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IntroductionPirtobrutinib, a highly selective, noncovalent (reversible) Bruton tyrosine kinase inhibitor, has demonstrated promising efficacy in B-cell malignancies and is associated with low rates of discontinuation and dose reduction. Pirtobrutinib is administered until disease progression or toxicity, necessitating an understanding of the safety profile in patients with extended treatment.MethodsHere we report the safety of pirtobrutinib in patients with relapsed/refractory B-cell malignancies with extended (≥12 months) drug exposure from the BRUIN trial. Assessments included median time-to-first-occurrence of adverse events (AEs), dose reductions, and discontinuations due to treatment-emergent AEs (TEAEs) and select AEs of interest (AESIs).ResultsOf 773 patients enrolled, 326 (42%) received treatment for ≥12 months. In the extended exposure cohort, the median time-on-treatment was 19 months. The most common all-cause TEAEs were fatigue (32%) and diarrhea (31%). TEAEs leading to dose reduction occurred in 23 (7%) and discontinuations in 11 (3%) extended exposure patients. One patient had a fatal treatment-related AE (COVID-19 pneumonia). Infections (73.0%) were the most common AESI with a median time-to-first-occurrence of 7.4 months. Majority of TEAEs and AESIs occurred during the first year of therapy.ConclusionsPirtobrutinib therapy continues to demonstrate an excellent safety profile amenable to long-term administration without evidence of new or worsening toxicity signals.

BackgroundHousehold contact investigations are effective for finding tuberculosis (TB) cases but are hindered by low referral uptake for clinic-based evaluation and testing. We assessed the acceptability and feasibility of in-home testing of household contacts (HHC) using the GeneXpert Edge platform.MethodsWe conducted a 2-arm, randomized study in Eastern Cape, South Africa. HHCs were verbally assessed using the World Health Organization-recommended 4-symptom screen. Households with ≥1 eligible symptomatic contact were randomized. Intervention households received in-home GeneXpert MTB/RIF molecular testing. GeneXpert-positive HHCs were referred for clinic-based treatment. Standard-of-care households were referred for clinic-based sputum collection and testing. We defined acceptability as agreeing to in-home testing and feasibility as generation of valid Xpert MTB/RIF results. The proportion and timeliness of test results received was compared between groups.ResultsEighty-four households were randomized (n = 42 per arm). Of 100 eligible HHCs identified, 98/100 (98%) provided consent. Of 51 HHCs allocated to the intervention arm, all accepted in-home testing; of those, 24/51 (47%) were sputum productive and 23/24 (96%) received their test results. Of 47 HCCs allocated to standard-of-care, 7 (15%) presented for clinic-based TB evaluation, 6/47 (13%) were tested, and 4/6 (67%) returned for their results. The median (interquartile range) number of days from screening to receiving test results was 0 (0) and 16.5 (11-15) in the intervention and standard-of-care arms, respectively.ConclusionsIn-home testing for TB was acceptable, feasible, and increased HHCs with a molecular test result. In-home testing mitigates a major limitation of household contact investigations (dependency on clinic-based referral), revealing new strategies for enhancing early case detection.

e14613 Background: Immune checkpoint inhibitors (ICIs) have notably changed the landscape of systemic treatment of advanced and metastatic cancers in the last decade. We previously estimated the eligibility for and responses to ICIs, but since then, there have been multiple approvals for additional indications. As such, we have updated these estimates to include all approvals, as of December 2023. Methods: For eligibility, we used death data from the American Cancer Society’s Cancer Facts and Figures. Deaths was used as a stand-in for eligibility because these therapies are often given in later lines. Data from the peer-reviewed literature was used to further refine specific tumor type estimates (e.g., the percentage of lung cancers that are non-small cell and small cell lung cancer). For each tumor type, we multiplied the response rates reported in the FDA’s package insert by the number of deaths for each respective tumor type. When multiple drugs were approved for the same indication, we used the highest response rate. Results: Eleven ICI drugs were approved for 88 different indication approvals in the advanced/metastatic setting between 2015 and 2023. These approvals were for 20 general tumor types. The estimated eligibility for ICIs increased from 1.54% in 2011 to 55.47% in 2023. The tumor types with the highest eligibility in 2023 were non-small cell lung cancer with PDL1 status of 50% (13.28%) or less and PDL1 status >50% (4.42%), solid tumors TMB-h (6.81%), and hepatocellular (4.82%). The estimated response to ICIs increased from 0.14% in 2011 to 19.60% in 2023. The tumor types with the highest contribution to response estimates in 2023 were non-small cell lung cancer with PDL1 status of 50% (4.78%) or less and PDL1 status >50% (1.99%), solid tumors TMB-h (2.72%), and renal cell carcinoma (1.95%). Conclusions: We find that the estimated eligibility for and response to ICIs has increased since their introduction in 2011. Our estimates are an upper bound since many people are not eligible due to contraindications or healthcare coverage. Both the response and eligibility were driven by certain tumor types, including non-small cell lung cancer (either PDL1 high or PDL1 low). Patients with these types of tumors made up about one-third of the estimated response and eligibility. Solid tumors TMB-h, hepatocellular and renal cell carcinoma were other tumors with high eligibility and response, but other tumor types had low eligibility and response. While many patients have benefited from ICIs, half of patients with advanced or metastatic cancers are either not eligible or likely do not respond. [Table: see text]

PurposeIn 2013, afatinib was approved for non-small-cell lung cancer with subsequent indication expansion. We investigated published afatinib clinical trials to assess risk and benefit profiles for the drug in its approved indication of non-small-cell lung cancer as well as in off-label uses. Previous literature demonstrates excessive patient burden and limited benefit as afatinib has spread into more indications. A trial analysis is needed to establish efficacy and risk.MethodsIn this investigation, we screened literature databases and clinical trial registries for trials of afatinib as monotherapy or in combination interventions for cancer treatment. We extracted participant demographics, adverse event characteristics, as well as clinical and surrogate endpoints for each trial. Studies were deemed positive, negative, or indeterminate based on their achieving of primary endpoints as well as their safety.ResultsOur search yielded 2444 articles; we excluded 2352 articles for a final inclusion of 92 trials of 8859 patients. Our sample had 49 (53%) positive trials, 27 (29%) negative trials, and 16 (17%) indeterminate trials. The most common off-label indications for afatinib were breast cancer and squamous cell carcinoma of head and neck. The median OS for all trials was 8.4 months, median PFS 3.4 months, and the total ORR was 29.6%. Our study found that trials performed in disease states beyond the initial indications were largely negative with little patient benefit. The adverse events within our trial sample appear to be in line with expectations for toxicity.ImplicationsThese results are consistent with other studies that present similar findings, such as in Carlisle et al which indicate limited efficacy in nonapproved indications. Future trials should keep this potential evidence and patient burden in mind before initiation of those trials. This study contributes to the understanding of afatinib's risk-benefit profile across many clinical applications.

AimTo develop a non-invasive prenatal test for beta-hemoglobinopathies based on analyzing maternal plasma by using next generation sequencing.MethodsWe applied next generation sequencing (NGS) of maternal plasma to the non-invasive prenatal testing (NIPT) of autosomal recessive diseases, sickle cell disease and beta-thalassemia. Using the Illumina MiSeq, we sequenced plasma libraries obtained via a Twist Bioscience probe capture panel covering 4 Kb of chromosome 11, including the beta-globin (HBB) gene and >450 genomic single-nucleotide polymorphisms (SNPs) used to estimate the fetal fraction (FF). The FF is estimated by counting paternally transmitted allelic sequence reads present in the plasma but absent in the mother. We inferred fetal beta-globin genotypes by comparing the observed mutation (Mut) and reference (Ref) read ratios to those expected for the three possible fetal genotypes (Mut/Mut; Mut/Ref; Ref/Ref), based on the FF.ResultsWe bioinformatically enriched the FF by excluding reads over a specified length via in-silico size selection (ISS), favoring the shorter fetal reads, which increased fetal genotype prediction accuracy. Finally, we determined the parental HBB haplotypes, which allowed us to use the read ratios observed at linked SNPs to help predict the fetal genotype at the mutation site(s). We determined HBB haplotypes via Oxford Nanopore MinION sequencing of a 2.2 kb amplicon and aligned these sequences using Soft Genetics' NextGENe LR software.ConclusionThe combined use of ISS and HBB haplotypes enabled us to correctly predict fetal genotypes in cases where the prediction based on variant read ratios alone was incorrect.

BackgroundExaggerated responses to sensory stimuli, a hallmark of Fragile X syndrome (FXS), contribute to anxiety and learning challenges. Sensory hypersensitivity is recapitulated in the Fmr1 knockout (KO) mouse model of FXS. Recent studies in Fmr1 KO mice have demonstrated differences in activity of cortical interneurons and a delayed switch in the polarity of GABA signaling during development. Previously, we reported that blocking the chloride transporter NKCC1 with the diuretic bumetanide, could rescue synaptic circuit phenotypes in primary somatosensory cortex (S1) of Fmr1 KO mice. However, it remains unknown whether bumetanide can rescue earlier circuit phenotypes or sensory hypersensitivity in Fmr1 KO mice.MethodsWe used acute and chronic systemic administration of bumetanide in Fmr1 KO mice and performed in vivo 2-photon calcium imaging to record neuronal activity, while tracking mouse behavior with high-resolution videos.ResultsWe demonstrate that layer (L) 2/3 pyramidal neurons in S1 of Fmr1 KO mice show a higher frequency of synchronous events at postnatal day (P) 6 compared to wild-type controls. This was reversed by acute administration of bumetanide. Furthermore, chronic bumetanide treatment (P5-P14) restored S1 circuit differences in Fmr1 KO mice, including reduced neuronal adaptation to repetitive whisker stimulation, and ameliorated tactile defensiveness. Bumetanide treatment also rectified the reduced feedforward inhibition of L2/3 neurons in S1 and boosted the circuit participation of parvalbumin interneurons.ConclusionsThis further supports the notion that synaptic, circuit, and sensory behavioral phenotypes in Fmr1 KO can be mitigated by inhibitors of NKCC1, such as the FDA-approved diuretic bumetanide.

7009 Background: The phase 2 CAPTIVATE study evaluated first-line ibrutinib (Ibr) + venetoclax (Ven) for CLL/SLL in 2 cohorts: minimal residual disease (MRD)-guided randomized discontinuation (MRD cohort) and Fixed Duration (FD cohort). Ibr±Ven retreatment was allowed in patients (pts) who had progressive disease (PD). Here, we report outcomes for pts with high-risk genomic features from the FD cohort and retreatment outcomes in pts from the FD cohort and MRD cohort placebo arm. Methods: Pts aged ≤70 y with previously untreated CLL/SLL without restriction on genomic risk factors received 3 cycles of Ibr, then 12 cycles of Ibr+Ven (Ibr, 420 mg/d orally; Ven, 5-wk ramp up to 400 mg/d orally). On-study retreatment included single-agent Ibr (FD cohort or MRD cohort placebo arm); pts with PD >2 y after end of treatment (EOT) could be retreated with FD Ibr+Ven (FD cohort). Results: In the FD cohort (n=159) with a median follow-up of 61.2 mo (range, 0.8–66.3), 5-y PFS and OS rates (95% CI) were 67% (59–74) and 96% (91–98), respectively. 5-y PFS rates were higher in pts with undetectable MRD at 3 mo after EOT in peripheral blood (83%) or bone marrow (84%) vs those without (48% and 50%, respectively). 5-y PFS rates (95% CI) in pts with genomic risk factors were: del(17p)/mutated TP53 41% (21–59), complex karyotype 57% (37–72), del(11q) 64% (30–85), and unmutated IGHV 68% (50–80) (Table). In total, 18 second malignancies occurred in 13 pts (10 events in 8 pts during FD Ibr+Ven, 6 events in 4 pts after EOT and before retreatment, and 2 events in 2 pts during retreatment). Of 202 pts who completed Ibr+Ven (FD cohort, n=159; MRD cohort placebo arm, n=43), 63 have had PD to date; PD occurred >2 y after EOT in 43/63 pts (68%). 32/63 (51%) pts initiated retreatment with Ibr (n=25) or Ibr+Ven (n=7). With a median time on Ibr retreatment of 21.9 mo (range, 0.03–50.4), ORR was 86% in 22 evaluable pts (best response: 1 CR; 1 nodular PR; 17 PR; 2 SD; 1 PD [Richter transformation]). With a median time on Ibr+Ven retreatment of 13.8 mo (range, 3.7–15.1), ORR was 71% in 7 evaluable pts (best response: 1 CR; 4 PR; 1 PR with lymphocytosis; 1 SD). Conclusions: With up to 5.5 y of follow-up, FD Ibr+Ven continues to provide clinically meaningful PFS in pts with high-risk genomic features, as well as in the overall population. Ibr-based retreatment provides promising responses in pts needing subsequent therapy after the all-oral FD regimen of Ibr+Ven. Clinical trial information: NCT02910583 . [Table: see text]

Splicing factor 3b subunit 1 (SF3B1) is the largest subunit and core component of the spliceosome. Inhibition of SF3B1 was associated with an increase in broad intron retention (IR) on most transcripts, suggesting that IR can be used as a marker of spliceosome inhibition in chronic lymphocytic leukemia (CLL) cells. Furthermore, we separately analyzed exonic and intronic mapped reads on annotated RNA-sequencing transcripts obtained from B cells (n = 98 CLL patients) and healthy volunteers (n = 9). We measured intron/exon ratio to use that as a surrogate for alternative RNA splicing (ARS) and found that 66% of CLL-B cell transcripts had significant IR elevation compared with normal B cells (NBCs) and that correlated with mRNA downregulation and low expression levels. Transcripts with the highest IR levels belonged to biological pathways associated with gene expression and RNA splicing. A >2-fold increase of active pSF3B1 was observed in CLL-B cells compared with NBCs. Additionally, when the CLL-B cells were treated with macrolides (pladienolide-B), a significant decrease in pSF3B1, but not total SF3B1 protein, was observed. These findings suggest that IR/ARS is increased in CLL, which is associated with SF3B1 phosphorylation and susceptibility to SF3B1 inhibitors. These data provide additional support to the relevance of ARS in carcinogenesis and evidence of pSF3B1 participation in this process.

Intratumor heterogeneity underlies cancer evolution and treatment resistance, but targetable mechanisms driving intratumor heterogeneity are poorly understood. Meningiomas are the most common primary intracranial tumors and are resistant to all medical therapies, and high-grade meningiomas have significant intratumor heterogeneity. Here we use spatial approaches to identify genomic, biochemical and cellular mechanisms linking intratumor heterogeneity to the molecular, temporal and spatial evolution of high-grade meningiomas. We show that divergent intratumor gene and protein expression programs distinguish high-grade meningiomas that are otherwise grouped together by current classification systems. Analyses of matched pairs of primary and recurrent meningiomas reveal spatial expansion of subclonal copy number variants associated with treatment resistance. Multiplexed sequential immunofluorescence and deconvolution of meningioma spatial transcriptomes using cell types from single-cell RNA sequencing show decreased immune infiltration, decreased MAPK signaling, increased PI3K-AKT signaling and increased cell proliferation, which are associated with meningioma recurrence. To translate these findings to preclinical models, we use CRISPR interference and lineage tracing approaches to identify combination therapies that target intratumor heterogeneity in meningioma cell co-cultures.

TDP-43 proteinopathy is a salient neuropathologic feature in a subset of frontotemporal lobar degeneration (FTLD-TDP), in amyotrophic lateral sclerosis (ALS-TDP), and in limbic-predominant age-related TDP-43 encephalopathy neuropathologic change (LATE-NC), and is associated with hippocampal sclerosis of aging (HS-A). We examined TDP-43-related pathology data in the National Alzheimer's Coordinating Center (NACC) in two parts: (I) availability of assessments, and (II) associations with clinical diagnoses and other neuropathologies in those with all TDP-43 measures available. Part I: Of 4326 participants with neuropathology data collected using forms that included TDP-43 assessments, data availability was highest for HS-A (97%) and ALS (94%), followed by FTLD-TDP (83%). Regional TDP-43 pathologic assessment was available for 77% of participants, with hippocampus the most common region. Availability for the TDP-43-related measures increased over time, and was higher in centers with high proportions of participants with clinical FTLD. Part II: In 2142 participants with all TDP-43-related assessments available, 27% of participants had LATE-NC, whereas ALS-TDP or FTLD-TDP (ALS/FTLD-TDP) was present in 9% of participants, and 2% of participants had TDP-43 related to other pathologies ("Other TDP-43"). HS-A was present in 14% of participants, of whom 55% had LATE-NC, 20% ASL/FTLD-TDP, 3% Other TDP-43, and 23% no TDP-43. LATE-NC, ALS/FTLD-TDP, and Other TDP-43, were each associated with higher odds of dementia, HS-A, and hippocampal atrophy, compared to those without TDP-43 pathology. LATE-NC was associated with higher odds for Alzheimer's disease (AD) clinical diagnosis, AD neuropathologic change (ADNC), Lewy bodies, arteriolosclerosis, and cortical atrophy. ALS/FTLD-TDP was associated with higher odds of clinical diagnoses of primary progressive aphasia and behavioral-variant frontotemporal dementia, and cortical/frontotemporal lobar atrophy. When using NACC data for TDP-43-related analyses, researchers should carefully consider the incomplete availability of the different regional TDP-43 assessments, the high frequency of participants with ALS/FTLD-TDP, and the presence of other forms of TDP-43 pathology.

PurposeMany survivors of rectal cancer experience persistent bowel dysfunction. There are few evidence-based symptom management interventions to improve bowel control. The purpose of this study is to describe recruitment and pre-randomization baseline sociodemographic, health status, and clinical characteristics for SWOG S1820, a trial of the Altering Intake, Managing Symptoms in Rectal Cancer (AIMS-RC) intervention.MethodsSWOG S1820 aimed to determine the preliminary efficacy, feasibility, and acceptability of AIMS-RC, a symptom management intervention for bowel health, comparing intervention to attention control. Survivors with a history of cancers of the rectosigmoid colon or rectum, within 6-24 months of primary treatment completion, with a post-surgical permanent ostomy or anastomosis, and over 18 years of age were enrolled. Outcomes included total bowel function, low anterior resection syndrome, quality of life, motivation for managing bowel health, self-efficacy for managing symptoms, positive and negative affect, and study feasibility and acceptability.ResultsThe trial completed accrual over a 29-month period and enrolled 117 participants from 34 institutions across 17 states and one US Pacific territory. At baseline, most enrolled participants reported self-imposed diet adjustments after surgery, persistent dietary intolerances, and bowel discomfort post-treatment, with high levels of constipation and diarrhea (grades 1-4).ConclusionsSWOG S1820 was able to recruit, in a timely manner, a study cohort that is demographically representative of US survivors of rectal cancer. Baseline characteristics illustrate the connection between diet/eating and bowel symptoms post-treatment, with many participants reporting diet adjustments and persistent inability to be comfortable with dietary intake.ClinicaltrialsGov registration date12/19/2019.ClinicaltrialsGov identifierNCT#04205955.

Tuberculous peritonitis (TB peritonitis) is one of the most challenging forms of extrapulmonary TB to diagnose. While tumor markers can be elevated in patients with TB peritonitis, FDG-PET/CT can aid in distinguishing TB peritonitis from malignancies, if an apron-like omentum pattern is seen. Laparoscopy is crucial for accurate and early diagnosis.

BackgroundAutoimmune diseases have been linked to an increased risk of pregnancy-related complications. A family history of autoimmune diseases may be related to the risk of childhood cancer based on similar histocompatibility antigens. We utilized data from national registries in Denmark to examine associations between maternal autoimmune disease and cancer in their offspring.MethodsWe linked data from several national registries in Denmark to identify childhood cancer cases in children

BackgroundMaternal solvent exposure has been suspected to increase offspring cancer risk. The study aimed to evaluate the associations between maternal residential exposure to solvents from industrial pollution during pregnancy and childhood cancer.MethodsThe present study included 15,744 cancer cases (aged 0-19 years at diagnosis) identified from California Cancer Registry and 283,141 controls randomly selected from California Birth Registry (20:1 frequency-matched by birth year: 1998-2016). We examined industrial releases of tetrachloroethylene and 1,1,1-trichloroethane within 3 km of the birth address, while we used a 5 km buffer for carbon disulfide. We calculated the total exposure from all linked Toxic Release Inventory sites during each index pregnancy and assigned "ever/never" and "high/low exposed/unexposed" exposure, using median values. We performed quadratic decay models to estimate cancer risks associated with maternal solvent exposure in pregnancy.Results1,1,1-Trichloroethane was associated with rhabdomyosarcoma (adjusted Odds Ratio (aOR): 1.96; 95% Confidence Interval (CI): 1.16, 3.32) in the "ever exposed" group. Ever exposure to carbon disulfide was associated with increased risks of medulloblastoma (OR = 1.85, 95% CI 1.01, 3.40) and ependymoma (OR = 1.63, 95% CI 0.97, 2.74).ConclusionsOverall, our findings suggested maternal residential exposure to solvents from industrial sources might be associated with elevated childhood cancer risks.

BackgroundAnti-MDA5 (Melanoma differentiation-associated protein-5) positive dermatomyositis (MDA5+-DM) is characterised by rapidly progressive interstitial lung disease (ILD) and high mortality. MDA5 is an RNA sensor and a key pattern recognition receptor for the SARS-CoV-2 virus.MethodsThis is a retrospective observational study of a surge in MDA5 autoimmunity, as determined using a 15 muscle-specific autoantibodies (MSAs) panel, between Janurary 2018 and December 2022 in Yorkshire, UK. MDA5-positivity was correlated with clinical features and outcome, and regional SARS-CoV-2 positivity and vaccination rates. Gene expression patterns in COVID-19 were compared with autoimmune lung disease and idiopathic pulmonary fibrosis (IPF) to gain clues into the genesis of the observed MDA5+-DM outbreak.FindingsSixty new anti-MDA5+, but not other MSAs surged between 2020 and 2022, increasing from 0.4% in 2019 to 2.1% (2020), 4.8% (2021) and 1.7% (2022). Few (8/60) had a prior history of confirmed COVID-19, peak rates overlapped with regional SARS-COV-2 community positivity rates in 2021, and 58% (35/60) had received anti-SARS-CoV-2 vaccines. 25/60 cases developed ILD which rapidly progression with death in 8 cases. Among the 35/60 non-ILD cases, 14 had myositis, 17 Raynaud phenomena and 10 had dermatomyositis spectrum rashes. Transcriptomic studies showed strong IFIH1 (gene encoding for MDA5) induction in COVID-19 and autoimmune-ILD, but not IPF, and IFIH1 strongly correlated with an IL-15-centric type-1 interferon response and an activated CD8+ T cell signature that is an immunologic hallmark of progressive ILD in the setting of systemic autoimmune rheumatic diseases. The IFIH1 rs1990760TT variant blunted such response.InterpretationA distinct pattern of MDA5-autoimmunity cases surged contemporaneously with circulation of the SARS-COV-2 virus during COVID-19. Bioinformatic insights suggest a shared immunopathology with known autoimmune lung disease mechanisms.FundingThis work was supported in part by the National Institute for Health Research (NIHR) Leeds Biomedical Research Centre (BRC), and in part by the National Institutes of Health (NIH) grant R01-AI155696 and pilot awards from the UC Office of the President (UCOP)-RGPO (R00RG2628, R00RG2642 and R01RG3780) to P.G. S.S was supported in part by R01-AI141630 (to P.G) and in part through funds from the American Association of Immunologists (AAI) Intersect Fellowship Program for Computational Scientists and Immunologists.

Glioblastoma is a clinically and molecularly heterogeneous disease, and new predictive biomarkers are needed to identify those patients most likely to respond to specific treatments. Through prospective genomic profiling of 459 consecutive primary treatment-naïve IDH-wildtype glioblastomas in adults, we identified a unique subgroup (2%, 9/459) defined by somatic hypermutation and DNA replication repair deficiency due to biallelic inactivation of a canonical mismatch repair gene. The deleterious mutations in mismatch repair genes were often present in the germline in the heterozygous state with somatic inactivation of the remaining allele, consistent with glioblastomas arising due to underlying Lynch syndrome. A subset of tumors had accompanying proofreading domain mutations in the DNA polymerase POLE and resultant "ultrahypermutation". The median age at diagnosis was 50 years (range 27-78), compared with 63 years for the other 450 patients with conventional glioblastoma (p

Novel mosquito genetic control tools, such as CRISPR-based gene drives, hold great promise in reducing the global burden of vector-borne diseases. As these technologies advance through the research and development pipeline, there is a growing need for modeling frameworks incorporating increasing levels of entomological and epidemiological detail in order to address questions regarding logistics and biosafety. Epidemiological predictions are becoming increasingly relevant to the development of target product profiles and the design of field trials and interventions, while entomological surveillance is becoming increasingly important to regulation and biosafety. We present MGDrivE 3 (Mosquito Gene Drive Explorer 3), a new version of a previously-developed framework, MGDrivE 2, that investigates the spatial population dynamics of mosquito genetic control systems and their epidemiological implications. The new framework incorporates three major developments: i) a decoupled sampling algorithm allowing the vector portion of the MGDrivE framework to be paired with a more detailed epidemiological framework, ii) a version of the Imperial College London malaria transmission model, which incorporates age structure, various forms of immunity, and human and vector interventions, and iii) a surveillance module that tracks mosquitoes captured by traps throughout the simulation. Example MGDrivE 3 simulations are presented demonstrating the application of the framework to a CRISPR-based homing gene drive linked to dual disease-refractory genes and their potential to interrupt local malaria transmission. Simulations are also presented demonstrating surveillance of such a system by a network of mosquito traps. MGDrivE 3 is freely available as an open-source R package on CRAN (https://cran.r-project.org/package=MGDrivE2) (version 2.1.0), and extensive examples and vignettes are provided. We intend the software to aid in understanding of human health impacts and biosafety of mosquito genetic control tools, and continue to iterate per feedback from the genetic control community.

AbstractThe transcription factor RUNX1 is a master regulator of hematopoiesis and is frequently mutated in myeloid malignancies. Mutations in its runt homology domain (RHD) frequently disrupt DNA binding and result in loss of RUNX1 function. However, it is not clearly understood how other RUNX1 mutations contribute to disease development. Here, we characterized RUNX1 mutations outside of the RHD. Our analysis of the patient data sets revealed that mutations within the C-terminus frequently occur in hematopoietic disorders. Remarkably, most of these mutations were nonsense or frameshift mutations and were predicted to be exempt from nonsense-mediated messenger RNA decay. Therefore, this class of mutation is projected to produce DNA-binding proteins that contribute to the pathogenesis in a distinct manner. To model this, we introduced the RUNX1R320∗ mutation into the endogenous gene locus and demonstrated the production of RUNX1R320∗ protein. Expression of RUNX1R320∗ resulted in the disruption of RUNX1 regulated processes such as megakaryocytic differentiation, through a transcriptional signature different from RUNX1 depletion. To understand the underlying mechanisms, we used Global RNA Interactions with DNA by deep sequencing (GRID-seq) to examine enhancer-promoter connections. We identified widespread alterations in the enhancer-promoter networks within RUNX1 mutant cells. Additionally, we uncovered enrichment of RUNX1R320∗ and FOXK2 binding at the MYC super enhancer locus, significantly upregulating MYC transcription and signaling pathways. Together, our study demonstrated that most RUNX1 mutations outside the DNA-binding domain are not subject to nonsense-mediated decay, producing protein products that act in concert with additional cofactors to dysregulate hematopoiesis through mechanisms distinct from those induced by RUNX1 depletion.

BackgroundSputum-based testing is a barrier to increasing access to molecular diagnostics for tuberculosis (TB). Many people with TB are unable to produce sputum, and sputum processing increases assay complexity and cost. Tongue swabs are emerging as an alternative to sputum, but performance limits are uncertain.MethodsFrom June 2022 to July 2023, we enrolled 397 consecutive adults with cough >2 weeks at 2 health centers in Kampala, Uganda. We collected demographic and clinical information, sputum for TB testing (Xpert MTB/RIF Ultra and 2 liquid cultures), and tongue swabs for same-day quantitative polymerase chain reaction (qPCR) testing. We evaluated tongue swab qPCR diagnostic accuracy versus sputum TB test results, quantified TB targets per swab, assessed the impact of serial swabbing, and compared 2 swab types (Copan FLOQSWAB and Steripack spun polyester).ResultsAmong 397 participants, 43.1% were female, median age was 33 years, 23.5% were diagnosed with human immunodeficiency virus, and 32.0% had confirmed TB. Sputum Xpert Ultra and tongue swab qPCR results were concordant for 98.2% (95% confidence interval [CI]: 96.2-99.1) of participants. Tongue swab qPCR sensitivity was 92.6% (95% CI: 86.5 to 96.0) and specificity was 99.1% (95% CI: 96.9 to 99.8) versus microbiological reference standard. A single tongue swab recovered a 7-log range of TB copies, with a decreasing recovery trend among 4 serial swabs. Swab types performed equivalently.ConclusionsTongue swabs are a promising alternative to sputum for molecular diagnosis of TB, with sensitivity approaching sputum-based molecular tests. Our results provide valuable insights for developing successful tongue swab-based TB diagnostics.

Craniosynostosis, defined by premature fusion of one or multiple cranial sutures, is a common congenital defect affecting more than 1/2000 infants and results in restricted brain expansion. Single gene mutations account for 15%-20% of cases, largely as part of a syndrome, but the majority are nonsyndromic with complex underlying genetics. We hypothesized that the two noncoding genomic regions identified by a GWAS for craniosynostosis contain distal regulatory elements for the risk genes BMPER and BMP2. To identify such regulatory elements, we surveyed conserved noncoding sequences from both risk loci for enhancer activity in transgenic Danio rerio. We identified enhancers from both regions that direct expression to skeletal tissues, consistent with the endogenous expression of bmper and bmp2. For each locus, we also found a skeletal enhancer that also contains a sequence variant associated with craniosynostosis risk. We examined the activity of each enhancer during craniofacial development and found that the BMPER-associated enhancer is active in the restricted region of cartilage closely associated with frontal bone initiation. The same enhancer is active in mouse skeletal tissues, demonstrating evolutionarily conserved activity. Using enhanced yeast one-hybrid assays, we identified transcription factors that bind each enhancer and observed differential binding between alleles, implicating multiple signaling pathways. Our findings help unveil the genetic mechanism of the two craniosynostosis risk loci. More broadly, our combined in vivo approach is applicable to many complex genetic diseases to build a link between association studies and specific genetic mechanisms.

Mycobacterium tuberculosis (Mtb) infects lung myeloid cells, but the specific Mtb-permissive cells and host mechanisms supporting Mtb persistence during chronic infection are incompletely characterized. We report that after the development of T cell responses, CD11clo monocyte-derived cells harbor more live Mtb than alveolar macrophages (AM), neutrophils, and CD11chi monocyte-derived cells. Transcriptomic and functional studies revealed that the lysosome pathway is underexpressed in this highly permissive subset, characterized by less lysosome content, acidification, and proteolytic activity than AM, along with less nuclear TFEB, a regulator of lysosome biogenesis. Mtb infection does not drive lysosome deficiency in CD11clo monocyte-derived cells but promotes recruitment of monocytes that develop into permissive lung cells, mediated by the Mtb ESX-1 secretion system. The c-Abl tyrosine kinase inhibitor nilotinib activates TFEB and enhances lysosome functions of macrophages in vitro and in vivo, improving control of Mtb infection. Our results suggest that Mtb exploits lysosome-poor lung cells for persistence and targeting lysosome biogenesis is a potential host-directed therapy for tuberculosis.

BACKGROUNDThe inclusion of adolescents in TB drug trials is essential for the development of safe, child-friendly regimens for the prevention and treatment of TB. TB Trials Consortium Study 31/AIDS Clinical Trials Group A5349 (S31/A5349) enrolled adolescents as young as 12 years old. We assessed investigator and coordinator described facilitators and barriers to adolescent recruitment, enrollment, and retention.METHODSInterviews were conducted with six investigators from sites that enrolled adolescent participants and six investigators from non-enrolling sites. Additionally, two focus groups were conducted with study coordinators from enrolling sites and two focus groups with non-enrolling sites. Discussions were transcribed, analyzed, summarized, and summaries were reviewed by Community Research Advisors Group members and research group representatives for content validity.RESULTSInvestigators and coordinators attributed the successful enrollment of adolescents to the establishment and cultivation of external partnerships, flexibility to accommodate adolescents' schedules, staff engagement, recruitment from multiple locations, dedicated recruitment staff working onsite to access potential participants, creation of youth-friendly environments, and effective communications. Non-enrolling sites were mainly hindered by regulations. Suggestions for improvement in future trials focused on study planning and site preparations.CONCLUSIONProactive partnerships and collaboration with institutions serving adolescents helped identify and reduce barriers to their inclusion in this trial..

Exocytosis is a critical factor for designing efficient nanocarriers and determining cytotoxicity. However, the research on the exocytosis mechanism of nanoparticles, especially the role of long non-coding RNAs (lncRNA), has not been reported. In this study, the exocytosis of AuNPs in the KYSE70 cells and the involved molecular pathways of exocytosis are analyzed. It demonstrates that nanoparticles underwent time-dependent release from the cells by exocytosis, and the release of β-hexosaminidase confirms that AuNPs are excreted through lysosomes. Mechanistic studies reveal that lncRNA ESCCAL-1 plays a vital role in controlling the exocytosis of AuNPs through activation of the MAPK pathway, including the phosphorylation of ERK and JNK. The study implies that the ESCCAL-1-mediated pathway plays an important role in the exocytosis of AuNPs in KYSE70 cells. This finding has implications for the role of ESCCAL-1 on the drug resistance of esophagus cancer by controlling lysosome-mediated exocytosis.

BACKGROUNDPatients hospitalized for COVID-19 exhibit diverse clinical outcomes, with outcomes for some individuals diverging over time even though their initial disease severity appears similar to that of other patients. A systematic evaluation of molecular and cellular profiles over the full disease course can link immune programs and their coordination with progression heterogeneity.METHODSWe performed deep immunophenotyping and conducted longitudinal multiomics modeling, integrating 10 assays for 1,152 Immunophenotyping Assessment in a COVID-19 Cohort (IMPACC) study participants and identifying several immune cascades that were significant drivers of differential clinical outcomes.RESULTSIncreasing disease severity was driven by a temporal pattern that began with the early upregulation of immunosuppressive metabolites and then elevated levels of inflammatory cytokines, signatures of coagulation, formation of neutrophil extracellular traps, and T cell functional dysregulation. A second immune cascade, predictive of 28-day mortality among critically ill patients, was characterized by reduced total plasma Igs and B cells and dysregulated IFN responsiveness. We demonstrated that the balance disruption between IFN-stimulated genes and IFN inhibitors is a crucial biomarker of COVID-19 mortality, potentially contributing to failure of viral clearance in patients with fatal illness.CONCLUSIONOur longitudinal multiomics profiling study revealed temporal coordination across diverse omics that potentially explain the disease progression, providing insights that can inform the targeted development of therapies for patients hospitalized with COVID-19, especially those who are critically ill.TRIAL REGISTRATIONClinicalTrials.gov NCT04378777.FUNDINGNIH (5R01AI135803-03, 5U19AI118608-04, 5U19AI128910-04, 4U19AI090023-11, 4U19AI118610-06, R01AI145835-01A1S1, 5U19AI062629-17, 5U19AI057229-17, 5U19AI125357-05, 5U19AI128913-03, 3U19AI077439-13, 5U54AI142766-03, 5R01AI104870-07, 3U19AI089992-09, 3U19AI128913-03, and 5T32DA018926-18); NIAID, NIH (3U19AI1289130, U19AI128913-04S1, and R01AI122220); and National Science Foundation (DMS2310836).

BackgroundNatural killer (NK) cells are key effector cells of antitumor immunity. However, tumors can acquire resistance programs to escape NK cell-mediated immunosurveillance. Identifying mechanisms that mediate this resistance enables us to define approaches to improve immune-mediate antitumor activity. In previous studies from our group, a genome-wide CRISPR-Cas9 screen identified Charged Multivesicular Body Protein 2A (CHMP2A) as a novel mechanism that mediates tumor intrinsic resistance to NK cell activity.MethodsHere, we use an immunocompetent mouse model to demonstrate that CHMP2A serves as a targetable regulator of not only NK cell-mediated immunity but also other immune cell populations. Using the recently characterized murine 4MOSC model system, a syngeneic, tobacco-signature murine head and neck squamous cell carcinoma model, we deleted mCHMP2A using CRISPR/Cas9-mediated knock-out (KO), following orthotopic transplantation into immunocompetent hosts.ResultsWe found that mCHMP2A KO in 4MOSC1 cells leads to more potent NK-mediated tumor cell killing in vitro in these tumor cells. Moreover, following orthotopic transplantation, KO of mCHMP2A in 4MOSC1 cells, but not the more immune-resistant 4MOSC2 cells enables both T cells and NK cells to better mediate antitumor activity compared with wild type (WT) tumors. However, there was no difference in tumor development between WT and mCHMP2A KO 4MOSC1 or 4MOSC2 tumors when implanted in immunodeficient mice. Mechanistically, we find that mCHMP2A KO 4MOSC1 tumors transplanted into the immunocompetent mice had significantly increased CD4+T cells, CD8+T cells. NK cell, as well as fewer myeloid-derived suppressor cells (MDSC).ConclusionsTogether, these studies demonstrate that CHMP2A is a targetable inhibitor of cellular antitumor immunity.

ObjectiveTo determine the feasibility and biologic correlations of dynamic susceptibility contrast (DSC), dynamic contrast enhanced (DCE), and quantitative maps derived from contrast leakage effects obtained simultaneously in gliomas using dynamic spin-and-gradient-echo echoplanar imaging (dynamic SAGE-EPI) during a single contrast injection.Materials and methodsThirty-eight patients with enhancing brain gliomas were prospectively imaged with dynamic SAGE-EPI, which was processed to compute traditional DSC metrics (normalized relative cerebral blood flow [nrCBV], percentage of signal recovery [PSR]), DCE metrics (volume transfer constant [Ktrans], extravascular compartment [ve]), and leakage effect metrics: ΔR2,ss* (reflecting T2*-leakage effects), ΔR1,ss (reflecting T1-leakage effects), and the transverse relaxivity at tracer equilibrium (TRATE, reflecting the balance between ΔR2,ss* and ΔR1,ss). These metrics were compared between patient subgroups (treatment-naïve [TN] vs recurrent [R]) and biological features (IDH status, Ki67 expression).ResultsIn IDH wild-type gliomas (IDHwt-i.e., glioblastomas), previous exposure to treatment determined lower TRATE (p = 0.002), as well as higher PSR (p = 0.006), Ktrans (p = 0.17), ΔR1,ss (p = 0.035), ve (p = 0.006), and ADC (p = 0.016). In IDH-mutant gliomas (IDHm), previous treatment determined higher Ktrans and ΔR1,ss (p = 0.026). In TN-gliomas, dynamic SAGE-EPI metrics tended to be influenced by IDH status (p ranging 0.09-0.14). TRATE values above 142 mM-1s-1 were exclusively seen in TN-IDHwt, and, in TN-gliomas, this cutoff had 89% sensitivity and 80% specificity as a predictor of Ki67 > 10%.ConclusionsDynamic SAGE-EPI enables simultaneous quantification of brain tumor perfusion and permeability, as well as mapping of novel metrics related to cytoarchitecture (TRATE) and blood-brain barrier disruption (ΔR1,ss), with a single contrast injection.Clinical relevance statementSimultaneous DSC and DCE analysis with dynamic SAGE-EPI reduces scanning time and contrast dose, respectively alleviating concerns about imaging protocol length and gadolinium adverse effects and accumulation, while providing novel leakage effect metrics reflecting blood-brain barrier disruption and tumor tissue cytoarchitecture.Key points• Traditionally, perfusion and permeability imaging for brain tumors requires two separate contrast injections and acquisitions. • Dynamic spin-and-gradient-echo echoplanar imaging enables simultaneous perfusion and permeability imaging. • Dynamic spin-and-gradient-echo echoplanar imaging provides new image contrasts reflecting blood-brain barrier disruption and cytoarchitecture characteristics.

A teenage girl presented with fever and altered mental status. MRI showed diffuse leptomeningeal enhancement of the brain and spine. She was diagnosed by a positive cerebrospinal fluid (CSF) culture with tuberculous (TB) meningitis and was started on anti-TB medications and corticosteroids. Her mental status improved, but she was noted to have proximal weakness of the lower extremities. In the course of tapering corticosteroids at week 11 of anti-TB therapy, she became acutely confused and febrile. MRI demonstrated interval development of tuberculomas in the brain and a mass lesion in the thoracic spine causing cord compression. Given the clinical picture was suggestive of a paradoxical reaction, the dose of corticosteroids was increased. Infliximab was added when repeat MRI revealed enlargement of the mass lesion in the spine with worsening cord compression. She was successfully tapered off of corticosteroids. Over several months, the patient's motor function recovered fully, and she returned to ambulating without assistance.

The cause of death in people affected by sickle cell disease (SCD) is often challenging to define as prior studies have used retrospective or administrative data for analysis. We used a prospective longitudinal registry to assess mortality and clinical co-morbidities among subjects enrolled in the Sickle Cell Disease Implementation Consortium (SCDIC) registry. At enrollment, we collected the following data: patient-reported demographics, SCD phenotype, baseline laboratory values, comorbidities, and current medications. Subjects were followed for a median of 4.7 years before the present analysis. The relationship of clinical co-morbidities (at time of enrollment) to mortality was determined using survival analysis, adjusting for SCD phenotype and gender. There was a total of 2439 people with SCD enrolled in the SCDIC registry. One hundred and twenty-eight participants (5%) died during the observation period (2017-2022). Six people died from trauma and were excluded from further analysis. Proximate cause of death was unwitnessed in 17% of the deaths, but commonest causes of death include cardiac (18%), acute chest or respiratory failure (11%), sudden unexplained death (8%). Enrollment characteristics of the individuals who died (n = 122) were compared to those of survivors (n = 2317). Several co-morbidities at enrollment increased the odds of death on univariate analysis. All co-morbidities were included in a multivariable model. After backward elimination, iron overload, pulmonary hypertension, and depression, remained statistically significant predictors of the risk of death. SCD reduces life expectancy. Improved comprehensive and supportive care to prevent end-organ damage and address comorbidities is needed for this population.

ObjectiveOur objective was to gauge adherence to nationally endorsed protocols in implementation of pulse oximetry (POx) screening for critical congenital heart disease (CCHD) in infants after mandate by all states and to assess associated characteristics.Study designBetween March and October 2019, an online questionnaire was administered to nurse supervisors who oversee personnel conducting POx screening. The questionnaire used eight questions regarding performance and interpretation of screening protocols to measure policy consistency, which is adherence to nationally endorsed protocols for POx screening developed by professional medical societies. Multilevel linear regression models evaluated associations between policy consistency and characteristics of hospitals and individuals, state of hospital location, early versus late mandate adopters, and state reporting requirements.ResultsResponses from 189 nurse supervisors spanning 38 states were analyzed. Only 17% received maximum points indicating full policy consistency, and 24% selected all four options for potential hypoxia that require a repeat screen. Notably, 33% did not recognize ≤90% SpO2 as an immediate failed screen and 31% responded that an infant with SpO2 of 89% in one extremity will be rescreened by nurses in an hour rather than receiving an immediate physician referral. Lower policy consistency was associated with lack of state reporting mandates (beta = -1.23 p = 0.01) and early adoption by states (beta = -1.01, p

Global control of infectious diseases depends on the continuous development and deployment of diverse vaccination strategies. Currently available live-attenuated and killed virus vaccines typically take a week or longer to activate specific protection by the adaptive immunity. The mosquito-transmitted Nodamura virus (NoV) is attenuated in mice by mutations that prevent expression of the B2 viral suppressor of RNA interference (VSR) and consequently, drastically enhance in vivo production of the virus-targeting small-interfering RNAs. We reported recently that 2 d after immunization with live-attenuated VSR-disabled NoV (NoVΔB2), neonatal mice become fully protected against lethal NoV challenge and develop no detectable infection. Using Rag1-/- mice that produce no mature B and T lymphocytes as a model, here we examined the hypothesis that adaptive immunity is dispensable for the RNAi-based protective immunity activated by NoVΔB2 immunization. We show that immunization of both neonatal and adult Rag1-/- mice with live but not killed NoVΔB2 induces full protection against NoV challenge at 2 or 14 d postimmunization. Moreover, NoVΔB2-induced protective antiviral immunity is virus-specific and remains effective in adult Rag1-/- mice 42 and 90 d after a single-shot immunization. We conclude that immunization with the live-attenuated VSR-disabled RNA virus vaccine activates rapid and long-lasting protective immunity against lethal challenges by a distinct mechanism independent of the adaptive immunity mediated by B and T cells. Future studies are warranted to determine whether additional animal and human viruses attenuated by VSR inactivation induce similar protective immunity in healthy and adaptive immunity-compromised individuals.

The aim of the present study is to provide a rational background for silencing the V-set and transmembrane domain containing 2 like (VSTM2L) in consort with recognising soluble VSTM2L against cholangiocarcinoma. A therapeutic target against cholangiocarcinoma was selected using iterative patient partitioning (IPP) calculation, and it was verified by in vitro and in silico analyses. VSTM2L was selected as a potential therapeutic target against cholangiocarcinoma. Silencing the VSTM2L expression significantly attenuated the viability and survival of cholangiocarcinoma cells through blockade of the intracellular signalling pathway. In silico analysis showed that VSTM2L affected the positive regulation of cell growth in cholangiocarcinoma. Liptak's z value revealed that the expression of VSTM2L worsened the prognosis of cholangiocarcinoma patients. In addition, soluble VSTM2L was significantly detected in the whole blood of cholangiocarcinoma patients compared with that of healthy donors. Our report reveals that VSTM2L might be the potential therapeutic target and a soluble prognostic biomarker against cholangiocarcinoma.

High-grade serous ovarian carcinoma (HGSOC), the deadliest form of ovarian cancer, is typically diagnosed after it has metastasized and often relapses after standard-of-care platinum-based chemotherapy, likely due to advanced tumor stage, heterogeneity, and immune evasion and tumor-promoting signaling from the tumor microenvironment. To understand how spatial heterogeneity contributes to HGSOC progression and early relapse, we profiled an HGSOC tissue microarray of patient-matched longitudinal samples from 42 patients. We found spatial patterns associated with early relapse, including changes in T cell localization, malformed tertiary lymphoid structure (TLS)-like aggregates, and increased podoplanin-positive cancer-associated fibroblasts (CAFs). Using spatial features to compartmentalize the tissue, we found that plasma cells distribute in two different compartments associated with TLS-like aggregates and CAFs, and these distinct microenvironments may account for the conflicting reports about the role of plasma cells in HGSOC prognosis.

Salivary glands have essential roles in maintaining oral health, mastication, taste and speech, by secreting saliva. Salivary glands are composed of several types of cells, and each cell type is predicted to be involved in the carcinogenesis of different types of cancers including adenoid cystic carcinoma (ACC), acinic cell carcinoma (AciCC), salivary duct carcinoma (SDC), myoepithelial carcinoma (MECA) and other histology. In our study, we performed single nucleus RNA-seq on three human salivary gland samples to clarify the gene expression profile of each complex cellular component of the salivary glands and related these expression patterns to expression found in salivary gland cancers (SGC) to infer cell of origin. By single nucleus RNA-seq, salivary gland cells were stratified into four clusters: acinar cells, ductal cells 1, ductal cells 2 and myoepithelial cells/stromal cells. The localization of each cell group was verified by IHC of each cluster marker gene, and one group of ductal cells was found to represent intercalated ductal cells labeled with HES1. Furthermore, in comparison with SGC RNA-seq data, acinar cell markers were upregulated in AciCC, but downregulated in ACC and ductal cell markers were upregulated in SDC but downregulated in MECA, suggesting that markers of origin are highly expressed in some SGC. Cell type expressions in specific SGC histology are similar to those found in normal salivary gland populations, indicating a potential etiologic relationship.

Melioidosis, caused by the intracellular bacterial pathogen and Tier 1 select agent Burkholderia pseudomallei (Bp), is a highly fatal disease endemic in tropical areas. No licensed vaccine against melioidosis exists. In preclinical vaccine studies, demonstrating protection against respiratory infection in the highly sensitive BALB/c mouse has been especially challenging. To address this challenge, we have used a safe yet potent live attenuated platform vector, LVS ΔcapB, previously used successfully to develop vaccines against the Tier 1 select agents of tularemia, anthrax, and plague, to develop a melioidosis vaccine. We have engineered melioidosis vaccines (rLVS ΔcapB/Bp) expressing multiple immunoprotective Bp antigens among type VI secretion system proteins Hcp1, Hcp2, and Hcp6, and membrane protein LolC. Administered intradermally, rLVS ΔcapB/Bp vaccines strongly protect highly sensitive BALB/c mice against lethal respiratory Bp challenge, but protection is overwhelmed at very high challenge doses. In contrast, administered intranasally, rLVS ΔcapB/Bp vaccines remain strongly protective against even very high challenge doses. Under some conditions, the LVS ΔcapB vector itself provides significant protection against Bp challenge, and consistent with this, both the vector and vaccines induce humoral immune responses to Bp antigens. Three-antigen vaccines expressing Hcp6-Hcp1-Hcp2 or Hcp6-Hcp1-LolC are among the most potent and provide long-term protection and protection even with a single intranasal immunization. Protection via the intranasal route was either comparable to or statistically significantly better than the single-deletional Bp mutant Bp82, which served as a positive control. Thus, rLVS ΔcapB/Bp vaccines are exceptionally promising safe and potent melioidosis vaccines.ImportanceMelioidosis, a major neglected disease caused by the intracellular bacterial pathogen Burkholderia pseudomallei, is endemic in many tropical areas of the world and causes an estimated 165,000 cases and 89,000 deaths in humans annually. Moreover, B. pseudomallei is categorized as a Tier 1 select agent of bioterrorism, largely because inhalation of low doses can cause rapidly fatal pneumonia. No licensed vaccine is available to prevent melioidosis. Here, we describe a safe and potent melioidosis vaccine that protects against lethal respiratory challenge with B. pseudomallei in a highly sensitive small animal model-even a single immunization is highly protective, and the vaccine gives long-term protection. The vaccine utilizes a highly attenuated replicating intracellular bacterium as a vector to express multiple key proteins of B. pseudomallei; this vector platform has previously been used successfully to develop potent vaccines against other Tier 1 select agent diseases including tularemia, anthrax, and plague.

Prion-like spread of disease-specific tau conformers is a hallmark of all tauopathies. A 19-residue probe peptide containing a P301L mutation and spanning the R2/R3 splice junction of tau folds and stacks into seeding-competent fibrils and induces aggregation of 4R, but not 3R tau. These tau peptide fibrils propagate aggregated intracellular tau over multiple generations, have a high β-sheet content, a colocalized lipid signal, and adopt a well-defined U-shaped fold found in 4R tauopathy brain-derived fibrils. Fully atomistic replica exchange molecular dynamics (MD) simulations were used to compute the free energy landscapes of the conformational ensemble of the peptide monomers. These identified an aggregation-prohibiting β-hairpin structure and an aggregation-competent U-fold unique to 4R tauopathy fibrils. Guided by MD simulations, we identified that the N-terminal-flanking residues to PHF6, which slightly vary between 4R and 3R isoforms, modulate seeding. Strikingly, when a single amino acid switch at position 305 replaced the serine of 4R tau with a lysine from the corresponding position in the first repeat of 3R tau, the seeding induced by the 19-residue peptide was markedly reduced. Conversely, a 4R tau mimic with three repeats, prepared by replacing those amino acids in the first repeat with those amino acids uniquely present in the second repeat, recovered aggregation when exposed to the 19-residue peptide. These peptide fibrils function as partial prions to recruit naive 4R tau-ten times the length of the peptide-and serve as a critical template for 4R tauopathy propagation. These results hint at opportunities for tau isoform-specific therapeutic interventions.

The efficacy of chimeric antigen receptor T cell (CAR-T) therapy has been limited against brain tumors to date. CAR-T cells infiltrating syngeneic intracerebral SB28 EGFRvIII gliomas revealed impaired mitochondrial ATP production and a markedly hypoxic status compared with ones migrating to subcutaneous tumors. Drug screenings to improve metabolic states of T cells under hypoxic conditions led us to evaluate the combination of the AMPK activator metformin and the mTOR inhibitor rapamycin (Met+Rap). Met+Rap-pretreated mouse CAR-T cells showed activated PPAR-γ coactivator 1α (PGC-1α) through mTOR inhibition and AMPK activation, and a higher level of mitochondrial spare respiratory capacity than those pretreated with individual drugs or without pretreatment. Moreover, Met+Rap-pretreated CAR-T cells demonstrated persistent and effective antiglioma cytotoxic activities in the hypoxic condition. Furthermore, a single intravenous infusion of Met+Rap-pretreated CAR-T cells significantly extended the survival of mice bearing intracerebral SB28 EGFRvIII gliomas. Mass cytometric analyses highlighted increased glioma-infiltrating CAR-T cells in the Met+Rap group, with fewer Ly6c+CD11b+ monocytic myeloid-derived suppressor cells in the tumors. Finally, human CAR-T cells pretreated with Met+Rap recapitulated the observations with murine CAR-T cells, demonstrating improved functions under in vitro hypoxic conditions. These findings advocate for translational and clinical exploration of Met+Rap-pretreated CAR-T cells in human trials.

Pancreatic ductal adenocarcinoma (PDAC) accounts for about 90% of all pancreatic cancer cases. Five-year survival rates have remained below 12% since the 1970s, in part due to the difficulty in detection prior to metastasis (migration and invasion into neighboring organs and glands). Mechanical memory is a concept that has emerged over the past decade that may provide a path toward understanding how invading PDAC cells "remember" the mechanical properties of their diseased ("stiff", elastic modulus, E ≈ 10 kPa) microenvironment even while invading a healthy ("soft", E ≈ 1 kPa) microenvironment. Here, we investigated the role of mechanical priming by culturing a dilute suspension of PDAC (FG) cells within a 3D, rheologically tunable microgel platform from hydrogels with tunable mechanical properties. We conducted a suite of acute (short-term) priming studies where we cultured PDAC cells in either a soft (E ≈ 1 kPa) or stiff (E ≈ 10 kPa) environment for 6 h, then removed and placed them into a new soft or stiff 3D environment for another 18 h. Following these steps, we conducted RNA-seq analyses to quantify gene expression. Initial priming in the 3D culture showed persistent gene expression for the duration of the study, regardless of the subsequent environments (stiff or soft). Stiff 3D culture was associated with the downregulation of tumor suppressors (LATS1, BCAR3, CDKN2C), as well as the upregulation of cancer-associated genes (RAC3). Immunofluorescence staining (BCAR3, RAC3) further supported the persistence of this cellular response, with BCAR3 upregulated in soft culture and RAC3 upregulated in stiff-primed culture. Stiff-primed genes were stratified against patient data found in The Cancer Genome Atlas (TCGA). Upregulated genes in stiff-primed 3D culture were associated with decreased survival in patient data, suggesting a link between patient survival and mechanical priming.

The Genome Aggregation Database (gnomAD), widely recognized as the gold-standard reference map of human genetic variation, has largely overlooked tandem repeat (TR) expansions, despite the fact that TRs constitute ∼6% of our genome and are linked to over 50 human diseases. Here, we introduce the TR-gnomAD (https://wlcb.oit.uci.edu/TRgnomAD), a biobank-scale reference of 0.86 million TRs derived from 338,963 whole-genome sequencing (WGS) samples of diverse ancestries (39.5% non-European samples). TR-gnomAD offers critical insights into ancestry-specific disease prevalence using disparities in TR unit number frequencies among ancestries. Moreover, TR-gnomAD is able to differentiate between common, presumably benign TR expansions, which are prevalent in TR-gnomAD, from those potentially pathogenic TR expansions, which are found more frequently in disease groups than within TR-gnomAD. Together, TR-gnomAD is an invaluable resource for researchers and physicians to interpret TR expansions in individuals with genetic diseases.

BackgroundNatural killer (NK) cells are cytotoxic cells capable of recognizing heterogeneous cancer targets without prior sensitization, making them promising prospects for use in cellular immunotherapy. Companion dogs develop spontaneous cancers in the context of an intact immune system, representing a valid cancer immunotherapy model. Previously, CD5 depletion of peripheral blood mononuclear cells (PBMCs) was used in dogs to isolate a CD5dim-expressing NK subset prior to co-culture with an irradiated feeder line, but this can limit the yield of the final NK product. This study aimed to assess NK activation, expansion, and preliminary clinical activity in first-in-dog clinical trials using a novel system with unmanipulated PBMCs to generate our NK cell product.MethodsStarting populations of CD5-depleted cells and PBMCs from healthy beagle donors were co-cultured for 14 days, phenotype, cytotoxicity, and cytokine secretion were measured, and samples were sequenced using the 3'-Tag-RNA-Seq protocol. Co-cultured human PBMCs and NK-isolated cells were also sequenced for comparative analysis. In addition, two first-in-dog clinical trials were performed in dogs with melanoma and osteosarcoma using autologous and allogeneic NK cells, respectively, to establish safety and proof-of-concept of this manufacturing approach.ResultsCalculated cell counts, viability, killing, and cytokine secretion were equivalent or higher in expanded NK cells from canine PBMCs versus CD5-depleted cells, and immune phenotyping confirmed a CD3-NKp46+ product from PBMC-expanded cells at day 14. Transcriptomic analysis of expanded cell populations confirmed upregulation of NK activation genes and related pathways, and human NK cells using well-characterized NK markers closely mirrored canine gene expression patterns. Autologous and allogeneic PBMC-derived NK cells were successfully expanded for use in first-in-dog clinical trials, resulting in no serious adverse events and preliminary efficacy data. RNA sequencing of PBMCs from dogs receiving allogeneic NK transfer showed patient-unique gene signatures with NK gene expression trends in response to treatment.ConclusionsOverall, the use of unmanipulated PBMCs appears safe and potentially effective for canine NK immunotherapy with equivalent to superior results to CD5 depletion in NK expansion, activation, and cytotoxicity. Our preclinical and clinical data support further evaluation of this technique as a novel platform for optimizing NK immunotherapy in dogs.

ImportancePreventive bilateral salpingo-oophorectomy is offered to women at high risk of ovarian cancer who carry a pathogenic variant in BRCA1 or BRCA2; however, the association of oophorectomy with all-cause mortality has not been clearly defined.ObjectiveTo evaluate the association between bilateral oophorectomy and all-cause mortality among women with a BRCA1 or BRCA2 sequence variation.Design, setting, and participantsIn this international, longitudinal cohort study of women with BRCA sequence variations, information on bilateral oophorectomy was obtained via biennial questionnaire. Participants were women with a BRCA1 or BRCA2 sequence variation, no prior history of cancer, and at least 1 follow-up questionnaire completed. Women were followed up from age 35 to 75 years for incident cancers and deaths. Cox proportional hazards regression was used to estimate the hazard ratios (HRs) and 95% CIs for all-cause mortality associated with a bilateral oophorectomy (time dependent). Data analysis was performed from January 1 to June 1, 2023.ExposuresSelf-reported bilateral oophorectomy (with or without salpingectomy).Main outcomes and measuresAll-cause mortality, breast cancer-specific mortality, and ovarian cancer-specific mortality.ResultsThere were 4332 women (mean age, 42.6 years) enrolled in the cohort, of whom 2932 (67.8%) chose to undergo a preventive oophorectomy at a mean (range) age of 45.4 (23.0-77.0) years. After a mean follow-up of 9.0 years, 851 women had developed cancer and 228 had died; 57 died of ovarian or fallopian tube cancer, 58 died of breast cancer, 16 died of peritoneal cancer, and 97 died of other causes. The age-adjusted HR for all-cause mortality associated with oophorectomy was 0.32 (95% CI, 0.24-0.42; P

Helicobacter pylori (H. pylori) infection is a known cause of many digestive diseases, including gastritis, peptic ulcers, and gastric cancer. However, the underlying mechanisms by which H. pylori infection triggers these disorders are still not clearly understood. Gastric cancer is a slow progressing disease, which makes it difficult to study. We have developed an accelerated disease progression mouse model, which leverages mice deficient in the myeloid differentiation primary response 88 gene (Myd88-/-) infected with Helicobacter felis (H. felis). Using this model and gastric biopsy samples from patients, we report that activation of the Toll/interleukin-1 receptor (TIR)-domain-containing adaptor inducing interferon-β (TRIF)-type I interferon (IFN-I) signaling pathway promotes Helicobacter-induced disease progression toward severe gastric pathology and gastric cancer development. Further, results implicated downstream targets of this pathway in disease pathogenesis. These findings may facilitate stratification of Helicobacter-infected patients and thus enable treatment prioritization of patients.

Chimeric antigen receptor T cell (CAR-T) therapy is an emerging strategy to improve treatment outcomes for recurrent high-grade glioma, a cancer that responds poorly to current therapies. Here we report a completed phase I trial evaluating IL-13Rα2-targeted CAR-T cells in 65 patients with recurrent high-grade glioma, the majority being recurrent glioblastoma (rGBM). Primary objectives were safety and feasibility, maximum tolerated dose/maximum feasible dose and a recommended phase 2 dose plan. Secondary objectives included overall survival, disease response, cytokine dynamics and tumor immune contexture biomarkers. This trial evolved to evaluate three routes of locoregional T cell administration (intratumoral (ICT), intraventricular (ICV) and dual ICT/ICV) and two manufacturing platforms, culminating in arm 5, which utilized dual ICT/ICV delivery and an optimized manufacturing process. Locoregional CAR-T cell administration was feasible and well tolerated, and as there were no dose-limiting toxicities across all arms, a maximum tolerated dose was not determined. Probable treatment-related grade 3+ toxicities were one grade 3 encephalopathy and one grade 3 ataxia. A clinical maximum feasible dose of 200 × 106 CAR-T cells per infusion cycle was achieved for arm 5; however, other arms either did not test or achieve this dose due to manufacturing feasibility. A recommended phase 2 dose will be refined in future studies based on data from this trial. Stable disease or better was achieved in 50% (29/58) of patients, with two partial responses, one complete response and a second complete response after additional CAR-T cycles off protocol. For rGBM, median overall survival for all patients was 7.7 months and for arm 5 was 10.2 months. Central nervous system increases in inflammatory cytokines, including IFNγ, CXCL9 and CXCL10, were associated with CAR-T cell administration and bioactivity. Pretreatment intratumoral CD3 T cell levels were positively associated with survival. These findings demonstrate that locoregional IL-13Rα2-targeted CAR-T therapy is safe with promising clinical activity in a subset of patients. ClinicalTrials.gov Identifier: NCT02208362 .

Hispanic/Latino children have the highest risk of acute lymphoblastic leukemia (ALL) in the US compared to other racial/ethnic groups, yet the basis of this remains incompletely understood. Through genetic fine-mapping analyses, we identified a new independent childhood ALL risk signal near IKZF1 in self-reported Hispanic/Latino individuals, but not in non-Hispanic White individuals, with an effect size of ∼1.44 (95% confidence interval = 1.33-1.55) and a risk allele frequency of ∼18% in Hispanic/Latino populations and