Pre-Clinical Model to Study Recurrent Venous Thrombosis in the Inferior Vena Cava.

BACKGROUND: Patients undergoing deep vein thrombosis (VT) have over 30% recurrence, directly increasing their risk of post-thrombotic syndrome. Current murine models of inferior vena cava (IVC) VT model host one thrombosis event. OBJECTIVE: We aimed to develop a murine model to study IVC recurrent VT in mice. MATERIALS AND METHODS: An initial VT was induced using the electrolytic IVC model (EIM) with constant blood flow. This approach takes advantage of the restored vein lumen 21 days after a single VT event in the EIM demonstrated by ultrasound. We then induced a second VT 21 days later, using either EIM or an IVC ligation model for comparison. The control groups were a sham surgery and, 21 days later, either EIM or IVC ligation. IVC wall and thrombus were harvested 2 days after the second insult and analysed for IVC and thrombus size, gene expression of fibrotic markers, histology for collagen and Western blot for citrullinated histone 3 (Cit-H3) and fibrin. RESULTS: Ultrasound confirmed the first VT and its progressive resolution with an anatomical channel allowing room for the second thrombus by day 21. As compared with a primary VT, recurrent VT has heavier walls with significant up-regulation of transforming growth factor-ß (TGF-ß), elastin, interleukin (IL)-6, matrix metallopeptidase 9 (MMP9), MMP2 and a thrombus with high citrullinated histone-3 and fibrin content. CONCLUSION: Experimental recurrent thrombi are structurally and compositionally different from the primary VT, with a greater pro-fibrotic remodelling vein wall profile. This work provides a VT recurrence IVC model that will help to improve the current understanding of the biological mechanisms and directed treatment of recurrent VT.

Changes in Caenorhabditis elegans gene expression following exposure to Photorhabdus luminescens strain TT01.

Photorhabdus bacteria enter into a mutualistic symbiosis with Heterorhabditis nematodes to infect insect larvae. However, they rapidly kill the model nematode Caenorhabditis elegans. One hypothesis for these divergent outcomes is that the nematode defense responses differ. To begin testing this hypothesis, we have systematically analyzed available data on the transcriptional response of C. elegans to P. luminescens strain Hb. From a starting pool of over 7000 differentially expressed genes, we carefully chose 21 Heterorhabditis-conserved genes to develop as comparative markers. Using newly designed and validated qRT-PCR primers, we measured expression of these genes in C. elegans exposed to the sequenced TT01 strain of P. luminescens, on two different media types. Almost all (18/21) of the genes showed a significant response to P. luminescens strain TT01. One response is dependent on media type, and a subset of genes may respond differentially to distinct strains. Overall, we have established useful resources and generated new hypotheses regarding how C. elegans responds to P. luminescens infection.

Death Becomes Them: Bacterial Community Dynamics and Stilbene Antibiotic Production in Cadavers of Galleria mellonella Killed by Heterorhabditis and Photorhabdus spp.

UNLABELLED: Insect larvae killed by entomopathogenic nematodes are thought to contain bacterial communities dominated by a single bacterial genus, that of the nematode's bacterial symbiont. In this study, we used next-generation sequencing to profile bacterial community dynamics in greater wax moth (Galleria mellonella) larvae cadavers killed by Heterorhabditis nematodes and their Photorhabdus symbionts. We found that, although Photorhabdus strains did initially displace an Enterococcus-dominated community present in uninfected G. mellonella insect larvae, the cadaver community was not static. Twelve days postinfection, Photorhabdus shared the cadaver with Stenotrophomonas species. Consistent with this result, Stenotrophomonas strains isolated from infected cadavers were resistant to Photorhabdus-mediated toxicity in solid coculture assays. We isolated and characterized a Photorhabdus-produced antibiotic from G. mellonella cadavers, produced it synthetically, and demonstrated that both the natural and synthetic compounds decreased G. mellonella-associated Enterococcus growth, but not Stenotrophomonas growth, in vitro Finally, we showed that the Stenotrophomonas strains described here negatively affected Photorhabdus growth in vitro Our results add an important dimension to a broader understanding of Heterorhabditis-Photorhabdus biology and also demonstrate that interspecific bacterial competition likely characterizes even a theoretically monoxenic environment, such as a Heterorhabditis-Photorhabdus-parasitized insect cadaver. IMPORTANCE: Understanding, and eventually manipulating, both human and environmental health depends on a complete accounting of the forces that act on and shape microbial communities. One of these underlying forces is hypothesized to be resource competition. A resource that has received little attention in the general microbiological literature, but likely has ecological and evolutionary importance, is dead/decaying multicellular organisms. Metazoan cadavers, including those of insects, are ephemeral and nutrient-rich environments, where resource competition might shape interspecific macrobiotic and microbiotic interactions. This study is the first to use a next-generation sequencing approach to study the community dynamics of bacteria within a model insect cadaver system: insect larvae parasitized by entomopathogenic nematodes and their bacterial symbionts. By integrating bioinformatic, biochemical, and classic in vitro microbiological approaches, we have provided mechanistic insight into how antibiotic-mediated bacterial interactions may shape community dynamics within insect cadavers.