High-Specificity CRISPR-Mediated Genome Engineering in Anti-BCMA Allogeneic CAR T Cells Suppresses Allograft Rejection in Preclinical Models.

Allogeneic chimeric antigen receptor (CAR) T cell therapies hold the potential to overcome many of the challenges associated with patient-derived (autologous) CAR T cells. Key considerations in the development of allogeneic CAR T cell therapies include prevention of graft-vs-host disease (GvHD) and suppression of allograft rejection. Here, we describe preclinical data supporting the ongoing first-in-human clinical study, the CaMMouflage trial (NCT05722418), evaluating CB-011 in patients with relapsed/refractory multiple myeloma. CB-011 is a hypoimmunogenic, allogeneic anti-B-cell maturation antigen (BCMA) CAR T cell therapy candidate. CB-011 cells feature 4 genomic alterations and were engineered from healthy donor-derived T cells using a Cas12a CRISPR hybrid RNA-DNA (chRDNA) genome-editing technology platform. To address allograft rejection, CAR T cells were engineered to prevent endogenous HLA class I complex expression and overexpress a single-chain polyprotein complex composed of beta-2 microglobulin (B2M) tethered to HLA-E. In addition, T-cell receptor (TCR) expression was disrupted at the TCR alpha constant locus in combination with the site-specific insertion of a humanized BCMA-specific CAR. CB-011 cells exhibited robust plasmablast cytotoxicity in vitro in a mixed lymphocyte reaction in cell cocultures derived from patients with multiple myeloma. In addition, CB-011 cells demonstrated suppressed recognition by and cytotoxicity from HLA-mismatched T cells. CB-011 cells were protected from natural killer cell-mediated cytotoxicity in vitro and in vivo due to endogenous promoter-driven expression of B2M-HLA-E. Potent antitumor efficacy, when combined with an immune-cloaking armoring strategy to dampen allograft rejection, offers optimized therapeutic potential in multiple myeloma. See related Spotlight by Caimi and Melenhorst, p. 385.

Signalling by senescent melanocytes hyperactivates hair growth.

Niche signals maintain stem cells in a prolonged quiescence or transiently activate them for proper regeneration1. Altering balanced niche signalling can lead to regenerative disorders. Melanocytic skin nevi in human often display excessive hair growth, suggesting hair stem cell hyperactivity. Here, using genetic mouse models of nevi2,3, we show that dermal clusters of senescent melanocytes drive epithelial hair stem cells to exit quiescence and change their transcriptome and composition, potently enhancing hair renewal. Nevus melanocytes activate a distinct secretome, enriched for signalling factors. Osteopontin, the leading nevus signalling factor, is both necessary and sufficient to induce hair growth. Injection of osteopontin or its genetic overexpression is sufficient to induce robust hair growth in mice, whereas germline and conditional deletions of either osteopontin or CD44, its cognate receptor on epithelial hair cells, rescue enhanced hair growth induced by dermal nevus melanocytes. Osteopontin is overexpressed in human hairy nevi, and it stimulates new growth of human hair follicles. Although broad accumulation of senescent cells, such as upon ageing or genotoxic stress, is detrimental for the regenerative capacity of tissue4, we show that signalling by senescent cell clusters can potently enhance the activity of adjacent intact stem cells and stimulate tissue renewal. This finding identifies senescent cells and their secretome as an attractive therapeutic target in regenerative disorders.

Allogeneic chimeric antigen receptor-T cells with CRISPR-disrupted programmed death-1 checkpoint exhibit enhanced functional fitness.

BACKGROUND AIMS: Therapeutic disruption of immune checkpoints has significantly advanced the armamentarium of approaches for treating cancer. The prominent role of the programmed death-1 (PD-1)/programmed death ligand-1 axis for downregulating T cell function offers a tractable strategy for enhancing the disease-modifying impact of CAR-T cell therapy. METHODS: To address checkpoint interference, primary human T cells were genome edited with a next-generation CRISPR-based platform (Cas9 chRDNA) by knockout of the PDCD1 gene encoding the PD-1 receptor. Site-specific insertion of a chimeric antigen receptor specific for CD19 into the T cell receptor alpha constant locus was implemented to drive cytotoxic activity. RESULTS: These allogeneic CAR-T cells (CB-010) promoted longer survival of mice in a well-established orthotopic tumor xenograft model of a B cell malignancy compared with identically engineered CAR-T cells without a PDCD1 knockout. The persistence kinetics of CB-010 cells in hematologic tissues versus CAR-T cells without PDCD1 disruption were similar, suggesting the robust initial debulking of established tumor xenografts was due to enhanced functional fitness. By single-cell RNA-Seq analyses, CB-010 cells, when compared with identically engineered CAR-T cells without a PDCD1 knockout, exhibited fewer Treg cells, lower exhaustion phenotypes and reduced dysfunction signatures and had higher activation, glycolytic and oxidative phosphorylation signatures. Further, an enhancement of mitochondrial metabolic fitness was observed, including increased respiratory capacity, a hallmark of less differentiated T cells. CONCLUSIONS: Genomic PD-1 checkpoint disruption in the context of allogeneic CAR-T cell therapy may provide a compelling option for treating B lymphoid malignancies.

Immediate myeloid depot for SARS-CoV-2 in the human lung.

In the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, considerable focus has been placed on a model of viral entry into host epithelial populations, with a separate focus upon the responding immune system dysfunction that exacerbates or causes disease. We developed a precision-cut lung slice model to investigate very early host-viral pathogenesis and found that SARS-CoV-2 had a rapid and specific tropism for myeloid populations in the human lung. Infection of alveolar macrophages was partially dependent upon their expression of ACE2, and the infections were productive for amplifying virus, both findings which were in contrast with their neutralization of another pandemic virus, Influenza A virus (IAV). Compared to IAV, SARS-CoV-2 was extremely poor at inducing interferon-stimulated genes in infected myeloid cells, providing a window of opportunity for modest titers to amplify within these cells. Endotracheal aspirate samples from humans with the acute respiratory distress syndrome (ARDS) from COVID-19 confirmed the lung slice findings, revealing a persistent myeloid depot. In the early phase of SARS-CoV-2 infection, myeloid cells may provide a safe harbor for the virus with minimal immune stimulatory cues being generated, resulting in effective viral colonization and quenching of the immune system.

Immediate myeloid depot for SARS-CoV-2 in the human lung.

In the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic1, considerable focus has been placed on a model of viral entry into host epithelial populations, with a separate focus upon the responding immune system dysfunction that exacerbates or causes disease. We developed a precision-cut lung slice model2,3 to investigate very early host-viral pathogenesis and found that SARS-CoV-2 had a rapid and specific tropism for myeloid populations in the human lung. Infection of alveolar macrophages was partially dependent upon their expression of ACE2, and the infections were productive for amplifying virus, both findings which were in contrast with their neutralization of another pandemic virus, Influenza A virus (IAV). Compared to IAV, SARS-CoV-2 was extremely poor at inducing interferon-stimulated genes in infected myeloid cells, providing a window of opportunity for modest titers to amplify within these cells. Endotracheal aspirate samples from humans with the acute respiratory distress syndrome (ARDS) from COVID-19 confirmed the lung slice findings, revealing a persistent myeloid depot. In the early phase of SARS-CoV-2 infection, myeloid cells may provide a safe harbor for the virus with minimal immune stimulatory cues being generated, resulting in effective viral colonization and quenching of the immune system.

Developing a granular scale environmental burden index (EBI) for diverse land cover types across the contiguous United States.

Critical to identifying the risk of environmentally driven disease is an understanding of the cumulative impact of environmental conditions on human health. Here we describe the methodology used to develop an environmental burden index (EBI). The EBI is calculated at U.S. census tract level, a finer scale than many similar national-level tools. EBI scores are also stratified by tract land cover type as per the National Land Cover Database (NLCD), controlling for urbanicity. The EBI was developed over the course of four stages: 1) literature review to identify potential indicators, 2) data source acquisition and indicator variable construction, 3) index creation, and 4) stratification by land cover type. For each potential indicator, data sources were assessed for completeness, update frequency, and availability. These indicators were: (1) particulate matter (PM2.5), (2) ozone, (3) Superfund National Priority List (NPL) locations, (4) Toxics Release Inventory (TRI) facilities, (5) Treatment, Storage, and Disposal (TSD) facilities, (6) recreational parks, (7) railways, (8) highways, (9) airports, and (10) impaired water sources. Indicators were statistically normalized and checked for collinearity. For each indicator, we computed and summed percentile ranking scores to create an overall ranking for each tract. Tracts having the same plurality of land cover type form a 'peer' group. We re-ranked the tracts into percentiles within each peer group for each indicator. The percentile scores were combined for each tract to obtain a stratified EBI. A higher score reveals a tract with increased environmental burden relative to other tracts of the same peer group. We compared our results to those of related indices, finding good convergent validity between the overall EBI and CalEnviroScreen 4.0. The EBI has many potential applications for research and use as a tool to develop public health interventions at a granular scale.

GenX in water: Interactions and self-assembly.

2,3,3,3-tetrafluoro-2-(heptafluoropropoxy) propanoate, a.k.a. "GenX", is a surfactant introduced as a safer alternative to replace perfluorooctanoate (PFOA) in the manufacturing of fluorinated polymers, however, GenX is shown to cause adverse health effects similar to, or even worse than, those of the legacy PFOA. With an overarching goal to understand the behavior of GenX molecules in aqueous media, we report here on GenX micelle formation and structure in aqueous solutions, on the basis of results obtained from a combination of experimental techniques such as surface tension, fluorescence, viscosity, and small-angle neutron scattering (SANS), and molecular dynamics (MD) simulations. To our best knowledge, this is the first report on GenX micelles. The critical micelle concentration (CMC) of GenX ammonium salt in water is 175 mM. GenX forms small micelles with association number 6-8 and 10 Å radius. GenX molecules prefer to align along the micelle surface, and the ether oxygen of GenX has very little interaction with and exposure to water. Information on the surfactant and interfacial properties of GenX is crucial, since such properties are manifestations of interactions between GenX molecules and between GenX and water molecules and, in turn, the amphiphilic character of GenX dictates its fate and transport in the aqueous environment, its interactions with various biomolecules, and its binding to adsorbent materials.

Ranking United States University-Based General Surgery Programs on the Academic Achievement of Surgery Department Faculty.

OBJECTIVE: Rankings of residency programs are highly influential and utilized by residency applicants. Existing ranking resources often use opaque criteria that may include bias or do not accurately represent the academic achievement of current faculty. This study aims to create an updated general surgery residency ranking list based on the academic achievements of their respective surgery department faculty members. DESIGN: One hundred and six general surgery residency programs were selected from the American Medical Association Residency & Fellowship Programs Database. The names of faculty members affiliated with the departments of surgery were manually obtained. Lifetime and five-year h-indexes, a sum of grant awards from the National Institute of Health and Veterans Affairs, and a tally of journal editorial board positions were collected for the faculty. Metrics were compared among surgical departments, and the corresponding residency programs were ranked accordingly. SETTING: The study evaluated university-based general surgery residency programs in the United States from 2017 to 2019 via assessing their respective institutions' departments of surgery. PARTICIPANTS: A total of 7568 faculty members were evaluated. Faculty were required to be full-time, clinical surgeons to meet inclusion criteria. RESULTS: Based on a composite of all measured criteria, the top overall surgery department was at the University of Michigan. Massachusetts General Hospital had the highest lifetime and five-year h-indexes. Brigham and Women's Hospital had the most National Institute of Health funding, and the University of Pittsburgh Medical Center had the most Veterans Affairs funding. Washington University in St. Louis/Barnes Jewish Hospital had the most editorial board positions in their department. CONCLUSIONS: The academic success of departments of surgery was evaluated to develop a ranking list of general surgery residency programs. Through utilizing standardized methods and several measures of academic achievement, this comprehensive general surgery residency classification system will allow residency applicants to make more informed decisions.

Internal herniation of the cecum through the foramen of Winslow-a case report.

Foramen of Winslow hernias are a rare, but dangerous form of internal hernia that can present in individuals with signs and symptoms of bowel obstruction. This case report details operative management of a cecal herniation through the foramen of Winslow in an elderly male with no prior history of intra-abdominal surgery. The patient presented with worsening abdominal pain, nausea, vomiting and obstipation. Due to the clinical picture of a complete bowel obstruction and subsequent imaging findings, an urgent abdominal exploration was performed. During the procedure, the cecum was found to be ischemic and strangulated in the lesser sac, herniated through the foramen of Winslow. Following operative reduction and right hemicolectomy, it was decided to close the foramen of Winslow to prevent recurrence and future complications. The patient had an uncomplicated postoperative course with resolution of symptoms.

Patient-derived xenograft culture-transplant system for investigation of human breast cancer metastasis.

Metastasis is a fatal disease where research progress has been hindered by a lack of authentic experimental models. Here, we develop a 3D tumor sphere culture-transplant system that facilitates the growth and engineering of patient-derived xenograft (PDX) tumor cells for functional metastasis assays in vivo. Orthotopic transplantation and RNA sequencing (RNA-seq) analyses show that PDX tumor spheres maintain tumorigenic potential, and the molecular marker and global transcriptome signatures of native tumor cells. Tumor spheres display robust capacity for lentiviral engineering and dissemination in spontaneous and experimental metastasis assays in vivo. Inhibition of pathways previously reported to attenuate metastasis also inhibit metastasis after sphere culture, validating our approach for authentic investigations of metastasis. Finally, we demonstrate a new role for the metabolic enzyme NME1 in promoting breast cancer metastasis, providing proof-of-principle that our culture-transplant system can be used for authentic propagation and engineering of patient tumor cells for functional studies of metastasis.

Associations between Perceptions and Measures of Weather and Walking, United States-2015.

INTRODUCTION: Weather can be a barrier to walking. Understanding how perceptions of weather as a barrier and measured temperature are associated with walking can inform monitoring and promotion strategies. The objective of this study is to examine the association between perceptions of weather as a barrier to walking and measured weather with the volume of leisure and transportation walking. METHODS: The 2015 National Health Interview Survey (NHIS) assessed participation in and volume of walking (transportation, leisure) in the past week and frequency of reporting weather as a barrier to walking. Data were collected over the entire year. In 2019, we merged month-specific temperature data from the PRISM climate group with individual NHIS records. We examined associations using logistic (participation) and linear regression models (volume). RESULTS: Participation in walking increased as frequency of reporting weather as a barrier to walking decreased, from 'almost always' (transportation: 23%, leisure: 42%) to 'a little of the time' (transportation: 40%, leisure: 67%). Among adults reporting walking, walking volume increased as frequency of reporting weather as a barrier decreased from 'almost always' (transportation: 51 min/week, leisure: 64 min/week) to 'never' (transportation: 69 min/week, leisure: 98 min/week). Month-specific temperature was significantly associated with leisure walking with lower participation at the lowest and highest temperature quintiles, although the strength of the association differed by frequency of reporting weather as a barrier. CONCLUSIONS: In general, prevalence and volume of leisure and transportation walking decreased as the perception of weather as a barrier increased. Low and high temperature conditions were also associated with leisure walking participation, particularly among adults with increased perceptions of weather as a barrier. Our findings highlight the importance of including strategies to help adults overcome perceived and actual weather-related barriers in walking promotion efforts.

An in vitro vascularized micro-tumor model of human colorectal cancer recapitulates in vivo responses to standard-of-care therapy.

Around 95% of anti-cancer drugs that show promise during preclinical study fail to gain FDA-approval for clinical use. This failure of the preclinical pipeline highlights the need for improved, physiologically-relevant in vitro models that can better serve as reliable drug-screening and disease modeling tools. The vascularized micro-tumor (VMT) is a novel three-dimensional model system (tumor-on-a-chip) that recapitulates the complex human tumor microenvironment, including perfused vasculature, within a transparent microfluidic device, allowing real-time study of drug responses and tumor-stromal interactions. Here we have validated this microphysiological system (MPS) platform for the study of colorectal cancer (CRC), the second leading cause of cancer-related deaths, by showing that gene expression, tumor heterogeneity, and treatment responses in the VMT more closely model CRC tumor clinicopathology than current standard drug screening modalities, including 2-dimensional monolayer culture and 3-dimensional spheroids.

Transcriptional diversity and bioenergetic shift in human breast cancer metastasis revealed by single-cell RNA sequencing.

Although metastasis remains the cause of most cancer-related mortality, mechanisms governing seeding in distal tissues are poorly understood. Here, we establish a robust method for the identification of global transcriptomic changes in rare metastatic cells during seeding using single-cell RNA sequencing and patient-derived-xenograft models of breast cancer. We find that both primary tumours and micrometastases display transcriptional heterogeneity but micrometastases harbour a distinct transcriptome program conserved across patient-derived-xenograft models that is highly predictive of poor survival of patients. Pathway analysis revealed mitochondrial oxidative phosphorylation as the top pathway upregulated in micrometastases, in contrast to higher levels of glycolytic enzymes in primary tumour cells, which we corroborated by flow cytometric and metabolomic analyses. Pharmacological inhibition of oxidative phosphorylation dramatically attenuated metastatic seeding in the lungs, which demonstrates the functional importance of oxidative phosphorylation in metastasis and highlights its potential as a therapeutic target to prevent metastatic spread in patients with breast cancer.

Tumour heterogeneity and metastasis at single-cell resolution.

Tumours comprise a heterogeneous collection of cells with distinct genetic and phenotypic properties that can differentially promote progression, metastasis and drug resistance. Emerging single-cell technologies provide a new opportunity to profile individual cells within tumours and investigate what roles they play in these processes. This Review discusses key technological considerations for single-cell studies in cancer, new findings using single-cell technologies and critical open questions for future applications.

Harvesting Nannochloris oculata by inorganic electrolyte flocculation: effect of initial cell density, ionic strength, coagulant dosage, and media pH.

Process variables affecting harvesting efficiency of Nannochloris oculata by AlCl(3) flocculation such as, cell density, ionic strength, coagulant dosage, media pH, and cell surface charge were investigated. Initial cell density and coagulant dosage had a significant effect on the removal efficiency; however, levels of ionic strength tested were not significant. Best flocculation conditions of investigated variables were: 0.0016 ng of AlCl(3)/cell, 3.0×10(7) cell/mL, and pH 5.3. Removal efficiency at optimum conditions and salt concentrations of: 0, 15, and 30 g/L NaCl was 96, 98, and 97 %, respectively. Low cell density cultures ∼10(6) cell/mL, required five times greater AlCl(3) dosage to achieve the same removal efficiency. Destabilization of algal cultures using 0.0032 ng of AlCl(3)/cell was observed by reducing the zeta potential to -22 mV. Acidification with HCl for conducting flocculation at pH 5.3 could be a significant cost burden unless is mitigated by selecting a low-buffering-capacity media.

Effect of lambda-carrageenan-induced inflammatory pain on brain uptake of codeine and antinociception.

This study investigated the potential clinical implications of lambda-carrageenan-induced inflammatory pain on brain uptake of a commonly used analgesic, codeine, in relation to the fundamental properties of the blood-brain barrier (BBB) correlated to its antinociceptive profile over a 168-h time course. BBB uptake of [14C]sucrose (a membrane impermeant marker) and [3H]codeine were investigated using an in situ brain perfusion model in the rat. Results demonstrated a significantly increased brain uptake of [14C]sucrose at 1, 3, 6 and 48 h (139+/-9%, 166+/-19%, 138+/-13% and 146+/-7% compared with control, respectively) and [3H]codeine at 3 and 48 h (179+/-6% and 179+/-12% compared with control, respectively). Capillary depletion analyses ensured that increased radioisotope associated with the brain was due to increased uptake rather than trapping in the cerebral vasculature. Antinociception studies using a radiant-heat tail flick analgesia method demonstrated that lambda-carrageenan-induced inflammatory pain enhanced the in vivo antinociceptive profile of i.p.-administered codeine (7 mg/kg) at 3 and 48 h (144+/-11% and 155+/-9% compared with control, respectively). This study demonstrated that brain uptake and antinociception of codeine are increased during lambda-carrageenan-induced inflammatory pain, suggesting that the presence of inflammatory pain may be an important consideration in therapeutic drug dosing, potential adverse effects and/or neurotoxicity.