Development of tumour imaging and therapy strategies utilising unengineered bacteria

The ability of systemically administered bacteria to target and replicate to high numbers within solid tumours is well established. Tumour localising bacteria can be exploited as biological vehicles for the delivery of nucleic acid, protein or therapeutic payloads to tumour sites and present researchers with a highly targeted and safe vehicle for tumour imaging and cancer therapy. This work aimed to utilise bacteria to activate imaging probes or prodrugs specifically within target tissue in order to facilitate the development of novel imaging and therapeutic strategies. The vast majority of existing bacterial-mediated cancer therapy strategies rely on the use of bacteria that have been genetically modified (GM) to express genes of interest. While these approaches have been shown to be effective in a preclinical setting, GM presents extra regulatory hurdles in a clinical context. Also, many strains of bacteria are not genetically tractably and hence cannot currently be engineered to express genes of interest. For this reason, the development of imaging and therapeutic systems that utilise unengineered bacteria for the activation of probes or drugs represents a significant improvement on the current gold standard. Endogenously expressed bacterial enzymes that are not found in mammalian cells can be used for the targeted activation of imaging probes or prodrugs whose activation is only achieved in the presence of these enzymes. Exploitation of the intrinsic enzymatic activity of bacteria allows the use of a wider range of bacteria and presents a more clinically relevant system than those that are currently in use. The nitroreductase (NTR) enzymes, found only in bacteria, represent one such option. Chapter 2 introduces the novel concept of utilising native bacterial NTRs for the targeted activation of the fluorophore CytoCy5S. Bacterial-mediated probe activation allowed for non-invasive fluorescence imaging of in vivo bacteria in models of infection and cancer. Chapter 3 extends the concept of using native bacterial enzymes to activate a novel luminescent, NTR activated probe. The use of luminescence based imaging improved the sensitivity of the system and provides researchers with a more accessible modality for preclinical imaging. It also represents an improvement over existing caged luciferin probe systems described to date. Chapter 4 focuses on the employment of endogenous bacterial enzymes for use in a therapeutic setting. Native bacterial enzymatic activity (including NTR enzymes) was shown to be capable of activating multiple prodrugs, in isolation and in combination, and eliciting therapeutic responses in murine models of cancer. Overall, the data presented in this thesis advance the fields of bacterial therapy and imaging and introduce novel strategies for disease diagnosis and treatment. These preclinical studies demonstrate potential for clinical translation in multiple fields of research and medicine.

Bifidobacterium longum 1714 as a translational psychobiotic: modulation of stress, electrophysiology and neurocognition in healthy volunteers

The emerging concept of psychobiotics—live microorganisms with a potential mental health benefit—represents a novel approach for the management of stress-related conditions. The majority of studies have focused on animal models. Recent preclinical studies have identified the B. longum 1714 strain as a putative psychobiotic with an impact on stress-related behaviors, physiology and cognitive performance. Whether such preclinical effects could be translated to healthy human volunteers remains unknown. We tested whether psychobiotic consumption could affect the stress response, cognition and brain activity patterns. In a within-participants design, healthy volunteers (N=22) completed cognitive assessments, resting electroencephalography and were exposed to a socially evaluated cold pressor test at baseline, post-placebo and post-psychobiotic. Increases in cortisol output and subjective anxiety in response to the socially evaluated cold pressor test were attenuated. Furthermore, daily reported stress was reduced by psychobiotic consumption. We also observed subtle improvements in hippocampus-dependent visuospatial memory performance, as well as enhanced frontal midline electroencephalographic mobility following psychobiotic consumption. These subtle but clear benefits are in line with the predicted impact from preclinical screening platforms. Our results indicate that consumption of B. longum 1714 is associated with reduced stress and improved memory. Further studies are warranted to evaluate the benefits of this putative psychobiotic in relevant stress-related conditions and to unravel the mechanisms underlying such effects.

Lost in translation? The potential psychobiotic Lactobacillus rhamnosus (JB-1) fails to modulate stress or cognitive performance in healthy male subjects

Background: Preclinical studies have identified certain probiotics as psychobiotics a live microorganisms with a potential mental health benefit. Lactobacillus rhamnosus (JB-1) has been shown to reduce stress-related behaviour, corticosterone release and alter central expression of GABA receptors in an anxious mouse strain. However, it is unclear if this single putative psychobiotic strain has psychotropic activity in humans. Consequently, we aimed to examine if these promising preclinical findings could be translated to healthy human volunteers. Objectives: To determine the impact of L. rhamnosus on stress-related behaviours, physiology, inflammatory response, cognitive performance and brain activity patterns in healthy male participants. An 8 week, randomized, placebo-controlled, cross-over design was employed. Twenty-nine healthy male volunteers participated. Participants completed self-report stress measures, cognitive assessments and resting electroencephalography (EEG). Plasma IL10, IL1β, IL6, IL8 and TNFα levels and whole blood Toll-like 4 (TLR-4) agonist-induced cytokine release were determined by multiplex ELISA. Salivary cortisol was determined by ELISA and subjective stress measures were assessed before, during and after a socially evaluated cold pressor test (SECPT). Results: There was no overall effect of probiotic treatment on measures of mood, anxiety, stress or sleep quality and no significant effect of probiotic over placebo on subjective stress measures, or the HPA response to the SECPT. Visuospatial memory performance, attention switching, rapid visual information processing, emotion recognition and associated EEG measures did not show improvement over placebo. No significant anti-inflammatory effects were seen as assessed by basal and stimulated cytokine levels. Conclusions: L. rhamnosus was not superior to placebo in modifying stress-related measures, HPA response, inflammation or cognitive performance in healthy male participants. These findings highlight the challenges associated with moving promising preclinical studies, conducted in an anxious mouse strain, to healthy human participants. Future interventional studies investigating the effect of this psychobiotic in populations with stress-related disorders are required.