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.

Time and temperature abuse of milk in conditions representing a school cafeteria share table does not meaningfully reduce microbial quality.

Share tables (ST) are tables or stations in school cafeterias where students can return unopened foods and beverages, providing an opportunity to access these items at no cost. Currently, research suggests that milk is among the most wasted items in breakfast and lunch programs in the United States. Share tables present a simple solution for reducing milk waste, but research is needed to understand the microbial spoilage potential of milk in ST. To this end, uninoculated milk cartons and milk cartons inoculated with 2 to 3 log10(cfu/mL) Pseudomonas poae, a fast-growing psychrotroph, was exposed to ambient temperature during winter (mean temperature = 20.3°C) and summer (23.1°C) for 125 min, repeated over 5 d (the length of a school week). Microbial counts in the inoculated milk cartons increased linearly, exceeding the spoilage threshold of 6.0 log10(cfu/mL) after d 3 and after d 4 in the winter and summer season trials, respectively. In the winter trial, the microbial counts for uninoculated milk cartons never exceeded the lower limit of detection, 2.31 log10(cfu/mL), and in the summer trials, microbial counts never reached the spoilage threshold, indicating that initial contamination is a driving factor of microbial milk spoilage. Regardless of sharing status or seasonality, the greatest changes in counts for inoculated milk cartons occurred during overnight refrigeration, ranging from 0.56 to 1.4 log10(cfu/mL), while during the share table ranged from no observable change up to 0.29 log10(cfu/mL), emphasizing that school nutrition personnel should focus efforts on tightly controlling refrigeration temperatures and returning milk to refrigeration as soon as possible. A previously developed model for school cafeteria share tables was adapted to understand the typical residence time of milk in a simulated cafeteria with an ambient temperature share table for the summer and winter seasons over 1,000 wk. Milk was predicted to have a very short mean residence time (85 min) regardless of sharing status or season, with 99.8% of milk consumed, discarded, or donated within the first 2 d. As a result, only 3 out of 451,410 and 6 out of 451,410 simulated milks spoiled in the winter and summer seasons, respectively. The data generated here can be used to inform science-based decision-making for including milk in share tables, or applied to any system where one might have to accept short-term unrefrigerated storage of milk to meet a waste reduction or food security goal.

Modeling Preharvest Cyclospora cayetanensis Sampling and Testing for Various Water and Produce Sampling Plans.

As of August 2023, the two U.S. Food and Drug Administration (FDA) official detection methods for C. cayetanensis are outlined in the FDA Bacteriological Analytical Manual (BAM) Chapters 19b (produce testing) and 19c (agricultural water testing). These newly developed detection methods have been shown to not always detect contamination when present at low levels. Yet, industry and regulators may choose to use these methods as part of their monitoring and verification activities while detection methods continue to be improved. This study uses simulation to better understand the performance of these methods for various produce and water sampling plans. To do so, we used published FDA test validation data to fit a logistic regression model that predicts the methods' detection rate given the number of oocysts present in a 10-L agricultural water or 25 g produce sample. By doing so, we were able to determine contamination thresholds at which different numbers of samples (n = 1, 2, 4, 8, 16, and 32) would be adequate for detecting contamination. Furthermore, to evaluate sampling plans in use cases, a simulation was developed to represent C. cayetanensis contamination in agricultural water and on cilantro throughout a 45-day growth cycle. The model included uncertainty around the contamination sources, including scenarios of unintentionally contaminated irrigation water or in-field contamination. The results demonstrate that in cases where irrigation water was the contamination source, frequent water testing proved to be more powerful than produce testing. In scenarios where contamination occurred in-field, conducting frequent produce testing or testing produce toward the end of the season more reliably detected contamination. This study models the power of C. cayetanensis detection methods to understand the sampling plan performance and how these methods can be better used to monitor this emerging food safety hazard.

Simulation Evaluation of Power of Sampling Plans to Detect Cronobacter in Powdered Infant Formula Production.

Cronobacteris a hazard in Powdered Infant Formula (PIF) products that is hard to detect due to localized and low-level contamination. We adapted a previously published sampling simulation to PIF sampling and benchmarked industry-relevant sampling plans across different numbers of grabs, total sample mass, and sampling patterns. We evaluated performance to detect published Cronobacter contamination profiles for a recalled PIF batch [42% prevalence, -1.8 ± 0.7 log(CFU/g)] and a reference, nonrecalled, PIF batch [1% prevalence, -2.4 ± 0.8 log(CFU/g)]. Simulating a range of numbers of grabs [n = 1-22,000 (representing testing every finished package)] with 300 g total composite mass showed that taking 30 or more grabs detected contamination reliably (<1% median probability to accept the recalled batch). Benchmarking representative sampling plans ([n = 30, mass grab = 10g], [n = 30, m = 25g], [n = 60, m = 25g], [n = 180, m = 25g]) showed that all plans would reject the recalled batch (<1% median probability to accept) but would rarely reject the reference batch (>50% median probability of acceptance, all plans). Overall, (i) systematic or stratified random sampling patterns are equal to or more powerful than random sampling of the same sample size and total sampled mass, and, (ii) taking more samples, even if smaller, can increase the power to detect contamination.

Leafy Green Farm-to-Customer Process Model Predicts Product Testing Is Most Effective at Detecting Contamination When Conducted Early in the System before Effective Interventions.

Commercial leafy green supply chains often are required to have test and reject (sampling) plans for specific microbial adulterants at primary production or finished product packing for market access. To better understand the impact of this type of sampling, this study simulated the effect of sampling (from preharvest to consumer) and processing interventions (such as produce wash with antimicrobial chemistry) on the microbial adulterant load reaching the system endpoint (customer). This study simulated seven leafy green systems, an optimal system (all interventions), a suboptimal system (no interventions), and five systems where single interventions were removed to represent single process failures, resulting in 147 total scenarios. The all-interventions scenario resulted in a 3.4 log reduction (95% confidence interval [CI], 3.3 to 3.6) of the total adulterant cells that reached the system endpoint (endpoint TACs). The most effective single interventions were washing, prewashing, and preharvest holding, 1.3 (95% CI, 1.2 to 1.5), 1.3 (95% CI, 1.2 to 1.4), and 0.80 (95% CI, 0.73 to 0.90) log reduction to endpoint TACs, respectively. The factor sensitivity analysis suggests that sampling plans that happen before effective processing interventions (preharvest, harvest, and receiving) were most effective at reducing endpoint TACs, ranging between 0.05 and 0.66 log additional reduction compared to systems with no sampling. In contrast, sampling postprocessing (finished product) did not provide meaningful additional reductions to the endpoint TACs (0 to 0.04 log reduction). The model suggests that sampling used to detect contamination was most effective earlier in the system before effective interventions. Effective interventions reduce undetected contamination levels and prevalence, reducing a sampling plan's ability to detect contamination. IMPORTANCE This study addresses the industry and academic need to understand the effect of test-and-reject sampling within a farm-to-customer food safety system. The model developed looks at product sampling beyond the preharvest stage by assessing sampling at multiple stages. This study shows that individual interventions and combined interventions substantially reduce the total adulterant cells reaching the system endpoint. When effective interventions occur during processing, sampling at earlier stages (preharvest, harvest, receiving) has more power to detect incoming contamination than postprocessing sampling, as prevalence and contamination levels are lower. This study reiterates that effective food safety interventions are crucial for food safety. When product sampling is used to test and reject a lot as a preventive control, it may detect critically high incoming contamination. However, if contamination levels and prevalence are low, typical sampling plans will fail to detect contamination.

Using Qualitative Interviews to Better Understand Differences in How Local Health Departments Inspect School Share Tables.

ABSTRACT: Environmental and health advocates are increasingly promoting food donations to reduce landfilled food waste and feed hungry people. Share tables are locations where students can put unwanted school food or beverage items, allowing their uneaten food items to be "shared" with other students and providing food donation opportunities for the 4.9 billion lunches served annually in the U.S. National School Lunch Program. The purpose of this qualitative study was to identify differences in health inspector interpretations of the Food Code as it relates to share table operations and risk mitigation techniques preferred by inspectors for preventing foodborne illness from recovered food. A snowball sampling technique was used to identify Illinois health inspectors (n = 13) engaged in share table inspections. Telephone interviews were audio recorded and transcribed verbatim. The transcripts were coded using a hybrid process of deductive and inductive content analysis. Participants considered contamination, rather than temperature abuse, to be the primary risk factor for foodborne illness. Those participants with permissive Food Code interpretations considered contamination risk in the context of the overall school environment. Participants had the lowest degree of consensus on whether to allow whole apple recovery via a share table. Participants also lacked consensus on reuse of unclaimed share table items in future meal programs (reservice). This lack of consensus indicates that further research is needed to develop data-driven strategies to assess and manage the microbial risks associated with share tables and ultimately to facilitate increased food recovery.

The Aggression Questionnaire: a validation study in student samples.

The goal of this study was to examine the psychometric properties of the Aggression Questionnaire (AQ) in Spain. The AQ is a 29-item instrument designed to measure the different dimensions of the hostility/anger/aggression construct. It consists of 4 subscales that assess: (a) anger, (b) hostility, (c) verbal aggression, and (d) physical aggression. In Study 1, reliability, construct validity, and convergent validity were evaluated in a group of 384 male and female university students. Test-retest reliability was evaluated using a group of 154 male and female university students. The results of the factor analysis were similar to the scale structure claimed for this instrument. The subscales also showed internal consistency and stability over time. The AQ and its subscales were also compared with the scales and subscales of the Spielberger State-Trait Anger Expression Inventory (STAXI), the Cook-Medley Hostility Scale (Ho), the Buss-Durkee Hostility Inventory (BDHI), and the Jenkins Activity Survey-Form H (JASE-H). The results show that the AQ evaluates some aspects of anger, such as Anger-Trait and Anger-Out, rather than other elements, such as Anger-In or Anger-State. In Study 2, two new male groups were used to evaluate the criterion validity of the AQ: 57 prison inmates and 93 university students, finding that this instrument discriminated between the scores obtained by common offenders and university students.