Sally R. Robinson

Gene expression profiling identifies inflammation and angiogenesis as distinguishing features of canine hemangiosarcoma

BackgroundThe etiology of hemangiosarcoma remains incompletely understood. Its common occurrence in dogs suggests predisposing factors favor its development in this species. These factors could represent a constellation of heritable characteristics that promote transformation events and/or facilitate the establishment of a microenvironment that is conducive for survival of malignant blood vessel-forming cells. The hypothesis for this study was that characteristic molecular features distinguish hemangiosarcoma from non-malignant endothelial cells, and that such features are informative for the etiology of this disease.MethodsWe first investigated mutations of VHL and Ras family genes that might drive hemangiosarcoma by sequencing tumor DNA and mRNA (cDNA). Protein expression was examined using immunostaining. Next, we evaluated genome-wide gene expression profiling using the Affymetrix Canine 2.0 platform as a global approach to test the hypothesis. Data were evaluated using routine bioinformatics and validation was done using quantitative real time RT-PCR.ResultsEach of 10 tumor and four non-tumor samples analyzed had wild type sequences for these genes. At the genome wide level, hemangiosarcoma cells clustered separately from non-malignant endothelial cells based on a robust signature that included genes involved in inflammation, angiogenesis, adhesion, invasion, metabolism, cell cycle, signaling, and patterning. This signature did not simply reflect a cancer-associated angiogenic phenotype, as it also distinguished hemangiosarcoma from non-endothelial, moderately to highly angiogenic bone marrow-derived tumors (lymphoma, leukemia, osteosarcoma).ConclusionsThe data show that inflammation and angiogenesis are important processes in the pathogenesis of vascular tumors, but a definitive ontogeny of the cells that give rise to these tumors remains to be established. The data do not yet distinguish whether functional or ontogenetic plasticity creates this phenotype, although they suggest that cells which give rise to hemangiosarcoma modulate their microenvironment to promote tumor growth and survival. We propose that the frequent occurrence of canine hemangiosarcoma in defined dog breeds, as well as its similarity to homologous tumors in humans, offers unique models to solve the dilemma of stem cell plasticity and whether angiogenic endothelial cells and hematopoietic cells originate from a single cell or from distinct progenitor cells.

Immune response to ORF5a protein immunization is not protective against Porcine Reproductive and Respiratory Syndrome Virus infection

Porcine reproductive and respiratory syndrome virus (PRRSV) is an enveloped RNA virus responsible for PRRS in swine, a disease with globally significant animal welfare and economic concerns. There is no specific treatment and variably effective immune protection. Molecular mechanisms responsible for virulence, pathogenesis and protective immune response remain poorly understood. These factors limit progress toward development of effective measures for prevention and treatment of PRRS. A novel PRRSV ORF5a protein, encoded in an open reading frame (ORF) that overlaps the major envelope glycoprotein GP5 ORF, was recently identified. Because ORF5a is highly conserved in diverse PRRSV isolates, is a structural protein in the virion, and elicits a specific antibody response in infected pigs, we investigated its potential role in immune protection against PRRSV infection. Pigs immunized with ORF5a protein had robust serologic responses. However, the antibodies did not neutralize virus, and immunity did not protect against challenge infection. We conclude from these findings that the ORF5a antibody response is neither neutralizing nor protective.

Broadly Neutralizing Antibodies Against the Rapidly Evolving Porcine Reproductive and Respiratory Syndrome Virus

Neutralizing antibodies are a critical part of the immune armory for defense against viruses, and the mechanism by which many effective vaccines work to protect against viral infections. However, infections by rapidly evolving and genetically diverse viruses are often characterized by ineffective neutralizing antibody responses. Porcine reproductive and respiratory syndrome virus (PRRSV) is a highly genetically diverse RNA virus that causes PRRS, the most significant disease of pigs worldwide. The prevailing view of immunity to PRRSV is characterized by delayed and ineffectual production of neutralizing antibodies lacking cross-reactivity that is necessary for vaccine efficacy. Using an ELISA-based neutralizing assay developed to analyze PRRSV growth in porcine alveolar macrophages, the naturally permissive cell of PRRSV, we showed that sera from previously infected commercial sows had high levels of neutralizing activity against diverse PRRSV strains, including across distinct genotypes of PRRSV. Fifty percent cross-neutralization titers in excess of 1/1024 were observed. Neutralizing activity was dose-dependent and was maintained in the immunoglobulin fraction. Presence of high-titer, anti-PRRSV antibody activity that cross-neutralizes diverse strains of virus has prompted reevaluation of the role of neutralizing antibodies for cross-protection against PRRSV under field conditions. Understanding conditions that favor development of cross-neutralizing activity will be crucial for improved strategies to enhance cross-protection against PRRSV. More detailed studies are expected to elucidate mechanisms of neutralizing antibody production and maturation and to investigate conserved epitope targets of cross-neutralization in this rapidly evolving virus.

Nicotine-mediated signals modulate cell death and survival of T lymphocytes

The capacity of nicotine to affect the behavior of non-neuronal cells through neuronal nicotinic acetylcholine receptors (nAChRs) has been the subject of considerable recent attention. Previously, we showed that exposure to nicotine activates the nuclear factor of activated T cells (NFAT) transcription factor in lymphocytes and endothelial cells, leading to alterations in cellular growth and vascular endothelial growth factor production. Here, we extend these studies to document effects of nicotine on lymphocyte survival. The data show that nicotine induces paradoxical effects that might alternatively enforce survival or trigger apoptosis, suggesting that depending on timing and context, nicotine might act both as a survival factor or as an inducer of apoptosis in normal or transformed lymphocytes, and possibly other non-neuronal cells. In addition, our results show that, while having overlapping functions, low and high affinity nAChRs also transmit signals that promote distinct outcomes in lymphocytes. The sum of our data suggests that selective modulation of nAChRs might be useful to regulate lymphocyte activation and survival in health and disease.

Purifying selection in porcine reproductive and respiratory syndrome virus ORF5a protein influences variation in envelope glycoprotein 5 glycosylation

Porcine reproductive and respiratory syndrome virus ORF5a protein is encoded in an alternate open reading frame upstream of the major envelope glycoprotein (GP5) in subgenomic mRNA5. Bioinformatic analysis of 3466 type 2 PRRSV sequences showed that the two proteins have co-evolved through a fine balance of purifying codon usage to maintain a conserved RQ-rich motif in ORF5a protein, while eliciting a variable N-linked glycosylation motif in the alternative GP5 reading frame. Conservation of the ORF5a protein RQ-motif also explains an anomalous uracil desert in GP5 hypervariable glycosylation region. The N-terminus of the mature GP5 protein was confirmed to start with amino acid 32, the hypervariable region of the ectodomain. Since GP5 glycosylation variability is assumed to result from immunological selection against neutralizing antibodies, these findings show that an alternative possibility unrelated to immunological selection not only exists, but provides a foundation for investigating previously unsuspected aspects of PRRSV biology. Understanding functional consequences of subtle nucleotide sequence modifications in the region responsible for critical function in ORF5a protein and GP5 glycosylation is essential for rational design of new vaccines against PRRS.

Porcine reproductive and respiratory syndrome virus neutralizing antibodies provide in vivo cross-protection to PRRSV1 and PRRSV2 viral challenge

Vaccine control and prevention of porcine reproductive and respiratory syndrome (PRRS), the most important disease of swine, is difficult to achieve. However, the discovery of broadly neutralizing antibody activity against porcine reproductive and respiratory syndrome virus (PRRSV) under typical field conditions opens the door to new immunologic approaches for robust protection. We show here that passive administration of purified immunoglobulins with neutralizing antibodies reduced PRRSV2 infection by up to 96%, and PRRSV1 infection by up to 87%, whereas immune immunoglobulins lacking neutralizing activity had no effect on viral infection. Hence, immune competence of passive immunoglobulin transfer was associated specifically with antibody neutralizing activity. Current models of PRRSV infection implicate a minor envelope glycoprotein (GP) complex including GP2, GP3, and GP4, as critical to permissive cell infection. However, conserved peptides comprising the putative cell attachment structure did not attenuate neutralization or viral infection. The results show that immunological approaches aimed at induction of broadly neutralizing antibodies may substantially enhance immune protection against PRRSV. The findings further show that naturally occurring viral isolates are able to induce protective humoral immunity against unrelated PRRSV challenge, thus removing a major conceptual barrier to vaccine development.