and TH17 generation were determined by flow cytometry. Transcriptional profiling was performed by NanoString and quantitative real-time PCR. RNA in situ hybridization was used to determine the quantities of various transcripts in intestinal mucosa. Results Analysis of 16 IL10- and IL10R-deficient patients demonstrated similar frequencies of peripheral blood and intestinal Tregs, compared to control subjects. In addition, in vitro Treg suppression of CD4+ T cell proliferation and generation of Treg were not dependent on IL10R signaling. However, IL10R-deficient T naïve cells exhibited significantly higher proliferative capacity, a strong TH17 signature and an increase in polarization towards TH17 cells, compared to controls. Moreover, the frequency of TH17 cells was increased in the colon and ileum of IL10R-deficient patients. Finally, we show that stimulation of IL10R-deficient Tregs in the presence of IL1 leads to enhanced production of IL17A. Conclusions IL10R signaling regulates TH17 polarization and T cell proliferation in humans, but is not required for the generation and in vitro suppression of Tregs. Therapies targeting the TH17 axis might be beneficial for IL10- and IL10R-deficient patients as a bridge to allogeneic hematopoietic stem cell transplantation.

Oncolytic vaccinia virus has been shown to induce a profound, rapid and tumor-specific vascular collapse in both preclinical models and clinical studies; however, a complete examination of the kinetics and levels of collapse and revascularization has not been described previously. Contrast-enhanced ultrasound was used to follow tumor perfusion levels in mouse tumor models at times after vaccinia therapy. It was observed that revascularization after viral therapy was dramatically delayed and did not occur until after viral clearance. This indicated that oncolytic vaccinia may possess a previously undescribed antiangiogenic potential that might synergize with the reported anti-vascular effects. Despite a rapid loss of perfusion and widespread hypoxia within the tumor, it was observed that VEGF levels in the tumor were suppressed throughout the period of active viral infection. Although tumor vasculature could eventually reform after the viral therapy was cleared in mouse models, anti-tumor effects could be significantly enhanced through additional combination with anti-VEGF therapies. This was initially examined using a gene therapy approach (Ad-Flk1-Fc) to target VEGF directly, demonstrating that the timing of application of the antiangiogenic therapy was critical. However, it is also known that oncolytic vaccinia sensitizes tumors to tyrosine kinase inhibitors (TKI) in the clinic through an unknown mechanism. It is possible this phenomenon may be mediated through the antiangiogenic effects of the TKIs. This was modeled in mouse tumors using sunitinib in combination with oncolytic vaccinia. It was observed that prevention of angiogenesis mediated by oncolytic vaccinia can be utilized to enhance the TKI therapy.

Pollution of various environmental matrices by antibiotic resistance genes (ARGs) has become a growing threat to human health. For the quantitative analysis of the presence of ARGs, there is a need for sensitive and robust qPCR assays which can detect various genes from different types of DNA extracts. Fourteen ARGs were selected as target genes in this study including: blaTEM, blaOXA-1 and blaCTX-M coded for resistance to β-lactams; ermB for macrolides; tetA, tetG, tetM, tetQ, tetW and tetX for tetracyclines; sul I and sul II for sulfonamides; drfA1 and drfA12 d for trimethoprim; and integron gene intI 1 and intI 2. Chemically synthesized double-stranded gene fragments were modified using molecular biology methods and used as real-time PCR standards as well as to establish in-house qPCR assays. The ermB gene from a naturally occurring plasmid was used to compare the performance of qPCR assay with the chemically synthesized ermB. Additionally, environmental water, soil and faeces samples were used to validate the established qPCR assays. Importantly, the study proves the usefulness of rapidly synthesized oligonucleotides serving as qPCR standards for ARG analysis and provides comparable sensitivity and reliability to a traditional amplicon standard.

The combination of an oncolytic virus, that directly destroys tumor cells and mediates an acute immune response, with an immune cell therapy, capable of further enlisting and enhancing the host immune response, has the potential to create a potent therapeutic effect. We have previously developed several strategies for optimizing the delivery of oncolytic vaccinia virus vectors to their tumor targets, including the use of immune cell-based carrier vehicles and the incorporation of mutations that increase production of the enveloped form of vaccinia (extracellular enveloped viral (EEV)) that is better adapted to spread within a host. Here, we initially combine these approaches to create a novel therapeutic, consisting of an immune cell (cytokine-induced killer, CIK) preloaded with an oncolytic virus that is EEV enhanced. This resulted in direct interaction between the viral and immune cell components with each assisting the other in directing the therapy to the tumor and so enhancing the antitumor effects. This effect could be further improved through CCL5 expression from the virus. The resulting multicomponent therapy displays the ability for synergistic crosstalk between components, so significantly enhancing tumor trafficking and antitumor effects.

This study explored how couples living with advanced multiple sclerosis approach planning for health changes together. A qualitative descriptive approach was used, with multiple in-depth interviews. Ten couples were interviewed, consisting of 10 participants with advanced multiple sclerosis and their 10 respective spouses. Interviews were transcribed verbatim and analyzed for themes related to dyad decision making. Particular focus was paid to how or if couples planned for worsening health, including advance care planning. Characteristics of couples who had made plans for worsening health included a desire to make decisions ahead of time, work in a healthcare field, and a life-threatening health event for the care-giving partner. Characteristics of couples who had not discussed plans for worsening health included diminished quality of communication between partners and hoping for a cure. Findings suggest that providers may need to assess quality of communication between partners, because strained communication may negatively affect the process of informed healthcare decision making.

People not only dehumanize others, they also dehumanize the self in response to their own harmful behavior. We examine this self-dehumanization effect across four studies. Studies 1 and 2 show that when participants are perpetrators of social ostracism, they view themselves as less human compared with when they engage in nonaversive interpersonal interactions. Perceived immorality of their behavior mediated this effect. Studies 3 and 4 highlight the behavioral consequences of self-dehumanization. The extent to which participants saw themselves as less human after perpetrating social ostracism predicted subsequent prosocial behavior. Studies 2 to 4 also demonstrate that consequences of self-dehumanization occur independently of any effects of self-esteem or mood. The findings are discussed in relation to previous work on dehumanization and self-perception. We conclude that in the context of immoral actions (self) dehumanization may be functional.

Genetic therapies, including transfected immune cells and viral vectors, continue to show clinical responses as systemically deliverable and targeted therapeutics, with the first such approaches having been approved for cancer treatment. The majority of these employ cytokine transgenes. However, expression of cytokines early after systemic delivery can result in increased toxicity and nonspecific induction of the immune response. In addition, premature immune-mediated clearance of the therapy may result, especially for viral-based approaches. Here, it was initially verified that cytokine (interleukin (IL)2) or chemokine (CCL5) expression from a systemically delivered oncolytic virus resulted in reduced oncolytic activity and suboptimal immune activation, while IL2 also resulted in increased toxicity. However, all these limitations could be overcome through incorporation of exogenous regulation of cytokine or chemokine transgene function through fusion of a small and externally controllable destabilizing domain to the protein of interest. Regulation allowed an initial phase without cytokine function, permitting enhanced delivery and oncolytic activity before activation of cytokine function and a subsequent phase of enhanced and tumor-targeted immunotherapeutic activity. As a result of this exogenous regulation of cytokine function, both oncolytic and immune-mediated mechanisms of action were optimized, greatly enhancing therapeutic activity, while toxicity was significantly reduced.

The entire collection of genetic information resides within the chromosomes, which themselves reside within almost every cell nucleus of eukaryotic organisms. Each individual chromosome is found to have its own preferred three-dimensional (3D) structure independent of the other chromosomes. The structure of each chromosome plays vital roles in controlling certain genome operations, including gene interaction and gene regulation. As a result, knowing the structure of chromosomes assists in the understanding of how the genome functions. Fortunately, the 3D structure of chromosomes proves possible to construct through computational methods via contact data recorded from the chromosome. We developed a unique computational approach based on optimization procedures known as adaptation, simulated annealing, and genetic algorithm to construct 3D models of human chromosomes, using chromosomal contact data. Our models were evaluated using a percentage-based scoring function. Analysis of the scores of the final 3D models demonstrated their effective construction from our computational approach. Specifically, the models resulting from our approach yielded an average score of 80.41 %, with a high of 91 %, across models for all chromosomes of a normal human B-cell. Comparisons made with other methods affirmed the effectiveness of our strategy. Particularly, juxtaposition with models generated through the publicly available method Markov chain Monte Carlo 5C (MCMC5C) illustrated the outperformance of our approach, as seen through a higher average score for all chromosomes. Our methodology was further validated using two consistency checking techniques known as convergence testing and robustness checking, which both proved successful. The pursuit of constructing accurate 3D chromosomal structures is fueled by the benefits revealed by the findings as well as any possible future areas of study that arise. This motivation has led to the development of our computational methodology. The implementation of our approach proved effective in constructing 3D chromosome models and proved consistent with, and more effective than, some other methods thereby achieving our goal of creating a tool to help advance certain research efforts. The source code, test data, test results, and documentation of our method, Gen3D, are available at our sourceforge site at: http://sourceforge.net/projects/gen3d/ .

PTEN hamartoma tumour syndrome (PHTS) is caused by heterozygous variants in PTEN and is characterised by tumour predisposition, macrocephaly, and cognition impairment. Bi-allelic loss of PTEN activity has not been reported so far and animal models suggest that bi-allelic loss of PTEN activity is embryonically lethal. Here, we report the identification of a novel homozygous variant in PTEN, NM_000314.4; c.545T>C; p.Leu182Ser, in two adolescent siblings with severe macrocephaly and mild intellectual disability. The variant is predicted to be damaging and is associated with significantly increased phospho-S6 downstream of PTEN. The absence of tumours in the two homozygous siblings as well as lack of symptoms of PHTS in the heterozygous carriers of the family suggest that this particular variant is functionally hypomorphic rather than deleterious.European Journal of Human Genetics advance online publication, 7 October 2015; doi:10.1038/ejhg.2015.209.

Divorce is not only a personal choice, but also a sign of the lack of confidence in the institution of marriage. The issue of divorce raises questions that are sociological, theological, experiential, philosophical and political. This article traces the arguments with particular reference to the situation in Taiwan where women have no right to initiate divorce. It surveys the biblical and theological arguments, locating issues of divorce in the area of gender justice within patriarchal systems. Marriage, as well as divorce, is complex. To impose a monolithic definition of divorce is to fail to recognize the diversity of marital conflict and breakdown, generally to the advantage of a patriarchal society. This was as true of Hebrew society as it is of modern Taiwan, with its history of political struggle. The article concludes by challenging the Taiwanese churches to provide insights for people in their relationships with one another and with God.

Oncolytic vaccinia virus has been shown to induce a profound, rapid and tumor-specific vascular collapse in both preclinical models and in clinical studies, however a complete examination of the kinetics and levels of collapse and revascularization has not been described previously. Contrast-enhanced ultrasound was used to follow tumor perfusion levels in mouse tumor models at times after vaccinia therapy. It was observed that re-vascularization after viral therapy was dramatically delayed and did not occur until after viral clearance. This indicated that oncolytic vaccinia may posses a previously undescribed anti-angiogenic potential that might synergize with the reported anti-vascular effects. Despite a rapid loss of perfusion and widespread hypoxia within the tumor it was observed that VEGF levels in the tumor were suppressed throughout the period of active viral infection. Although tumor vasculature could eventually reform after the viral therapy was cleared in mouse models, anti-tumor effects could be significantly enhanced through additional combination with anti-VEGF therapies. This was initially examined using a gene therapy approach (Ad-Flk1-Fc) to target VEGF directly, demonstrating that the timing of application of the anti-angiogenic therapy was critical. However, it is also known that oncolytic vaccinia sensitizes tumors to tyrosine kinase inhibitors (TKI) in the clinic through an unknown mechanism. It is possible this phenomenon may be mediated through the anti-angiogenic effects of the TKIs. This was modeled in mouse tumors using sunitinib in combination with oncolytic vaccinia. It was observed that prevention of angiogenesis mediated by oncolytic vaccinia can be utilized to enhance the TKI therapy.

Traditional approaches for translating observations of molecular events into the context of a living organism have suffered from the requirements for either sacrificing animals at multiple time points prior to labor-intensive analyses of multiple tissues, or have relied on subjective observations or measurements of the animals over time. Recently an explosion of dedicated animal imaging modalities and the release of modified clinical imaging devices dedicated for animal imaging have allowed for the design of quantitative real time experiments incorporating fewer animals and providing whole animal analyses. Of these modalities, optical imaging (bioluminescence and fluorescence) has emerged as a powerful research tool, allowing investigators with limited whole animal imaging expertise to rapidly and inexpensively translate models produced in cellular assays into the context of a living animal. Here we will outline the steps necessary for translation of models established in culture systems into rodents.

The endothelium imposes a structural barrier to the extravasation of systemically delivered oncolytic adenovirus (Ad). Here, we introduced a transendothelial route of delivery in order to increase tumor accumulation of virus particles (vp) beyond that resulting from convection-dependent extravasation alone. This was achieved by engineering an Ad encoding a syncytium-forming protein, gibbon ape leukemia virus (GALV) fusogenic membrane glycoprotein (FMG). The expression of GALV was regulated by a hybrid viral enhancer-human promoter construct comprising the human cytomegalovirus (CMV) immediate-early enhancer and the minimal human endothelial receptor tyrosine kinase promoter (“eTie1”). Endothelial cell-selectivity of the resulting Ad-eTie1-GALV vector was demonstrated by measuring GALV mRNA transcript levels. Furthermore, Ad-eTie1-GALV selectively induced fusion between infected endothelial cells and uninfected epithelial cells in vitro and in vivo, allowing transendothelial virus penetration. Heterofusion of infected endothelium to human embryonic kidney 293 (HEK 293) cells, in mixed in vitro cultures or in murine xenograft models, permitted fusion-dependent transactivation of the replication-deficient Ad-eTie1-GALV, due to enabled access to viral E1 proteins derived from the HEK 293 cytoplasm. These data provide evidence to support our proposed use of GALV to promote Ad penetration through tumor-associated vasculature, an approach that may substantially improve the efficiency of systemic delivery of oncolytic viruses to disseminated tumors.

Replicating viruses have broad applications in biomedicine, notably in cancer virotherapy and in the design of attenuated vaccines; however, uncontrolled virus replication in vulnerable tissues can give pathology and often restricts the use of potent strains. Increased knowledge of tissue-selective microRNA expression now affords the possibility of engineering replicating viruses that are attenuated at the RNA level in sites of potential pathology, but retain wild-type replication activity at sites not expressing the relevant microRNA. To assess the usefulness of this approach for the DNA virus adenovirus, we have engineered a hepatocyte-safe wild-type adenovirus 5 (Ad5), which normally mediates significant toxicity and is potentially lethal in mice. To do this, we have included binding sites for hepatocyte-selective microRNA mir-122 within the 3′ UTR of the E1A transcription cassette. Imaging versions of these viruses, produced by fusing E1A with luciferase, showed that inclusion of mir-122 binding sites caused up to 80-fold decreased hepatic expression of E1A following intravenous delivery to mice. Animals administered a ten-times lethal dose of wild-type Ad5 (5×1010 viral particles/mouse) showed substantial hepatic genome replication and extensive liver pathology, while inclusion of 4 microRNA binding sites decreased replication 50-fold and virtually abrogated liver toxicity. This modified wild-type virus retained full activity within cancer cells and provided a potent, liver-safe oncolytic virus. In addition to providing many potent new viruses for cancer virotherapy, microRNA control of virus replication should provide a new strategy for designing safe attenuated vaccines applied across a broad range of viral diseases., Author Summary Attenuated viruses have found important applications in medicine, including their use as vaccines (notably for measles, mumps, polio, influenza, and chicken pox) and their experimental development as selective cancer-killing agents, so-called “virotherapy.” Wild-type versions are often most effective in both of these settings; however, attenuated viruses have usually been developed to decrease the risk of significant viral pathology. Recent advances in understanding regulation of gene expression by microRNA now afford the possibility to design viruses that are “selectively attenuated” in sites of potential pathology, by engineering them for inhibition by microRNA molecules that are expressed there. Here we have engineered wild-type adenovirus for recognition by a microRNA expressed in hepatocytes, producing a virus that retains wild-type infection and replication at sites of therapeutic activity (such as cancer cells) but is severely attenuated in hepatocytes, both in vitro and in vivo. This virus caused no significant liver toxicity to mice even when applied at ten times the lethal dose of wild-type virus. The ability to produce replication-competent viruses with key toxicities removed should provide a new platform for development of improved cancer treatments and better vaccines for a broad range of viral diseases.

A report on the symposium 'In vivo barriers to gene delivery', Cold Spring Harbor, USA, 26-29 November 2007. Many different obstacles impede the in vivo efficiency of both viral and non-viral agents for gene delivery. A recent symposium at the Cold Spring Harbor Laboratory, New York, addressed the latest approaches to overcoming these barriers. Topics encompassed diverse technologies for preventing vector neutralization, enhancing the persistence of transgene expression and the use of modified measles coat proteins to allow receptor specific HIV-1 pseudotyping.