MiR-34a regulates the invasive capacity of canine osteosarcoma cell lines.

BACKGROUND: Osteosarcoma (OSA) is the most common bone tumor in children and dogs; however, no substantial improvement in clinical outcome has occurred in either species over the past 30 years. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression and play a fundamental role in cancer. The purpose of this study was to investigate the potential contribution of miR-34a loss to the biology of canine OSA, a well-established spontaneous model of the human disease. METHODOLOGY AND PRINCIPAL FINDINGS: RT-qPCR demonstrated that miR-34a expression levels were significantly reduced in primary canine OSA tumors and canine OSA cell lines as compared to normal canine osteoblasts. In canine OSA cell lines stably transduced with empty vector or pre-miR-34a lentiviral constructs, overexpression of miR-34a inhibited cellular invasion and migration but had no effect on cell proliferation or cell cycle distribution. Transcriptional profiling of canine OSA8 cells possessing enforced miR-34a expression demonstrated dysregulation of numerous genes, including significant down-regulation of multiple putative targets of miR-34a. Moreover, gene ontology analysis of down-regulated miR-34a target genes showed enrichment of several biological processes related to cell invasion and motility. Lastly, we validated changes in miR-34a putative target gene expression, including decreased expression of KLF4, SEM3A, and VEGFA transcripts in canine OSA cells overexpressing miR-34a and identified KLF4 and VEGFA as direct target genes of miR-34a. Concordant with these data, primary canine OSA tumor tissues demonstrated increased expression levels of putative miR-34a target genes. CONCLUSIONS: These data demonstrate that miR-34a contributes to invasion and migration in canine OSA cells and suggest that loss of miR-34a may promote a pattern of gene expression contributing to the metastatic phenotype in canine OSA.

Advanced age is associated with worsened outcomes and a unique genomic response in severely injured patients with hemorrhagic shock.

INTRODUCTION: We wished to characterize the relationship of advanced age to clinical outcomes and to transcriptomic responses after severe blunt traumatic injury with hemorrhagic shock. METHODS: We performed epidemiological, cytokine, and transcriptomic analyses on a prospective, multi-center cohort of 1,928 severely injured patients. RESULTS: We found that there was no difference in injury severity between the aged (age ≥55, n = 533) and young (age <55, n = 1395) cohorts. However, aged patients had more comorbidities. Advanced age was associated with more severe organ failure, infectious complications, ventilator days, and intensive care unit length of stay, as well as, an increased likelihood of being discharged to skilled nursing or long-term care facilities. Additionally, advanced age was an independent predictor of a complicated recovery and 28-day mortality. Acutely after trauma, blood neutrophil genome-wide expression analysis revealed an attenuated transcriptomic response as compared to the young; this attenuated response was supported by the patients' plasma cytokine and chemokine concentrations. Later, these patients demonstrated gene expression changes consistent with simultaneous, persistent pro-inflammatory and immunosuppressive states. CONCLUSIONS: We concluded that advanced age is one of the strongest non-injury related risk factors for poor outcomes after severe trauma with hemorrhagic shock and is associated with an altered and unique peripheral leukocyte genomic response. As the general population's age increases, it will be important to individualize prediction models and therapeutic targets to this high risk cohort.

Persistent inflammation, immunosuppression, and catabolism syndrome after severe blunt trauma.

BACKGROUND: We recently proffered that a new syndrome persistent inflammation, immunosuppression, and catabolism syndrome (PICS) has replaced late multiple-organ failure as a predominant phenotype of chronic critical illness. Our goal was to validate this by determining whether severely injured trauma patients with complicated outcomes have evidence of PICS at the genomic level. METHODS: We performed a secondary analysis of the Inflammation and Host Response to Injury database of adults with severe blunt trauma. Patients were classified into complicated, intermediate, and uncomplicated clinical trajectories. Existing genomic microarray data were compared between cohorts using Ingenuity Pathways Analysis. Epidemiologic data and outcomes were also analyzed between cohorts on admission, Day 7, and Day 14. RESULTS: Complicated patients were older, were sicker, and required increased ventilator days compared with the intermediate/uncomplicated patients. They also had persistent leukocytosis as well as low lymphocyte and albumin levels compared with uncomplicated patients. Total white blood cell leukocyte analysis in complicated patients showed that overall genome-wide expression patterns and those patterns on Days 7 and 14 were more aberrant from control subjects than were patterns from uncomplicated patients. Complicated patients also had significant down-regulation of adaptive immunity and up-regulation of inflammatory genes on Days 7 and 14 (vs. magnitude in fold change compared with control and in magnitude compared with uncomplicated patients). On Day 7, complicated patients had significant changes in functional pathways involved in the suppression of myeloid cell differentiation, increased inflammation, decreased chemotaxis, and defective innate immunity compared with uncomplicated patients and controls. Subset analysis of monocyte, neutrophil, and T-cells supported these findings. CONCLUSION: Genomic analysis of patients with complicated clinical outcomes exhibit persistent genomic expression changes consistent with defects in the adaptive immune response and increased inflammation. Clinical data showed persistent inflammation, immunosuppression, and protein depletion. Overall, the data support the hypothesis that patients with complicated clinical outcomes are exhibiting PICS. LEVEL OF EVIDENCE: Epidemiologic study, level III.

Genomic responses in mouse models poorly mimic human inflammatory diseases.

A cornerstone of modern biomedical research is the use of mouse models to explore basic pathophysiological mechanisms, evaluate new therapeutic approaches, and make go or no-go decisions to carry new drug candidates forward into clinical trials. Systematic studies evaluating how well murine models mimic human inflammatory diseases are nonexistent. Here, we show that, although acute inflammatory stresses from different etiologies result in highly similar genomic responses in humans, the responses in corresponding mouse models correlate poorly with the human conditions and also, one another. Among genes changed significantly in humans, the murine orthologs are close to random in matching their human counterparts (e.g., R(2) between 0.0 and 0.1). In addition to improvements in the current animal model systems, our study supports higher priority for translational medical research to focus on the more complex human conditions rather than relying on mouse models to study human inflammatory diseases.

Partitioning transcript variation in Drosophila: abundance, isoforms, and alleles.

Multilevel analysis of transcription is facilitated by a new array design that includes modules for assessment of differential expression, isoform usage, and allelic imbalance in Drosophila. The ∼2.5 million feature chip incorporates a large number of controls, and it contains 18,769 3' expression probe sets and 61,919 exon probe sets with probe sequences from Drosophila melanogaster and 60,118 SNP probe sets focused on Drosophila simulans. An experiment in D. simulans identified genes differentially expressed between males and females (34% in the 3' expression module; 32% in the exon module). These proportions are consistent with previous reports, and there was good agreement (κ = 0.63) between the modules. Alternative isoform usage between the sexes was identified for 164 genes. The SNP module was verified with resequencing data. Concordance between resequencing and the chip design was greater than 99%. The design also proved apt in separating alleles based upon hybridization intensity. Concordance between the highest hybridization signals and the expected alleles in the genotype was greater than 96%. Intriguingly, allelic imbalance was detected for 37% of 6579 probe sets examined that contained heterozygous SNP loci. The large number of probes and multiple probe sets per gene in the 3' expression and exon modules allows the array to be used in D. melanogaster and in closely related species. The SNP module can be used for allele specific expression and genotyping of D. simulans.

A limited innate immune response is induced by a replication-defective herpes simplex virus vector following delivery to the murine central nervous system.

Herpes simplex virus type 1 (HSV-1)-based vectors readily transduce neurons and have a large payload capacity, making them particularly amenable to gene therapy applications within the central nervous system (CNS). Because aspects of the host responses to HSV-1 vectors in the CNS are largely unknown, we compared the host response of a nonreplicating HSV-1 vector to that of a replication-competent HSV-1 virus using microarray analysis. In parallel, HSV-1 gene expression was tracked using HSV-specific oligonucleotide-based arrays in order to correlate viral gene expression with observed changes in host response. Microarray analysis was performed following stereotactic injection into the right hippocampal formation of mice with either a replication-competent HSV-1 or a nonreplicating recombinant of HSV-1, lacking the ICP4 gene (ICP4-). Genes that demonstrated a significant change (P < .001) in expression in response to the replicating HSV-1 outnumbered those that changed in response to mock or nonreplicating vector by approximately 3-fold. Pathway analysis revealed that both the replicating and nonreplicating vectors induced robust antigen presentation but only mild interferon, chemokine, and cytokine signaling responses. The ICP4- vector was restricted in several of the Toll-like receptor-signaling pathways, indicating reduced stimulation of the innate immune response. These array analyses suggest that although the nonreplicating vector induces detectable activation of immune response pathways, the number and magnitude of the induced response is dramatically restricted compared to the replicating vector, and with the exception of antigen presentation, host gene expression induced by the nonreplicating vector largely resembles mock infection.