Intracellular \u3cem\u3eSalmonella\u3c/em\u3e delivery of an exogenous immunization antigen refocuses CD8 T cells against cancer cells, eliminates pancreatic tumors and forms antitumor immunity

Introduction: Immunotherapies have shown great promise, but are not effective for all tumors types and are effective in less than 3% of patients with pancreatic ductal adenocarcinomas (PDAC). To make an immune treatment that is effective for more cancer patients and those with PDAC specifically, we genetically engineered Salmonella to deliver exogenous antigens directly into the cytoplasm of tumor cells. We hypothesized that intracellular delivery of an exogenous immunization antigen would activate antigen-specific CD8 T cells and reduce tumors in immunized mice. Methods: To test this hypothesis, we administered intracellular delivering (ID) Salmonella that deliver ovalbumin as a model antigen into tumor-bearing, ovalbumin-vaccinated mice. ID Salmonella delivers antigens by autonomously lysing in cells after the induction of cell invasion. Results: We showed that the delivered ovalbumin disperses throughout the cytoplasm of cells in culture and in tumors. This delivery into the cytoplasm is essential for antigen cross-presentation. We showed that co-culture of ovalbumin-recipient cancer cells with ovalbumin-specific CD8 T cells triggered a cytotoxic T cell response. After the adoptive transfer of OT-I CD8 T cells, intracellular delivery of ovalbumin reduced tumor growth and eliminated tumors. This effect was dependent on the presence of the ovalbumin-specific T cells. Following vaccination with the exogenous antigen in mice, intracellular delivery of the antigen cleared 43% of established KPC pancreatic tumors, increased survival, and prevented tumor re-implantation. Discussion: This response in the immunosuppressive KPC model demonstrates the potential to treat tumors that do not respond to checkpoint inhibitors, and the response to re-challenge indicates that new immunity was established against intrinsic tumor antigens. In the clinic, ID Salmonella could be used to deliver a protein antigen from a childhood immunization to refocus pre-existing T cell immunity against tumors. As an off-the-shelf immunotherapy, this bacterial system has the potential to be effective in a broad range of cancer patients

The Effect of Dietary Tartrazine on Brain Dopamine and the Behavioral Symptoms of Attention Deficit Hyperactivity Disorder

Attention Deficit Hyperactivity Disorder is a neurodevelopmental disorder correlated with a decrease in brain dopamine and an increase in behavioral symptoms of hyperactivity and impulsivity. This experiment explored how tartrazine (Yellow #5) impacts these symptoms. After tartrazine administration to Spontaneously Hypertensive Rats (SHR), dopamine concentrations in regions of brain tissue were measured using Enzyme-Linked Immunosorbent Assay analysis. Behavioral testing with a T-maze and open field test measured impulsivity and hyperactivity, respectively. Results indicate that dietary tartrazine increases hyperactive behaviors in the SHR. However, results do not indicate a relationship between dietary tartrazine and brain dopamine. No conclusions regarding the relationship between dietary tartrazine and impulsivity were drawn

Intracellular delivery of protein drugs with an autonomously lysing bacterial system reduces tumor growth and metastases

Critical cancer pathways often cannot be targeted because of limited efficiency crossing cell membranes. Here we report the development of a Salmonella-based intracellular delivery system to address this challenge. We engineer genetic circuits that (1) activate the regulator flhDC to drive invasion and (2) induce lysis to release proteins into tumor cells. Released protein drugs diffuse from Salmonella containing vacuoles into the cellular cytoplasm where they interact with their therapeutic targets. Control of invasion with flhDC increases delivery over 500 times. The autonomous triggering of lysis after invasion makes the platform self-limiting and prevents drug release in healthy organs. Bacterial delivery of constitutively active caspase-3 blocks the growth of hepatocellular carcinoma and lung metastases, and increases survival in mice. This success in targeted killing of cancer cells provides critical evidence that this approach will be applicable to a wide range of protein drugs for the treatment of solid tumors

ENGINEERING SALMONELLA AS A DELIVERY VEHICLE FOR NUCLEAR ACTING ANTI-CANCER THERAPY

The greatest opportunity for the treatment of cancer lies with intranuclear protein and localized viral delivery. Conventional macromolecular therapies fail to selectively accumulate in tumors, are membrane impermeable, and inactive in the nucleus. The only clinical viral therapy is approved for intratumoral injection as the virus is cleared prior to colonization. As such, an intranuclear delivery vehicle is needed to overcome the limitations faced by traditional therapies. Salmonella is a beneficial delivery vehicle for anti-cancer therapies. Non-pathogenic Salmonella colonize and grow within tumors at ratios greater than ten thousand to one over other organs. The highly motile bacteria invade epithelial cells and activate a set of genes to control for intracellular survival. Salmonella have been engineered to lyse intracellularly and deliver protein therapeutics to the cytoplasm of cancerous cells. This thesis had two purposes: (1) to demonstrate that bacteria can deliver protein and plasmid DNA to the nucleus of cancerous cells for therapeutic effect and (2) increase the safety of therapeutic bacteria by utilizing a failsafe genetic circuit. We hypothesized that bacterially delivered proteins reach the nucleus for a therapeutic effect and that a delivered plasmid can express a virus. To test this, bacteria were engineered to release protein, and the location of said protein was determined using cell-based infection assays. To test the delivery of plasmid, both GFP and a viral genome were placed on a plasmid and delivered to cancer cells. Delivered plasmids resulted in protein expression by the cancer cells. Salmonella delivery of protein and plasmid reach the nucleus for therapeutic effect and protein expression. To enhance the safety of therapeutic non-pathogenic Salmonella, we hypothesized that the creation of a failsafe genetic circuit would enable the clearance of bacteria following treatment. To test this hypothesis, we engineered a genetic circuit that controls the expression of a vital bacterial gene, aspartate semialdehyde dehydrogenase, under the control of a small molecule inducer, sodium propionate. In the presence of sodium propionate, Salmonella grow and replicate; when sodium propionate is removed, bacteria lyse. These results demonstrates that an engineered genetic circuit can control the growth of Salmonella to ensure bacterial clearance following treatment to prevent subsequent infection

Intracellular delivery of oncolytic viruses with engineered Salmonella causes viral replication and cell death

Summary: As therapies, oncolytic viruses regress tumors and have the potential to induce antitumor immune responses that clear hard-to-treat and late-stage cancers. Despite this promise, clearance from the blood prevents treatment of internal solid tumors. To address this issue, we developed virus-delivering Salmonella (VDS) to carry oncolytic viruses into cancer cells. The VDS strain contains the PsseJ-lysE delivery circuit and has deletions in four homologous recombination genes (ΔrecB, ΔsbcB, ΔsbcCD, and ΔrecF) to preserve essential hairpins in the viral genome required for replication and infectivity. VDS delivered the genome for minute virus of mice (MVMp) to multiple cancers, including breast, pancreatic, and osteosarcoma. Viral delivery produced functional viral particles that are cytotoxic and infective to neighboring cells. The release of mature virions initiated new rounds of infection and amplified the infection. Using Salmonella for delivery will circumvent the limitations of oncolytic viruses and will provide a new therapy for many cancers

Centering Digital Health Equity During Technology Innovation: Protocol for a Comprehensive Scoping Review of Evidence-Based Tools and Approaches

BackgroundIn the rush to develop health technologies for the COVID-19 pandemic, the unintended consequence of digital health inequity or the inability of priority communities to access, use, and receive equal benefits from digital health technologies was not well examined. ObjectiveThis scoping review will examine tools and approaches that can be used during digital technology innovation to improve equitable inclusion of priority communities in the development of digital health technologies. The results from this study will provide actionable insights for professionals in health care, health informatics, digital health, and technology development to proactively center equity during innovation. MethodsBased on the Arksey and O’Malley framework, this scoping review will consider priority communities’ equitable involvement in digital technology innovation. Bibliographic databases in health, medicine, computing, and information sciences will be searched. Retrieved citations will be double screened against the inclusion and exclusion criteria using Covidence (Veritas Health Innovation). Data will be charted using a tailored extraction tool and mapped to a digital health innovation pathway defined by the Centre for eHealth Research roadmap for eHealth technologies. An accompanying narrative synthesis will describe the outcomes in relation to the review’s objectives. ResultsThis scoping review is currently in progress. The search of databases and other sources returned a total of 4868 records. After the initial screening of titles and abstracts, 426 studies are undergoing dual full-text review. We are aiming to complete the full-text review stage by May 30, 2024, data extraction in October 2024, and subsequent synthesis in December 2024. Funding was received on October 1, 2023, from the Centre for Health Equity Incubator Grant Scheme, University of Melbourne, Australia. ConclusionsThis paper will identify and recommend a series of validated tools and approaches that can be used by health care stakeholders and IT developers to produce equitable digital health technology across the Centre for eHealth Research roadmap. Identified evidence gaps, possible implications, and further research will be discussed. International Registered Report Identifier (IRRID)DERR1-10.2196/5385

Transcription-controlled gene therapy against tumor angiogenesis

A major drawback of current approaches to antiangiogenic gene therapy is the lack of tissue-specific targeting. The aim of this work was to trigger endothelial cell–specific apoptosis, using adenoviral vector–mediated delivery of a chimeric death receptor derived from the modified endothelium-specific pre-proendothelin-1 (PPE-1) promoter. In the present study, we constructed an adenovirus-based vector that targets tumor angiogenesis. Transcriptional control was achieved by use of a modified endothelium-specific promoter. Expression of a chimeric death receptor, composed of Fas and TNF receptor 1, resulted in specific apoptosis of endothelial cells in vitro and sensitization of cells to the proapoptotic effect of TNF-α. The antitumoral activity of the vectors was assayed in two mouse models. In the model of B16 melanoma, a single systemic injection of virus to the tail vein caused growth retardation of tumor and reduction of tumor mass with central tumor necrosis. When the Lewis lung carcinoma lung-metastasis model was applied, i.v. injection of vector resulted in reduction of lung-metastasis mass, via an antiangiogenic mechanism. Moreover, by application of the PPE-1–based transcriptional control, a humoral immune response against the transgene was avoided. Collectively, these data provide evidence that transcriptionally controlled, angiogenesis-targeted gene therapy is feasible