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.

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.

Outbreak of Foodborne Botulism Associated with a Commercially Produced Multipack Potato Product, Colorado: September 2019.

During September 2019, public health authorities in El Paso County, Colorado, were notified of four patients who had presented to nearby hospitals with clinical features consistent with botulism, a paralytic illness caused by botulinum neurotoxin. One patient died soon after presentation; the other three patients required intensive care but recovered after receiving botulism antitoxin. Botulinum toxin type A was detected in serum from all patients. On further investigation, all four patients had shared a meal that included commercially prepared roasted potatoes from an individual package without refrigeration instructions that had been left unrefrigerated for 15 d. Storage of the product at ambient temperature likely allowed botulism spores to produce botulinum toxin, resulting in severe illness and death. The manufacturer improved labeling in response to this outbreak. Public health officials should consider unrefrigerated potato products as a potential source of botulism; clinicians should consider botulism as a possible cause of paralytic illness.

Health effects in people relocating between environments of differing ambient air pollution concentrations: A literature review.

People who relocate to a new environment may experience health effects from a change in ambient air pollution. We undertook a literature review of studies of such relocations and health effects and report the results as a narrative analysis. Fifteen articles of heterogeneous designs met the inclusion criteria. Four short-term (relocation duration less than six months) and three long-term (relocation duration six months or greater) studies reported evidence of the effect of relocation on physiological outcome, biomarkers or symptoms. All had potential weaknesses of design or analysis but, as a whole, their results are broadly consistent in suggesting short-term adverse effects of air pollutants or their reversibility. One long-term study provided evidence that changes in air pollution exposure during adolescence have a measurable effect on lung function growth. Four cohort studies were also identified that used relocation to strengthen evidence of air-pollution-exposure relationships by using a design that incorporates effective randomization of exposure or the use of relocation to improve exposure classification. However, three studies of relocation during pregnancy provided limited evidence to conclude an effect of relocation-related change in exposure on pregnancy outcome. Overall, most relocation studies are consistent with short- or long-term adverse effects of air pollution on biological function or mortality, but many studies of change in exposure have design weaknesses that limit the robustness of interpretation. We outline principles for improved design and analysis to help strengthen future studies for the insights they can provide from their quasi-experimental designs, including on the nature and timing of functional changes of relocation-related changes in exposure to ambient air pollution.

Mild Botulism From Illicitly Brewed Alcohol in a Large Prison Outbreak in Mississippi.

Botulism is typically described as a rapidly progressing, severe neuroparalytic disease. Foodborne botulism is transmitted through consuming food or drink that has been contaminated with botulinum toxin. During a botulism outbreak linked to illicitly brewed alcohol (also known as "hooch" or "pruno") in a prison, 11 (35%) of 31 inmates that consumed contaminated hooch had mild illnesses. This includes 2 inmates with laboratory confirmed botulism. The most frequently reported signs and symptoms among the 11 patients with mild illness included dry mouth (91%), hoarse voice (91%), difficulty swallowing (82%), fatigue (82%), and abdominal pain (82%). Foodborne botulism is likely underdiagnosed and underreported in patients with mild illness. Botulism should be considered on the differential diagnosis for patients with cranial nerve palsies.

Foodborne Botulism Outbreaks in the United States, 2001-2017.

Foodborne botulism is an intoxication caused by ingestion of food containing botulinum neurotoxin. Cases of foodborne botulism are usually sporadic (single, unrelated) but outbreaks of two or more cases occur. In this mini-review we will examine the following for the period 2001-2017, in the United States: botulism surveillance data, outbreaks of botulism affecting 10 or more people, and the public health preparedness and response approach.

Personal exposure monitoring of PM2.5 among US diplomats in Kathmandu during the COVID-19 lockdown, March to June 2020.

The 2019 Novel Coronavirus SARS-CoV 2 (COVID-191) pandemic has severely impacted global health, safety, economic development and diplomacy. The government of Nepal issued a lockdown order in the Kathmandu Valley for 80 days from 24 March to 11 June 2020. This paper reports associated changes in ambient PM2.5 measured at fixed-site monitors and changes in personal exposure to PM2.5 monitored by APT Minima by four American diplomats who completed monitoring before and during lockdown (24 h for each period per person, 192 person-hours in total). Time activities and use of home air pollution mitigation measures (use of room air cleaners (RACs), sealing of homes) were recorded by standardized diary. We compared PM2.5 exposure level by microenvironment (home (cooking), home (other activities), at work, commuting, other outdoor environment) in terms of averaged PM2.5 concentration and the contribution to cumulative personal exposure (the product of PM2.5 concentration and time spent in each microenvironment). Ambient PM2.5 measured at fixed-sites in the US Embassy and in Phora Durbar were 38.2% and 46.7% lower than during the corresponding period in 2017-2019. The mean concentration of PM2.5 to which US diplomats were exposed was very much lower than the concentrations of ambient levels measured at fixed site monitors in the city both before and during lockdown. Within-person comparisons suggest personal PM2.5 exposure was 50.0% to 76.7% lower during lockdown than before it. Time spent outdoors and cooking at home were large contributors to cumulative personal exposure. Low indoor levels of PM2.5 were achieved at work and home through use of RACs and measures to seal homes against the ingress of polluted air from outside. Our observations indicate the potential reduction in exposure to PM2.5 with large-scale changes to mainly fossil-fuel related emissions sources and through control of indoor environments and activity patterns.

Wound Botulism Among Persons Who Inject Black Tar Heroin in New Mexico, 2016.

Outbreaks of wound botulism are rare, but clinicians and health departments should maintain suspicion for signs, symptoms, and risk factors of wound botulism among persons who inject drugs in order to initiate treatment quickly. This report describes an outbreak of three wound botulism cases among persons in two adjacent counties who injected drugs. Provisional information about these cases was previously published in the CDC National Botulism Surveillance Summary. All three cases in this outbreak were laboratory-confirmed, including one case with detection of botulinum toxin type A in a wound culture sample taken 43 days after last possible heroin exposure. Findings highlight the delay in diagnosis which led to prolonged hospitalization and the persistence of botulinum toxin in one patient.

Synergistic action of substituted indole derivatives and clinically used antibiotics against drug-resistant bacteria.

Aim: The current report describes the discovery of indole derivatives that synergize with standard antibiotics. Materials & methods: The antibacterial activities were determined using an optimized time-kill method, while viability of mammalian cells was assessed using the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Results: The synergy is observed with methicillin- and vancomycin-resistant Staphylococcus aureus bacterial strains, against which the standard antibiotics show no activities of their own. Our indole derivatives in combination with antibiotics lack toxicity toward mammalian cells, do not promote the evolution of resistance of S. aureus in comparison to clinically established antibiotics, and likely work by permeabilizing bacterial cell membranes. Conclusion: The above-mentioned findings demonstrate the potential clinical applications of our indole derivatives.

Harnessing type I CRISPR-Cas systems for genome engineering in human cells.

Type I CRISPR-Cas systems are the most abundant adaptive immune systems in bacteria and archaea1,2. Target interference relies on a multi-subunit, RNA-guided complex called Cascade3,4, which recruits a trans-acting helicase-nuclease, Cas3, for target degradation5-7. Type I systems have rarely been used for eukaryotic genome engineering applications owing to the relative difficulty of heterologous expression of the multicomponent Cascade complex. Here, we fuse Cascade to the dimerization-dependent, non-specific FokI nuclease domain8-11 and achieve RNA-guided gene editing in multiple human cell lines with high specificity and efficiencies of up to ~50%. FokI-Cascade can be reconstituted via an optimized two-component expression system encoding the CRISPR-associated (Cas) proteins on a single polycistronic vector and the guide RNA (gRNA) on a separate plasmid. Expression of the full Cascade-Cas3 complex in human cells resulted in targeted deletions of up to ~200 kb in length. Our work demonstrates that highly abundant, previously untapped type I CRISPR-Cas systems can be harnessed for genome engineering applications in eukaryotic cells.

Photoactivated 2,3-distyrylindoles kill multi-drug resistant bacteria.

Compounds based on the 2,3-distyrylindole scaffold were found to exhibit bactericidal properties upon irradiation with white light. At the concentration of 1 µM, the lead compound 1 completely (ca. 109 CFU/mL) eradicated such Gram-positive organisms as S. aureus (MRSA, MSSA), E. faecalis (VRE), S. pyogenes and S. mutans when irradiated with white light for 2 min. At the concentration of 5 µM and in the presence of polymyxin E at non-bactericidal 1.25 µg/mL concentration, 1 also showed a 7-log to 9-log reductions in bacterial counts of such Gram-negative organisms as multi-drug resistant (MDR) A. baumannii, MDR P. aeruginosa, E. coli and Klebsiella pneumoniae (CRE: KPC and NDM-1), also when irradiated with white light for 2 min. The structure-activity relationship studies revealed that unsubstituted at benzene rings 2,3-distyrylindole 2 was most potent and gave a 5-order of magnitude eradication of a MRSA strain at the concentration of 30 nM upon irradiation with white light. Initial mechanistic experiments revealed the disruption of bacterial cell membrane, but indicated that singlet oxygen production, which is commonly associated with photodynamic therapy, may not play a role in the bactericidal effects of the 2,3-distyrylindoles.

Mapping the genomic landscape of CRISPR-Cas9 cleavage.

RNA-guided CRISPR-Cas9 endonucleases are widely used for genome engineering, but our understanding of Cas9 specificity remains incomplete. Here, we developed a biochemical method (SITE-Seq), using Cas9 programmed with single-guide RNAs (sgRNAs), to identify the sequence of cut sites within genomic DNA. Cells edited with the same Cas9-sgRNA complexes are then assayed for mutations at each cut site using amplicon sequencing. We used SITE-Seq to examine Cas9 specificity with sgRNAs targeting the human genome. The number of sites identified depended on sgRNA sequence and nuclease concentration. Sites identified at lower concentrations showed a higher propensity for off-target mutations in cells. The list of off-target sites showing activity in cells was influenced by sgRNP delivery, cell type and duration of exposure to the nuclease. Collectively, our results underscore the utility of combining comprehensive biochemical identification of off-target sites with independent cell-based measurements of activity at those sites when assessing nuclease activity and specificity.

Frequency of influenza H3N2 intra-subtype reassortment: attributes and implications of reassortant spread.

BACKGROUND: Increasing evidence suggests that influenza reassortment not only contributes to the emergence of new human pandemics but also plays an important role in seasonal influenza epidemics, disease severity, evolution, and vaccine efficacy. We studied this process within 2091 H3N2 full genomes utilizing a combination of the latest reassortment detection tools and more conventional phylogenetic analyses. RESULTS: We found that the amount of H3N2 intra-subtype reassortment depended on the number of sampled genomes, occurred with a steady frequency of 3.35%, and was not affected by the geographical origins, evolutionary patterns, or previous reassortment history of the virus. We identified both single reassortant genomes and reassortant clades, each clade representing one reassortment event followed by successful spread of the reassorted variant in the human population. It was this spread that was mainly responsible for the observed high presence of H3N2 intra-subtype reassortant genomes. The successfully spread variants were generally sampled within one year of their formation, highlighting the risk of their rapid spread but also presenting an opportunity for their rapid detection. Simultaneous spread of several different reassortant lineages was observed, and despite their limited average lifetime, second and third generation reassortment was detected, as well as reassortment between viruses belonging to different vaccine-associated clades, likely displaying differing antigenic properties. Some of the spreading reassortants remained confined to certain geographical regions, while others, sharing common properties in amino acid positions of the HA, NA, and PB2 segments, were found throughout the world. CONCLUSIONS: Detailed surveillance of seasonal influenza reassortment patterns and variant properties may provide unique information needed for prediction of spread and construction of future influenza vaccines.

Distribution of influenza and other acute respiratory viruses during the first year after the 2009-2010 influenza pandemic in the English- and Dutch-speaking Caribbean countries.

BACKGROUND: Limited specimen collection and testing for influenza occurred in the English and Dutch-speaking Caribbean countries prior to the 2009/2010 influenza pandemic. Caribbean Epidemiology Centre (CAREC) member countries rapidly mobilized to collect specimens during the pandemic and a vast majority of confirmed cases during the pandemic period were influenza A(H1N1)pdm09. OBJECTIVES: To describe the aetiology and distribution of acute respiratory illness (ARI) among laboratory confirmed cases during the first year after the 2009/2010 influenza pandemic in the English- and Dutch-speaking Caribbean. RESULTS: In total, 774 specimens were tested and 394 (52.7%) cases had positive laboratory confirmation. Respiratory syncytial virus (RSV) (28.4%) and influenza A(H3N2) (23.1%) were most frequently detected. RSV activity peaked in July 2011 while influenza A(H3N2) peaked in October 2010. Influenza was responsible for illness in greater numbers in persons 15-64 years while RSV was seen in primarily in children<5 years and adults>65 years. Other agents confirmed include rhinovirus (12.9%), influenza B (10.9%) and influenza A(H1N1)pdm09 (9.4%). CONCLUSIONS: RSV and influenza A(H3N2) were the most common viruses identified during the first year after the influenza A(H1N1)pdm09 pandemic. Influenza was detected every month with peak activity corresponding to that typically seen in North America (October to March). In order to determine the seasonality of influenza and RSV, laboratory data from subsequent years and increased specimen submission is needed.

The EDLL motif: a potent plant transcriptional activation domain from AP2/ERF transcription factors.

In plants, the ERF/EREBP family of transcriptional regulators plays a key role in adaptation to various biotic and abiotic stresses. These proteins contain a conserved AP2 DNA-binding domain and several uncharacterized motifs. Here, we describe a short motif, termed 'EDLL', that is present in AtERF98/TDR1 and other clade members from the same AP2 sub-family. We show that the EDLL motif, which has a unique arrangement of acidic amino acids and hydrophobic leucines, functions as a strong activation domain. The motif is transferable to other proteins, and is active at both proximal and distal positions of target promoters. As such, the EDLL motif is able to partly overcome the repression conferred by the AtHB2 transcription factor, which contains an ERF-associated amphiphilic repression (EAR) motif. We further examined the activation potential of EDLL by analysis of the regulation of flowering time by NF-Y (nuclear factor Y) proteins. Genetic evidence indicates that NF-Y protein complexes potentiate the action of CONSTANS in regulation of flowering in Arabidopsis; we show that the transcriptional activation function of CONSTANS can be substituted by direct fusion of the EDLL activation motif to NF-YB subunits. The EDLL motif represents a potent plant activation domain that can be used as a tool to confer transcriptional activation potential to heterologous DNA-binding proteins.

Cardiorespiratory instability before and after implementing an integrated monitoring system.

OBJECTIVES: Cardiorespiratory instability may be undetected in monitored step-down unit patients. We explored whether using an integrated monitoring system that continuously amalgamates single noninvasive monitoring parameters (heart rate, respiratory rate, blood pressure, and peripheral oxygen saturation) into AN instability index value (INDEX) correlated with our single-parameter cardiorespiratory instability concern criteria, and whether nurse response to INDEX alert for patient attention was associated with instability reduction. DESIGN: Prospective, longitudinal evaluation in sequential 8-, 16-, and 8-wk phases (phase I, phase II, and phase III, respectively). SETTING: A 24-bed trauma step-down unit in single urban tertiary care center. PATIENTS: All monitored patients. INTERVENTIONS: Phase I: Patients received continuous single-channel monitoring (heart rate, respiratory rate, blood pressure, and peripheral oxygen saturation) and standard care; INDEX background was recorded but not displayed. Phase II: INDEX was background-recorded; staff was educated on use. Phase III: Staff used a clinical response algorithm for INDEX alerts. MEASUREMENT AND MAIN RESULTS: Any monitored parameters even transiently beyond local cardiorespiratory instability concern triggers (heart rate of <40 or >140 beats/min, respiratory rate of <8 or >36 breaths/min, systolic blood pressure of <80 or >200 mm Hg, diastolic blood pressure of >110 mm Hg, and peripheral oxygen saturation of <85%) defined INSTABILITYmin. INSTABILITYmin further judged as both persistent and serious defined INSTABILITYfull. The INDEX alert states were defined as INDEXmin and INDEXfull by using same classification. Phase I and phase III admissions (323 vs. 308) and monitoring (18,258 vs. 18,314 hrs) were similar. INDEXmin and INDEXfull correlated significantly with INSTABILITYmin and INSTABILITYfull (r = .713 and r = .815, respectively, p < .0001). INDEXmin occurred before INSTABILITYmin in 80% of cases (mean advance time 9.4 ± 9.2 mins). Phase I and phase III admissions were similarly likely to develop INSTABILITYmin (35% vs. 33%), but INSTABILITYmin duration/admission decreased from phase I to phase III (p = .018). Both INSTABILITYfull episodes/admission (p = .03) and INSTABILITYfull duration/admission (p = .05) decreased in phase III. CONCLUSION: The integrated monitoring system INDEX correlated significantly with cardiorespiratory instability concern criteria, usually occurred before overt instability, and when coupled with a nursing alert was associated with decreased cardiorespiratory instability concern criteria in step-down unit patients.

Defining the incidence of cardiorespiratory instability in patients in step-down units using an electronic integrated monitoring system.

BACKGROUND: To our knowledge, detection of cardiorespiratory instability using noninvasive monitoring via electronic integrated monitoring systems (IMSs) in intermediate or step-down units (SDUs) has not been described. We undertook this study to characterize respiratory status in an SDU population, to define features of cardiorespiratory instability, and to evaluate an IMS index value that should trigger medical emergency team (MET) activation. METHODS: This descriptive, prospective, single-blinded, observational study evaluated all patients in a 24-bed SDU in a university medical center during 8 weeks from November 16, 2006, to January 11, 2007. An IMS (BioSign; OBS Medical, Carmel, Indiana) was inserted into the standard noninvasive hardwired monitoring system and used heart rate, blood pressure, respiratory rate, and peripheral oxygen saturation by pulse oximetry to develop a single neural networked signal, or BioSign Index (BSI). Data were analyzed for cardiorespiratory instability according to BSI trigger value and local MET activation criteria. Staff were blinded to BSI data collected in 326 patients (total census). RESULTS: Data for 18 248 hours of continuous monitoring were captured. Data for peripheral oxygen saturation by pulse oximetry were absent in 30% of monitored hours despite being a standard of care. Cardiorespiratory status in most patients (243 of 326 [74.5%]) was stable throughout their SDU stay, and instability in the remaining patients (83 of 326 [25%]) was exhibited infrequently. We recorded 111 MET activation criteria events caused by cardiorespiratory instability in 59 patients, but MET activation for this cause occurred in only 7 patients. All MET events were detected by BSI in advance (mean, 6.3 hours) in a bimodal distribution (>6 hours and < or =45 minutes). CONCLUSIONS: Cardiorespiratory instability, while uncommon and often unrecognized, was preceded by elevation of the IMS index. Continuous noninvasive monitoring augmented by IMS provides sensitive detection of early instability in patients in SDUs.