New and improved demonstrations, each illustrating a single scientific paper

Thesis (Ed.M.)--Boston Universit

Bacillus Calmette-Guerin (BCG) induces superior anti-tumour responses by Vδ2 + T-cells compared to the aminobisphosphonate drug Zoledronic acid.

Vδ2 + T-cells can recognise malignantly transformed cells as well as those infected with mycobacteria. This cross-reactivity supports the idea of using mycobacteria to manipulate Vδ2 + T-cells in cancer immunotherapy. To date, therapeutic interventions using Vδ2 + T-cells in cancer have involved expanding these cells in or ex vivo using zoledronic acid (ZA). Here, we show that the mycobacterium Bacillus Calmette-Guérin (BCG) also causes Vδ2 + T-cell expansion in vitro and that resulting Vδ2 + cell populations are cytotoxic towards tumour cell lines. We show that both ZA and BCG-expanded Vδ2+ cells effectively killed both Daudi and THP-1 cells. THP-1 cell killing by both ZA and BCG-expanded Vδ2+ cells was enhanced by treatment of targets cells with ZA. Although no difference in cytotoxic activity between ZA- and BCG-expanded Vδ2+ cells was observed, BCG-expanded cells degranulated more and produced a more diverse range of cytokines upon tumour cell recognition compared to ZA-expanded cells. ZA-expanded Vδ2+ cells were shown to upregulate exhaustion marker CD57 to a greater extent than BCG-expanded Vδ2+ cells. Furthermore, ZA expansion was associated with upregulation of inhibitory markers PD-1 and TIM3 in a dose-dependent manner whereas PD-1 expression was not increased following expansion using BCG. Intradermal BCG vaccination of rhesus macaques caused in vivo expansion of Vδ2 + cells. In combination with the aforementioned in vitro data, this finding suggests that BCG treatment could induce expansion of Vδ2 + T-cells with enhanced anti-tumour potential compared to ZA treatment and that either ZA or BCG could be used intratumourally as a means to potentiate stronger anti-tumour Vδ2 + T-cell responses

Persistent ER Stress Induces the Spliced Leader RNA Silencing Pathway (SLS), Leading to Programmed Cell Death in Trypanosoma brucei

Trypanosomes are parasites that cycle between the insect host (procyclic form) and mammalian host (bloodstream form). These parasites lack conventional transcription regulation, including factors that induce the unfolded protein response (UPR). However, they possess a stress response mechanism, the spliced leader RNA silencing (SLS) pathway. SLS elicits shut-off of spliced leader RNA (SL RNA) transcription by perturbing the binding of the transcription factor tSNAP42 to its cognate promoter, thus eliminating trans-splicing of all mRNAs. Induction of endoplasmic reticulum (ER) stress in procyclic trypanosomes elicits changes in the transcriptome similar to those induced by conventional UPR found in other eukaryotes. The mechanism of up-regulation under ER stress is dependent on differential stabilization of mRNAs. The transcriptome changes are accompanied by ER dilation and elevation in the ER chaperone, BiP. Prolonged ER stress induces SLS pathway. RNAi silencing of SEC63, a factor that participates in protein translocation across the ER membrane, or SEC61, the translocation channel, also induces SLS. Silencing of these genes or prolonged ER stress led to programmed cell death (PCD), evident by exposure of phosphatidyl serine, DNA laddering, increase in reactive oxygen species (ROS) production, increase in cytoplasmic Ca2+, and decrease in mitochondrial membrane potential, as well as typical morphological changes observed by transmission electron microscopy (TEM). ER stress response is also induced in the bloodstream form and if the stress persists it leads to SLS. We propose that prolonged ER stress induces SLS, which serves as a unique death pathway, replacing the conventional caspase-mediated PCD observed in higher eukaryotes

Linking water-balance simulation and multiobjective programming: land-use plan design in Hawaii

In the Hawaiian Islands, planners and public officials have decided recently to raise the permissible level of urban development in central Oahu. The decision is opposed by many on the grounds that it threatens agricultural land as well as the sustainability of groundwater supply. A two-part procedure is presented for exploring the impacts of such development and designing urban-expansion patterns that minimize them. First, a water-balance simulation model is used to calculate groundwater recharge as it varies with land use and location within the area. The difference between recharge and withdrawal is computed, and any changes are then estimated for different land uses. This information is then incorporated into optimization models having objectives related to agricultural land retention, groundwater balance, and residential population growth. The models generate alternative land-use expansion plans and show the trade-offs among objectives. The consideration of slightly suboptimal (dominated) solutions allows a significant expansion in the range of such alternatives. The results suggest that, if future agricultural development does not occur on currently nonagricultural land, then both agricultural land and groundwater sustainability will suffer significant adverse effects under the new population limits.

Releasing the restraints of Vγ9Vδ2 T-cells in cancer immunotherapy.

OBJECTIVES: Vγ9Vδ2 T-cells are a subset of T-cells with a crucial role in immunosurveillance which can be activated and expanded by multiple means to stimulate effector responses. Little is known about the expression of checkpoint molecules on this cell population and whether the ligation of these molecules can regulate their activity. The aim of this study was to assess the expression of both activatory and inhibitory receptors on Vγ9Vδ2 T-cells to assess potential avenues of regulation to target with immunotherapy. METHODS: Expression of various activatory and inhibitory receptors was assessed on Vγ9Vδ2 T-cells by flow cytometry following activation and expansion using zoledronic acid (ZA) and Bacillus Calmette-Guérin (BCG). Expression of these markers and production of effector molecules was also examined following co-culture with various tumour cell targets. The effect of immune checkpoint blockade on Vγ9Vδ2 T-cells was also explored. RESULTS: Vγ9Vδ2 T-cells expressed high levels of activatory markers both at baseline and following stimulation. Vγ9Vδ2 T-cells expressed variable levels of inhibitory checkpoint receptors with many being upregulated following stimulation. Expression of these markers is further modulated upon co-culture with tumour cells with changes reflecting activation and effector functions. Despite their high expression of inhibitory receptors when cultured with tumour cells expressing cognate ligands there was no effect on Vδ2+ T-cell cytotoxic capacity or cytokine production with immune checkpoint blockade. CONCLUSIONS: Our work suggests the expression of checkpoint receptors present on Vγ9Vδ2 T-cells which may provide a mechanism with the potential to be utilised by tumour cells to subvert Vγ9Vδ2 T-cell cytotoxicity. This work suggests important candidates for blockade by ICI therapy in order to increase the successful use of Vγ9Vδ2 T-cells in immunotherapy

A comparison of DEF x-ray film and a photodiode array (reticon) as detectors for an x-ray crystal spectrometer

A crystal spectrometer with a photodiode array (PDA) detector was tested for a range of x-ray energies between 1 and 2 keV. A laser-produced plasma has been used as an x-ray source and was generated by the high-power (Vulcan) glass laser system at the SERC Rutherford Appleton Laboratory, UK. The performance of the array was directly compared with the response of Kodak DEF x-ray film. In order to compare quantitatively the performances of the PDA and the film, detective quantum efficiency (DQE) considerations are presented for both devices. It is demonstrated that the PDA has a useful dynamic range which is approximately seven times greater than that of film, a peak DQE of approximately six times that of film, and a greatly superior low-signal performance. The operational characteristics of the PDA are discussed. © 1989