Generation of allogeneic CAR-NKT cells from hematopoietic stem and progenitor cells using a clinically guided culture method.

Cancer immunotherapy with autologous chimeric antigen receptor (CAR) T cells faces challenges in manufacturing and patient selection that could be avoided by using 'off-the-shelf' products, such as allogeneic CAR natural killer T (AlloCAR-NKT) cells. Previously, we reported a system for differentiating human hematopoietic stem and progenitor cells into AlloCAR-NKT cells, but the use of three-dimensional culture and xenogeneic feeders precluded its clinical application. Here we describe a clinically guided method to differentiate and expand IL-15-enhanced AlloCAR-NKT cells with high yield and purity. We generated AlloCAR-NKT cells targeting seven cancers and, in a multiple myeloma model, demonstrated their antitumor efficacy, expansion and persistence. The cells also selectively depleted immunosuppressive cells in the tumor microenviroment and antagonized tumor immune evasion via triple targeting of CAR, TCR and NK receptors. They exhibited a stable hypoimmunogenic phenotype associated with epigenetic and signaling regulation and did not induce detectable graft versus host disease or cytokine release syndrome. These properties of AlloCAR-NKT cells support their potential for clinical translation.

Profiling ovarian cancer tumor and microenvironment during disease progression for cell-based immunotherapy design.

Ovarian cancer (OC) is highly lethal due to late detection and frequent recurrence. Initial treatments, comprising surgery and chemotherapy, lead to disease remission but are invariably associated with subsequent relapse. The identification of novel therapies and an improved understanding of the molecular and cellular characteristics of OC are urgently needed. Here, we conducted a comprehensive analysis of primary tumor cells and their microenvironment from 16 chemonaive and 10 recurrent OC patient samples. Profiling OC tumor biomarkers allowed for the identification of potential molecular targets for developing immunotherapies, while profiling the microenvironment yielded insights into its cellular composition and property changes between chemonaive and recurrent samples. Notably, we identified CD1d as a biomarker of the OC microenvironment and demonstrated its targeting by invariant natural killer T (iNKT) cells. Overall, our study presents a comprehensive immuno-profiling of OC tumor and microenvironment during disease progression, guiding the development of immunotherapies for OC treatment, especially for recurrent disease.

Foodborne Illness Outbreaks at Retail Food Establishments - National Environmental Assessment Reporting System, 25 State and Local Health Departments, 2017-2019.

Problem/Condition: Each year, state and local public health departments report hundreds of foodborne illness outbreaks associated with retail food establishments (e.g., restaurants or caterers) to CDC. Typically, investigations involve epidemiology, laboratory, and environmental health components. Health departments voluntarily report epidemiologic and laboratory data from their foodborne illness outbreak investigations to CDC through the National Outbreak Reporting System (NORS); however, minimal environmental health data from outbreak investigations are reported to NORS. This report summarizes environmental health data collected during outbreak investigations and reported to the National Environmental Assessment Reporting System (NEARS). Period Covered: 2017-2019. Description of System: In 2014, CDC launched NEARS to complement NORS surveillance and to use these data to enhance prevention efforts. State and local health departments voluntarily enter data from their foodborne illness outbreak investigations of retail food establishments into NEARS. These data include characteristics of foodborne illness outbreaks (e.g., etiologic agent and factors contributing to the outbreak), characteristics of establishments with outbreaks (e.g., number of meals served daily), and food safety policies in these establishments (e.g., ill worker policy requirements). NEARS is the only available data source that collects environmental characteristics of retail establishments with foodborne illness outbreaks. Results: During 2017-2019, a total of 800 foodborne illness outbreaks associated with 875 retail food establishments were reported to NEARS by 25 state and local health departments. Among outbreaks with a confirmed or suspected agent (555 of 800 [69.4%]), the most common pathogens were norovirus and Salmonella, accounting for 47.0% and 18.6% of outbreaks, respectively. Contributing factors were identified in 62.5% of outbreaks. Approximately 40% of outbreaks with identified contributing factors had at least one reported factor associated with food contamination by an ill or infectious food worker. Investigators conducted an interview with an establishment manager in 679 (84.9%) outbreaks. Of the 725 managers interviewed, most (91.7%) said their establishment had a policy requiring food workers to notify their manager when they were ill, and 66.0% also said these policies were written. Only 23.0% said their policy listed all five illness symptoms workers needed to notify managers about (i.e., vomiting, diarrhea, jaundice, sore throat with fever, and lesion with pus). Most (85.5%) said that their establishment had a policy restricting or excluding ill workers from working, and 62.4% said these policies were written. Only 17.8% said their policy listed all five illness symptoms that would require restriction or exclusion from work. Only 16.1% of establishments with outbreaks had policies addressing all four components relating to ill or infectious workers (i.e., policy requires workers to notify a manager when they are ill, policy specifies all five illness symptoms workers need to notify managers about, policy restricts or excludes ill workers from working, and policy specifies all five illness symptoms requiring restriction or exclusion from work). Interpretation: Norovirus was the most commonly identified cause of outbreaks reported to NEARS, and contamination of food by ill or infectious food workers contributed to approximately 40% of outbreaks with identified contributing factors. These findings are consistent with findings from other national outbreak data sets and highlight the role of ill workers in foodborne illness outbreaks. Although a majority of managers reported their establishment had an ill worker policy, often these policies were missing components intended to reduce foodborne illness risk. Contamination of food by ill or infectious food workers is an important cause of outbreaks; therefore, the content and enforcement of existing policies might need to be re-examined and refined. Public Health Action: Retail food establishments can reduce viral foodborne illness outbreaks by protecting food from contamination through proper hand hygiene and excluding ill or infectious workers from working. Development and implementation of policies that prevent contamination of food by workers are important to foodborne outbreak reduction. NEARS data can help identify gaps in food safety policies and practices, particularly those concerning ill workers. Future analyses of stratified data linking specific outbreak agents and foods with outbreak contributing factors can help guide the development of effective prevention approaches by describing how establishments' characteristics and food safety policies and practices relate to foodborne illness outbreaks.

Genetic engineering strategies to enhance antitumor reactivity and reduce alloreactivity for allogeneic cell-based cancer therapy.

The realm of cell-based immunotherapy holds untapped potential for the development of next-generation cancer treatment through genetic engineering of chimeric antigen receptor (CAR)-engineered T (CAR-T) cell therapies for targeted eradication of cancerous malignancies. Such allogeneic "off-the-shelf" cell products can be advantageously manufactured in large quantities, stored for extended periods, and easily distributed to treat an exponential number of cancer patients. At current, patient risk of graft-versus-host disease (GvHD) and host-versus-graft (HvG) allorejection severely restrict the development of allogeneic CAR-T cell products. To address these limitations, a variety of genetic engineering strategies have been implemented to enhance antitumor efficacy, reduce GvHD and HvG onset, and improve the overall safety profile of T-cell based immunotherapies. In this review, we summarize these genetic engineering strategies and discuss the challenges and prospects these approaches provide to expedite progression of translational and clinical studies for adoption of a universal cell-based cancer immunotherapy.

Characteristics associated with successful foodborne outbreak investigations involving United States retail food establishments (2014-2016).

This study examined relationships between foodborne outbreak investigation characteristics, such as the epidemiological methods used, and the success of the investigation, as determined by whether the investigation identified an outbreak agent (i.e. pathogen), food item and contributing factor. This study used data from the Centers for Disease Control and Prevention's (CDC) National Outbreak Reporting System and National Environmental Assessment Reporting System to identify outbreak investigation characteristics associated with outbreak investigation success. We identified investigation characteristics that increase the probability of successful outbreak investigations: a rigorous epidemiology investigation method; a thorough environmental assessment, as measured by number of visits to complete the assessment; and the collection of clinical samples. This research highlights the importance of a comprehensive outbreak investigation, which includes epidemiology, environmental health and laboratory personnel working together to solve the outbreak.

Development of an Empirically Derived Measure of Food Safety Culture in Restaurants.

A poor food safety culture has been described as an emerging risk factor for foodborne illness outbreaks, yet there has been little research on this topic in the retail food industry. The purpose of this study was to identify and validate conceptual domains around food safety culture and develop an assessment tool that can be used to assess food workers' perceptions of their restaurant's food safety culture. The study, conducted from March 2018 through March 2019, surveyed restaurant food workers for their level of agreement with 28 statements. We received 579 responses from 331 restaurants spread across eight different health department jurisdictions. Factor analysis and structural equation modeling supported a model composed of four primary constructs. The highest rated construct was Resource Availability (x¯=4.69, sd=0.57), which assessed the availability of resources to maintain good hand hygiene. The second highest rated construct was Employee Commitment (x¯=4.49, sd=0.62), which assessed workers' perceptions of their coworkers' commitment to food safety. The last two constructs were related to management. Leadership (x¯=4.28, sd=0.69) assessed the existence of food safety policies, training, and information sharing. Management Commitment (x¯=3.94, sd=1.05) assessed whether food safety was a priority in practice. Finally, the model revealed one higher-order construct, Worker Beliefs about Food Safety Culture (x¯=4.35, sd=0.53). The findings from this study can support efforts by the restaurant industry, food safety researchers, and health departments to examine the influence and effects of food safety culture within restaurants.

Graft-versus-Host Disease Modulation by Innate T Cells.

Allogeneic cell therapies, defined by genetically mismatched transplantation, have the potential to become a cost-effective solution for cell-based cancer immunotherapy. However, this type of therapy is often accompanied by the development of graft-versus-host disease (GvHD), induced by the mismatched major histocompatibility complex (MHC) between healthy donors and recipients, leading to severe complications and death. To address this issue and increase the potential for allogeneic cell therapies in clinical practice, minimizing GvHD is a crucial challenge. Innate T cells, encompassing subsets of T lymphocytes including mucosal-associated invariant T (MAIT) cells, invariant natural killer T (iNKT) cells, and gamma delta T (γδ T) cells, offer a promising solution. These cells express MHC-independent T-cell receptors (TCRs), allowing them to avoid MHC recognition and thus GvHD. This review examines the biology of these three innate T-cell populations, evaluates research on their roles in GvHD modulation and allogeneic stem cell transplantation (allo HSCT), and explores the potential futures for these therapies.

Mucosal-associated invariant T cells for cancer immunotherapy.

Human mucosal-associated invariant T (MAIT) cells are characterized by their expression of an invariant TCR α chain Vα7.2-Jα33/Jα20/Jα12 paired with a restricted TCR ß chain. MAIT cells recognize microbial peptides presented by the highly conserved MHC class I-like molecule MR1 and bridge the innate and acquired immune systems to mediate augmented immune responses. Upon activation, MAIT cells rapidly proliferate, produce a variety of cytokines and cytotoxic molecules, and trigger efficient antitumor immunity. Administration of a representative MAIT cell ligand 5-OP-RU effectively activates MAIT cells and enhances their antitumor capacity. In this review, we introduce MAIT cell biology and their importance in antitumor immunity, summarize the current development of peripheral blood mononuclear cell-derived and stem cell-derived MAIT cell products for cancer treatment, and discuss the potential of genetic engineering of MAIT cells for off-the-shelf cancer immunotherapy.

Fidgetin-like 2 depletion enhances cell migration by regulating GEF-H1, RhoA, and FAK.

The microtubule (MT) cytoskeleton and its dynamics play an important role in cell migration. Depletion of the microtubule-severing enzyme Fidgetin-like 2 (FL2), a regulator of MT dynamics at the leading edge of migrating cells, leads to faster and more efficient cell migration. Here we examine how siRNA knockdown of FL2 increases cell motility. Förster resonance energy transfer biosensor studies shows that FL2 knockdown decreases activation of the p21 Rho GTPase, RhoA, and its activator GEF-H1. Immunofluorescence studies reveal that GEF-H1 is sequestered by the increased MT density resulting from FL2 depletion. Activation of the Rho GTPase, Rac1, however, does not change after FL2 knockdown. Furthermore, FL2 depletion leads to an increase in focal adhesion kinase activation at the leading edge, as shown by immunofluorescence studies, but no change in actin dynamics, as shown by fluorescence recovery after photobleaching. We believe these results expand our understanding of the role of MT dynamics in cell migration and offer new insights into RhoA and Rac1 regulation.

Estimating the Prevalence of Hypercholesterolemia in Indigenous Populations: A Systematic Review and Meta-Analysis.

Background: Hypercholesterolemia is a common condition characterized by elevated levels of low-density lipoprotein cholesterol (LDL-C) and increased risk of atherosclerotic cardiovascular disease (ASCVD). Indigenous populations experience disproportionate rates of ASCVD, however, the extent to which hypercholesterolemia contributes to this burden is unknown. Objectives: This study aimed to estimate the prevalence of hypercholesterolemia, severe hypercholesterolemia, and familial hypercholesterolemia (FH) in Indigenous populations in Canada, the United States, Australia, and New Zealand. Methods: We searched MEDLINE, EMBASE, Web of Science, Native Health Database, Cochrane Central Register of Controlled Trials, and Cochrane Database of Systematic Reviews for peer-reviewed studies reporting on hypercholesterolemia and elevated LDL-C in Indigenous populations. All diagnostic criteria used to classify hypercholesterolemia were included. Pooled prevalence and 95% CIs were calculated using a random-effects model. Results: There were no studies reporting the prevalence of FH and one study reporting the prevalence of severe hypercholesterolemia in Indigenous populations. The pooled prevalence of hypercholesterolemia was 28.9% or ∼1 in 3 to 1 in 4 individuals (95% CI: 22.4%-36.4%) and 12.6% (95% CI: 7.7%-19.9%) using an LDL-C cutoff of ≥3.5 mmol/L (135 mg/dL). The pooled prevalence in Indigenous populations in North America was 24.3% (95% CI: 17.1%-33.3%) compared with 40.0% (95% CI: 31.3%-49.3%) in Australia. Meta-regression showed diabetes had a significant effect on prevalence (P = 0.022). Conclusions: Hypercholesterolemia is prevalent in Indigenous communities and may contribute to the high burden of ASCVD these populations face. There is insufficient research on FH and severe hypercholesterolemia in Indigenous populations worldwide.

Off-the-shelf third-party HSC-engineered iNKT cells for ameliorating GvHD while preserving GvL effect in the treatment of blood cancers.

Allo-HSCT is a curative therapy for hematologic malignancies owing to GvL effect mediated by alloreactive T cells; however, the same T cells also mediate GvHD, a severe side effect limiting the widespread application of allo-HSCT in clinics. Invariant natural killer T (iNKT) cells can ameliorate GvHD while preserving GvL effect, but the clinical application of these cells is restricted by their scarcity. Here, we report the successful generation of third-party HSC-engineered human iNKT (3rdHSC-iNKT) cells using a method combining HSC gene engineering and in vitro HSC differentiation. The 3rdHSC-iNKT cells closely resembled the CD4-CD8-/+ subsets of endogenous human iNKT cells in phenotype and functionality. These cells displayed potent anti-GvHD functions by eliminating antigen-presenting myeloid cells in vitro and in xenograft models without negatively impacting tumor eradication by allogeneic T cells in preclinical models of lymphoma and leukemia, supporting 3rdHSC-iNKT cells as a promising off-the-shelf cell therapy candidate for GvHD prophylaxis.

Tumor-Localized Administration of α-GalCer to Recruit Invariant Natural Killer T Cells and Enhance Their Antitumor Activity against Solid Tumors.

Invariant natural killer T (iNKT) cells have the capacity to mount potent anti-tumor reactivity and have therefore become a focus in the development of cell-based immunotherapy. iNKT cells attack tumor cells using multiple mechanisms with a high efficacy; however, their clinical application has been limited because of their low numbers in cancer patients and difficulties in infiltrating solid tumors. In this study, we aimed to overcome these critical limitations by using α-GalCer, a synthetic glycolipid ligand specifically activating iNKT cells, to recruit iNKT to solid tumors. By adoptively transferring human iNKT cells into tumor-bearing humanized NSG mice and administering a single dose of tumor-localized α-GalCer, we demonstrated the rapid recruitment of human iNKT cells into solid tumors in as little as one day and a significantly enhanced tumor killing ability. Using firefly luciferase-labeled iNKT cells, we monitored the tissue biodistribution and pharmacokinetics/pharmacodynamics (PK/PD) of human iNKT cells in tumor-bearing NSG mice. Collectively, these preclinical studies demonstrate the promise of an αGC-driven iNKT cell-based immunotherapy to target solid tumors with higher efficacy and precision.

Targeting Immunosuppressive Tumor-Associated Macrophages Using Innate T Cells for Enhanced Antitumor Reactivity.

The field of T cell-based and chimeric antigen receptor (CAR)-engineered T (CAR-T) cell-based antitumor immunotherapy has seen substantial developments in the past decade; however, considerable issues, such as graft-versus-host disease (GvHD) and tumor-associated immunosuppression, have proven to be substantial roadblocks to widespread adoption and implementation. Recent developments in innate immune cell-based CAR therapy have opened several doors for the expansion of this therapy, especially as it relates to allogeneic cell sources and solid tumor infiltration. This study establishes in vitro killing assays to examine the TAM-targeting efficacy of MAIT, iNKT, and γδT cells. This study also assesses the antitumor ability of CAR-engineered innate T cells, evaluating their potential adoption for clinical therapies. The in vitro trials presented in this study demonstrate the considerable TAM-killing abilities of all three innate T cell types, and confirm the enhanced antitumor abilities of CAR-engineered innate T cells. The tumor- and TAM-targeting capacity of these innate T cells suggest their potential for antitumor therapy that supplements cytotoxicity with remediation of tumor microenvironment (TME)-immunosuppression.

An Ex Vivo 3D Tumor Microenvironment-Mimicry Culture to Study TAM Modulation of Cancer Immunotherapy.

Tumor-associated macrophages (TAMs) accumulate in the solid tumor microenvironment (TME) and have been shown to promote tumor growth and dampen antitumor immune responses. TAM-mediated suppression of T-cell antitumor reactivity is considered to be a major obstacle for many immunotherapies, including immune checkpoint blockade and adoptive T/CAR-T-cell therapies. An ex vivo culture system closely mimicking the TME can greatly facilitate the study of cancer immunotherapies. Here, we report the development of a 3D TME-mimicry culture that is comprised of the three major components of a human TME, including human tumor cells, TAMs, and tumor antigen-specific T cells. This TME-mimicry culture can readout the TAM-mediated suppression of T-cell antitumor reactivity, and therefore can be used to study TAM modulation of T-cell-based cancer immunotherapy. As a proof-of-principle, the studies of a PD-1/PD-L1 blockade therapy and a MAO-A blockade therapy were performed and validated.

A Case of Clear Cell Hidradenoma Found During Abdominal Cyst Excision.

Clear cell hidradenoma (CCH), a rare yet benign finding, is a tumor that originates from sweat glands. It mainly presents as a slow-growing cystic nodule on the scalp or trunk. We report a case of a 60-year-old man who presented with an abdominal subcutaneous mass. This mass was excised and pathology confirmed the presence of CCH, with cytology negative for malignant cells. Although benign, CCHs cannot be confirmed as such until excision and pathologic analysis, which is the standard treatment of choice.

Major adverse cardiovascular events in homozygous familial hypercholesterolaemia: a systematic review and meta-analysis.

AIMS: Homozygous familial hypercholesterolaemia (HoFH) is a genetic condition characterized by extremely elevated levels of low-density lipoprotein cholesterol and premature atherosclerotic cardiovascular disease and death. Due to its rarity, accurate assessment of cardiovascular outcomes associated with HoFH and how they have changed over time has been challenging. The goal of this study was to assess the prevalence and age-of-onset of major adverse cardiovascular events (MACE) among patients with HoFH. METHODS AND RESULTS: We searched MEDLINE, EMBASE, Pubmed, Cochrane Central Register of Controlled Trials, Scopus, Africa-Wide, Google Scholar, Open Grey, and various clinical trial registries from inception to February 2020 to identify studies reporting on MACE in HoFH patients. We determined the pooled prevalence and mean age-of-onset of MACE outcomes individually using a random effects inverse variance model. We identified 94 studies that met our eligibility criteria. Myocardial infarction and coronary revascularization were common with a prevalence of 15.1% [95% confidence interval (95% CI) 10.7-20.0] and 28.3% (95% CI 22.5-34.3), respectively. The mean age-of-onset was 24.5 (95% CI 19.2-29.8) years for myocardial infarction and 32.2 (95% CI 26.6-37.8) years for revascularization. Sub-group analyses based on the year of publication revealed significant delays in the onset of MACE outcomes post-1990 compared to pre-1990. Egger's regression suggested possible bias, likely due to small study effects. CONCLUSIONS: Atherosclerotic cardiovascular disease is common among HoFH patients and occurs at a young age. Age-of-onset of myocardial infarction was delayed by more than a decade from pre-1990 to post-1990, likely attributable to widespread use of statins and other therapies, reflecting substantial progress in the management of this rare but severe disorder.

The design and rationale of the Advancing Cardiac Care Unit-based Rapid Assessment and Treatment of hypErcholesterolemia (ACCURATE) study.

Familial hypercholesterolemia (FH) is an inherited condition characterized by elevated low-density lipoprotein cholesterol (LDL-C) levels and premature atherosclerotic cardiovascular disease (ASCVD). Despite being the most common inherited cardiovascular disorder, it is still highly underdiagnosed and undertreated worldwide. We designed the Advancing Cardiac Care Unit-based Rapid Assessment and Treatment of hypErcholesterolemia (ACCURATE) study to test the hypothesis that opportunistic genetic testing for FH among patients hospitalized for acute coronary syndrome (ACS) will increase the diagnosis of FH and improve patient outcomes. ACCURATE is a non-randomized, controlled trial of patients <60 years old admitted to an acute cardiac unit with ACS and elevated LDL-C levels. The first cohort will consist of a control group of patients presenting with ACS who will be treated according to usual standard-of-care. The second cohort will consist of patients presenting with ACS in whom research-based genetic testing for FH will be performed during hospitalization and the results returned to the treating physicians. The primary endpoint will be the number of patients with a new diagnosis of FH. The secondary endpoints will be the proportion of patients who undergo intensification of lipid-lowering therapy, the lowest LDL-C level achieved, and the proportion of patients reaching guideline recommended lipid targets in the 12 months after the index ACS. To our knowledge, ACCURATE represents the first clinical trial of genetic testing for FH in the acute cardiac care setting and is expected to help identify optimal approaches to increase the diagnosis and treatment of FH.

Engineering stem cells for cancer immunotherapy.

Engineering stem cells presents an attractive paradigm for cancer immunotherapy. Stem cells engineered to stably express various chimeric antigen receptors (CARs) or T-cell receptors (TCRs) against tumor-associated antigens are showing increasing promise in the treatment of solid tumors and hematologic malignancies. Stem cells engraft for long-term immune cell generation and serve as a sustained source of tumor-specific effector cells to maintain remissions. Furthermore, engineering stem cells provides 'off-the-shelf' cellular products, obviating the need for a personalized and patient-specific product that plagues current autologous cell therapies. Herein, we summarize recent progress of stem cell-engineered cancer therapies, and discuss the utility, impact, opportunities, and challenges of cellular engineering that may facilitate the translational and clinical research.

Resection of an Asymptomatic Lymphangioma in a 76-Year-Old Male.

Lymphangiomas are benign congenital malformation comprised of the lymphatic system. They typically present in the head, neck, and axillary regions of children with <1% being described in the small bowel mesentery. We report a case of a 76-year-old man who presented with incidental large (9x6 cm) multiloculated cystic mass in the right upper quadrant (RUQ) on a CT scan performed for nephrolithiasis. He was asymptomatic at the presentation. We performed a diagnostic laparoscopy which was converted to an open procedure due to the mesenteric mass extending deeply toward the mesenteric root. The depth of invasion required small bowel resection with primary side-to-side anastomosis. Pathology confirmed a lymphangioma of the small bowel mesentery with histopathological analysis and cytology negative for malignant cells. Lymphangiomas are benign masses, however, their complete resection, including the resection of the involved organs is necessary. Incomplete resection or drainage is no longer used in management due to high rates of recurrence. Mesenteric lymphangiomas, while typically benign congenital malformations, can progress and impact surrounding structures via mass effect. Definitive treatment of lymphangiomas, even when asymptomatic, should be complete resection.

Restaurant Date-Marking Practices Concerning Ready-to-Eat Food Requiring Time and Temperature Control for Safety.

Certain foods are more vulnerable to foodborne pathogen growth and formation of toxins than others. Lack of time and temperature control for these foods can result in the growth of pathogens, such as Listeria monocytogenes, and lead to foodborne outbreaks. The Food and Drug Administration's (FDA) Food Code classifies these foods as time/temperature control for safety (TCS) foods and details safe cooking, holding, and storing temperatures for these foods. The FDA Food Code also includes a date-marking provision for ready-to-eat TCS foods that are held for >24 h. The provision states that these foods should not be held in refrigeration for >7 days and should be marked with the date or day by which the food should be "consumed on the premises, sold, or discarded." To learn more about restaurants' date-marking practices, the Centers for Disease Control and Prevention's Environmental Health Specialists Network (EHS-Net) conducted observations and manager interviews in 359 restaurants in 8 EHS-Net jurisdictions. Managers reported that they date marked ready-to-eat TCS foods more often than data collectors observed this practice (91% vs. 77%). Observation data showed almost a quarter of study restaurants did not date-mark ready-to-eat TCS foods. In addition, restaurants with an internal date-marking policy date marked 1.25 times more often than restaurants without such a policy and chain restaurants date marked 5.02 times more often than independently owned restaurants. These findings suggest that regulators and the retail food industry may improve food safety and lower the burden of foodborne illness in the United States if they target interventions to independent restaurants and encourage strong date-marking policies.