Induced pluripotent stem cell modeling of malaria

Malaria is one of the oldest parasitic diseases known to man, and the disease has played a role in shaping civilizations and the success of human populations over many centuries. While the malaria is well studied, it still remains a worldwide killer--claiming about 600,000 lives annually with children under the age of five representing a disproportionate population of those lethally infected. Malaria is caused by the protozoan parasite Plasmodium, which is introduced to the human body through the bite of a female Anopheles mosquito. The most lethal form of the disease is carried by the parasite Plasmodium falciparum, while the most widespread form of malaria is caused by Plasmodium vivax, the latter of which has a specific mode of entry and life cycle that makes it difficult to eradicate. The entry of P. vivax into human reticulocytes is based on the presence of the Duffy antigen chemokine receptor (DARC), which is uniquely absent in two-thirds of the Black population and populations of immediate African descent making it rare in the African region while endemic in Western and Asian countries. Inability to culture the parasite P. vivax in vitro and exhaustible tissue samples makes an accurate model of P. vivax malaria difficult to maintain ex vivo. The current study focuses on overcoming those limitations by modeling the mode of entry of P. vivax into patient-specific, induced pluripotent stem cell (iPSC)-derived erythrocyte-lineage cells by showing firstly that DARC is a measurable marker of susceptibility in vitro via FACS analysis, and that secondly, P. vivax cell culture limitations can be bypassed by creating a lentivirus designed to specifically infect DARC-expressing cells. To demonstrate the potency of this system, we show that a virus expressing the conserved region of the Duffy binding ligand, Duffy binding protein II (DBPII), can selectively infect peripheral blood mononuclear cells (PBMCs) that express DARC. Moreover, our current study focuses on the development of an iPSC-based disease model using patient samples derived from DARC expressing patients (DARC+) and DARC negative Sickle Cell Disease (SCD) patients (DARC-). We show that DARC+ iPSC-derived erythroid lineage cells express a transient population of DARC-expressing cells via FACS analysis, and we explore different protocols to stabilize this unique population. We hypothesize that DARC is a stage-specific marker for erythrocyte maturation, and we believe that any subset of cells expressing DARC consists of more mature erythrocyte-lineage cells. This study then, provides a novel platform by which to study malaria infection in a patient-specific manner while bypassing the limitations of culturing P. vivax in an in vitro culture system, as well as introducing a new way to measure erythrocyte maturation. Successful establishment of such a disease model has great implications for in-depth drug screenings for novel therapeutics that target the blood stage of the parasitic disease that were previously difficult to validate due to the limitations of currently existing models

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Specific recognition of an FGFR2 fusion by tumor infiltrating lymphocytes from a patient with metastatic cholangiocarcinoma

Background Metastatic cholangiocarcinoma (CC), a form of gastrointestinal cancer that originates from the bile ducts, cannot be cured by currently available therapies, and is associated with dismal prognosis. In a previous case report, adoptive transfer of autologous tumor infiltrating lymphocytes (TILs), the majority of which recognized a tumor-specific point mutation, led to a profound and durable cancer regression in a patient with metastatic CC. Thus, more effective treatment for patients with this disease may be developed by using TILs that target cancer-specific mutations, but also other genetic aberrations such as gene fusions. In this context, fusions that involve fibroblast growth factor receptor 2 (FGFR2) and function as oncogenes in a subset of patients with intrahepatic CC (ICC) represent particularly attractive targets for adoptive cell therapy. However, no study to date has explored whether FGFR2 fusions can be recognized by patients’ T cells.Method To address whether FGFR2 fusions can be recognized by patients’ T cells, we tested TILs from four patients with FGFR2 fusion-positive ICC for recognition of peptides and minigenes that represented the breakpoint regions of these fusions, which were unique to each of the four patients.Results We found that CD4+ TILs from one patient specifically recognized the breakpoint region of a unique FGFR2-TDRD1 (tudor domain-containing 1) fusion, and we isolated a T-cell receptor responsible for its recognition.Conclusions This finding suggests that FGFR2 fusion-reactive TILs can be isolated from some patients with metastatic ICC, and thus provides a rationale for future exploration of T cell-based therapy targeting FGFR2 fusions in patients with cancer. Furthermore, it augments the rationale for extending such efforts to other types of solid tumors hallmarked by oncogenic gene fusions

Neoantigen Identification and Response to Adoptive Cell Transfer in anti PD-1 Naïve and Experienced Patients with Metastatic Melanoma

Immune checkpoint blockade (ICB) agents and adoptive cell transfer (ACT) of tumor infiltrating lymphocytes (TIL) are prominent immunotherapies used for the treatment of advanced melanoma. Both therapies rely on activation of lymphocytes that target shared tumor antigens or neoantigens. Recent analysis of patients with metastatic melanoma who underwent treatment with TIL ACT at the National Cancer Institute (NCI) demonstrated decreased responses in patients previously treated with anti PD-1 agents. We aimed to find a basis for the difference in response rates between anti PD-1 naïve and experienced patients. We examined the tumor mutational burden (TMB) of resected tumors and the repertoire of neoantigens targeted by autologous TIL in a cohort of 112 anti PD-1 naïve and 69 anti PD-1 experienced patients. Anti PD-1 naïve patients were found to have higher TMBs (352.0 vs. 213.5, p = 0.005) and more neoantigens (2 vs. 1, p = 0.003) compared to anti PD-1 experienced patients. Among patients treated with TIL ACT, TMB and number of neoantigens identified were higher in ACT responders than ACT non-responders in both anti PD-1naïve and experienced patients. Among patients with comparable TMBs and predicted neoantigen loads, patients naïve to anti PD-1 therapy were more likely to have identifiable neoantigens and tumor specific lymphocytes in their treatment products (2.5 vs. 1, p = 0.02). These results indicate that decreases in TMB and targeted neoantigens only partially account for the difference in response to ACT and that additional factors likely influence responses in these patients

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field