Three-dimensional organotypic co-culture model of intestinal epithelial cells and macrophages to study Salmonella enterica colonization patterns

Three-dimensional models of human intestinal epithelium mimic the differentiated form and function of parental tissues often not exhibited by two-dimensional monolayers and respond to Salmonella in key ways that reflect in vivo infections. To further enhance the physiological relevance of three-dimensional models to more closely approximate in vivo intestinal microenvironments encountered by Salmonella, we developed and validated a novel three-dimensional co-culture infection model of colonic epithelial cells and macrophages using the NASA Rotating Wall Vessel bioreactor. First, U937 cells were activated upon collagen-coated scaffolds. HT-29 epithelial cells were then added and the three-dimensional model was cultured in the bioreactor until optimal differentiation was reached, as assessed by immunohistochemical profiling and bead uptake assays. The new co-culture model exhibited in vivo-like structural and phenotypic characteristics, including three-dimensional architecture, apical-basolateral polarity, well-formed tight/adherens junctions, mucin, multiple epithelial cell types, and functional macrophages. Phagocytic activity of macrophages was confirmed by uptake of inert, bacteria-sized beads. Contribution of macrophages to infection was assessed by colonization studies of Salmonella pathovars with different host adaptations and disease phenotypes (Typhimurium ST19 strain SL1344 and ST313 strain D23580; Typhi Ty2). In addition, Salmonella were cultured aerobically or microaerobically, recapitulating environments encountered prior to and during intestinal infection, respectively. All Salmonella strains exhibited decreased colonization in co-culture (HT-29-U937) relative to epithelial (HT-29) models, indicating antimicrobial function of macrophages. Interestingly, D23580 exhibited enhanced replication/survival in both models following invasion. Pathovar-specific differences in colonization and intracellular co-localization patterns were observed. These findings emphasize the power of incorporating a series of related three-dimensional models within a study to identify microenvironmental factors important for regulating infection

Demonstrating the potential of low-cost GPS units for the remote measurement of tides and water levels using interferometric reflectometry

A low-cost [$30 (U.S. dollars)] consumer grade GPS receiver with a sideways-mounted antenna has been applied to measure tidal water levels at a mesotidal coastal site using an interferometric reflectometry approach. The proof-of-concept system was installed approximately 16 m above mean sea level in close proximity to a conventional bubbler tide gauge that provided validation data. The received signal-to-noise ratios (SNR) for the satellites in view were recorded for several months during two successive years and the observed frequencies of the interferometric oscillations used to calculate the difference in elevation between the receiver and the water surface. Comparisons with concurrent and historic in situ tide gauge data at the site initially helped to identify a calibration issue with the in situ gauge. The GPS-based measurements were shown to be in excellent agreement with the corrected in situ gauge, exhibiting a root-mean-square difference of 5.7 cm over a tidal range exceeding 3 m at spring tides and a daily averaged RMS of 1.7 cm. The SNR data from the low-cost GPS receivers are shown to provide significantly higher-quality data for this purpose compared with high-end geodetic grade receivers at similar sites. This low-cost, widely available technology has the potential to be applied globally for monitoring water levels in a wide variety of circumstances and applications that would otherwise be cost or situation prohibitive. It could also be applied as an independent cross check and quality control measure for conventional water-level gauges

Plate-based diversity subset screening generation 2: An improved paradigm for high throughput screening of large compound files

High throughput screening (HTS) is an effective method for lead and probe discovery that is widely used in industry and academia to identify novel chemical matter and to initiate the drug discovery process. However, HTS can be time-consuming and costly and the use of subsets as an efficient alternative to screening these large collections has been investigated. Subsets may be selected on the basis of chemical diversity, molecular properties, biological activity diversity, or biological target focus. Previously we described a novel form of subset screening: plate-based diversity subset (PBDS) screening, in which the screening subset is constructed by plate selection (rather than individual compound cherry-picking), using algorithms that select for compound quality and chemical diversity on a plate basis. In this paper, we describe a second generation approach to the construction of an updated subset: PBDS2, using both plate and individual compound selection, that has an improved coverage of the chemical space of the screening file, whilst only selecting the same number of plates for screening. We describe the validation of PBDS2 and its successful use in hit and lead discovery. PBDS2 screening became the default mode of singleton (one compound per well) HTS for lead discovery in Pfizer

Socio-oceanography: an opportunity to integrate marine social and natural sciences

Marine natural sciences have been instrumental in helping society understand how ocean systems operate and the threats they face. However, there is a growing realisation that the societal challenges related to the marine environment can only be addressed through more effective integration with all aspects of social sciences. Nevertheless, to date, social sciences remain insufficiently integrated into marine research. Recognising historical weaknesses and drawing on the authors’ own experience of interdisciplinary research, albeit writing primarily from a natural marine science perspective, we propose a series of steps to promote integrated marine research inclusive of strong social science. We suggest that changing the perspectives and attitudes of natural scientists is key. The inherent interconnectivity between the ocean and society ensures that nearly everything we do in the marine natural sciences has the potential to influence and, perhaps address, ongoing and future societal challenges. Consequently, a key challenge for natural scientists is to recognise and communicate this in an accessible manner outside their own disciplines. To attempt to address these issues, we introduce the concept of “Socio-oceanography” which we define as an area of research that takes a “whole system” approach to the marine environment. It focuses on the challenges which require advancement of both natural and social science components, especially on those where the feedbacks between social and natural components are beginning to emerge. Here, we discuss its scope, challenges to its effective application and key steps to catalyse interdisciplinary approaches using this concept

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival