CAR19 monitoring by peripheral blood immunophenotyping reveals histology-specific expansion and toxicity.

Chimeric antigen receptor (CAR) T cells directed against CD19 (CAR19) are a revolutionary treatment for B-cell lymphomas. CAR19 cell expansion is necessary for CAR19 function but is also associated with toxicity. To define the impact of CAR19 expansion on patient outcomes, we prospectively followed a cohort of 236 patients treated with CAR19 (brexucabtagene autoleucel or axicabtagene ciloleucel) for mantle cell (MCL), follicular (FL), and large B-cell lymphoma (LBCL) over the course of five years and obtained CAR19 expansion data using peripheral blood immunophenotyping for 188 of these patients. CAR19 expansion was higher in patients with MCL compared to other lymphoma histologic subtypes. Notably, patients with MCL had increased toxicity and required four-fold higher cumulative steroid doses than patients with LBCL. CAR19 expansion was associated with the development of cytokine release syndrome (CRS), immune effector cell associated neurotoxicity syndrome (ICANS), and the requirement for granulocyte colony stimulating factor (GCSF) after day 14 post-infusion. Younger patients and those with elevated lactate dehydrogenase (LDH) had significantly higher CAR19 expansion. In general, no association between CAR19 expansion and LBCL treatment response was observed. However, when controlling for tumor burden, we found that lower CAR19 expansion in conjunction with low LDH was associated with improved outcomes in LBCL. In sum, this study finds CAR19 expansion principally associates with CAR-related toxicity. Additionally, CAR19 expansion as measured by peripheral blood immunophenotyping may be dispensable to favorable outcomes in LBCL.

In situ incubation of iron(II)-bearing minerals and Fe(0) reveals insights into metabolic flexibility of chemolithotrophic bacteria in a nitrate polluted karst aquifer.

Groundwater nitrate pollution is a major reason for deteriorating water quality and threatens human and animal health. Yet, mitigating groundwater contamination naturally is often complicated since most aquifers are limited in bioavailable carbon. Since metabolically flexible microbes might have advantages for survival, this study presents a detailed description and first results on our modification of the BacTrap© method, aiming to determine the prevailing microbial community's potential to utilize chemolithotrophic pathways. Our microbial trapping devices (MTDs) were amended with four different iron sources and incubated in seven groundwater monitoring wells for ∼3 months to promote growth of nitrate-reducing Fe(II)-oxidizing bacteria (NRFeOxB) in a nitrate-contaminated karst aquifer. Phylogenetic analysis based on 16S rRNA gene sequences implies that the identity of the iron source influenced the microbial community's composition. In addition, high throughput amplicon sequencing revealed increased relative 16S rRNA gene abundances of OTUs affiliated to genera such as Thiobacillus, Rhodobacter, Pseudomonas, Albidiferax, and Sideroxydans. MTD-derived enrichments set up with Fe(II)/nitrate/acetate to isolate potential NRFeOxB, were dominated by e.g., Acidovorax spp., Paracoccus spp. and Propionivibrio spp. MTDs are a cost-effective approach for investigating microorganisms in groundwater and our data not only solidifies the MTD's capacity to provide insights into the metabolic flexibility of the aquifer's microbial community, but also substantiates its metabolic potential for anaerobic Fe(II) oxidation.

Monitoring redox stress in human airway epithelial cells exposed to woodsmoke at an air-liquid interface.

Wildland fires contribute significantly to the ambient air pollution burden worldwide, causing a range of adverse health effects in exposed populations. The toxicity of woodsmoke, a complex mixture of gases, volatile organic compounds, and particulate matter, is commonly studied in vitro using isolated exposures of conventionally cultured lung cells to either resuspended particulate matter or organic solvent extracts of smoke, leading to incomplete toxicity evaluations. This study aimed to improve our understanding of the effects of woodsmoke inhalation by building an advanced in vitro exposure system that emulates human exposure of the airway epithelium. We report the development and characterization of an innovative system that permits live-cell monitoring of the intracellular redox status of differentiated primary human bronchial epithelial cells cultured at an air-liquid interface (pHBEC-ALI) as they are exposed to unfractionated woodsmoke generated in a tube furnace in real time. pHBEC-ALI exposed to freshly generated woodsmoke showed oxidative changes that were dose-dependent and reversible, and not attributable to carbon monoxide exposure. These findings show the utility of this novel system for studying the molecular initiating events underlying woodsmoke-induced toxicity in a physiologically relevant in vitro model, and its potential to provide biological plausibility for risk assessment and public health measures.

A human lymphoma organoid model for evaluating and targeting the follicular lymphoma tumor immune microenvironment.

Heterogeneity in the tumor microenvironment (TME) of follicular lymphomas (FLs) can affect clinical outcomes. Current immunotherapeutic strategies, including antibody- and cell-based therapies, variably overcome pro-tumorigenic mechanisms for sustained disease control. Modeling the intact FL TME, with its native, syngeneic tumor-infiltrating leukocytes, is a major challenge. Here, we describe an organoid culture method for cultivating patient-derived lymphoma organoids (PDLOs), which include cells from the native FL TME. We define the robustness of this method by successfully culturing cryopreserved FL specimens from diverse patients and demonstrate the stability of TME cellular composition, tumor somatic mutations, gene expression profiles, and B/T cell receptor dynamics over 3 weeks. PDLOs treated with CD3:CD19 and CD3:CD20 therapeutic bispecific antibodies showed B cell killing and T cell activation. This stable system offers a robust platform for advancing precision medicine efforts in FL through patient-specific modeling, high-throughput screening, TME signature identification, and treatment response evaluation.

Distinct Hodgkin lymphoma subtypes defined by noninvasive genomic profiling.

The scarcity of malignant Hodgkin and Reed-Sternberg cells hampers tissue-based comprehensive genomic profiling of classic Hodgkin lymphoma (cHL). By contrast, liquid biopsies show promise for molecular profiling of cHL due to relatively high circulating tumour DNA (ctDNA) levels1-4. Here we show that the plasma representation of mutations exceeds the bulk tumour representation in most cases, making cHL particularly amenable to noninvasive profiling. Leveraging single-cell transcriptional profiles of cHL tumours, we demonstrate Hodgkin and Reed-Sternberg ctDNA shedding to be shaped by DNASE1L3, whose increased tumour microenvironment-derived expression drives high ctDNA concentrations. Using this insight, we comprehensively profile 366 patients, revealing two distinct cHL genomic subtypes with characteristic clinical and prognostic correlates, as well as distinct transcriptional and immunological profiles. Furthermore, we identify a novel class of truncating IL4R mutations that are dependent on IL-13 signalling and therapeutically targetable with IL-4Rα-blocking antibodies. Finally, using PhasED-seq5, we demonstrate the clinical value of pretreatment and on-treatment ctDNA levels for longitudinally refining cHL risk prediction and for detection of radiographically occult minimal residual disease. Collectively, these results support the utility of noninvasive strategies for genotyping and dynamic monitoring of cHL, as well as capturing molecularly distinct subtypes with diagnostic, prognostic and therapeutic potential.

Making good on the promise of genomics in healthcare: the NSW Health perspective.

NSW Health is implementing genomics as a mainstream component of clinical care. The strategic, holistic approach is considering infrastructure, data governance and management, workforce, education, service planning and delivery. This work is generating insights about how to realise the promise of genomics in healthcare, highlighting the need for strong foundations, real-world application, accessibility and a focus on people using genomic information in clinical care.

Unveiling the nongenomic actions of thyroid hormones in adult mammalian brain: The legacy of Mary B. Dratman.

A comprehensive review was conducted to compile the contributions of Mary B. Dratman and studies by other researchers in the field of nongenomic actions of thyroid hormones in adult mammalian brain. Dratman and her collaborators authored roughly half of the papers in this area. It has been almost fifty years since Dratman introduced the novel concept of thyroid hormones as neurotransmitters for the first time. The characterization of unique brain-region specific accumulation of thyroid hormones within the nerve terminals in adult mammals was a remarkable contribution by Dratman. It suggested a neurotransmitter- or neuromodulator-like role of thyroid hormone and/or its derivative, 3-iodothyronamine within adrenergic systems in adult mammalian brain. Several studies by other researchers using synaptosomes as a model system, have contributed to the concept of direct nongenomic actions of thyroid hormones at synaptic regions by establishing that thyroid hormones or their derivatives can bind to synaptosomal membranes, alter membrane functions including enzymatic activities and ion transport, elicit Ca2+/NO-dependent signaling pathways and induce substrate-protein phosphorylation. Such findings can help to explain the physiological and pathophysiological roles of thyroid hormone in psychobehavioral control in adult mammalian brain. However, the exact mode of nongenomic actions of thyroid hormones at nerve terminals in adult mammalian brain awaits further study.

Nongenomic roles of thyroid hormones and their derivatives in adult brain: are these compounds putative neurotransmitters?

We review the evidence regarding the nongenomic (or non-canonical) actions of thyroid hormones (thyronines) and their derivatives (including thyronamines and thyroacetic acids) in the adult brain. The paper seeks to evaluate these compounds for consideration as candidate neurotransmitters. Neurotransmitters are defined by their (a) presence in the neural tissue, (b) release from neural tissue or cell, (c) binding to high-affinity and saturable recognition sites, (d) triggering of a specific effector mechanism and (e) inactivation mechanism. Thyronines and thyronamines are concentrated in brain tissue and show distinctive patterns of distribution within the brain. Nerve terminals accumulate a large amount of thyroid hormones in mature brain, suggesting a synaptic function. However, surprisingly little is known about the potential release of thyroid hormones at synapses. There are specific binding sites for thyroid hormones in nerve-terminal fractions (synaptosomes). A notable cell-membrane binding site for thyroid hormones is integrin αvß3. Furthermore, thyronines bind specifically to other defined neurotransmitter receptors, including GABAergic, catecholaminergic, glutamatergic, serotonergic and cholinergic systems. Here, the thyronines tend to bind to sites other than the primary sites and have allosteric effects. Thyronamines also bind to specific membrane receptors, including the trace amine associated receptors (TAARs), especially TAAR1. The thyronines and thyronamines activate specific effector mechanisms that are short in latency and often occur in subcellular fractions lacking nuclei, suggesting nongenomic actions. Some of the effector mechanisms for thyronines include effects on protein phosphorylation, Na+/K+ ATPase, and behavioral measures such as sleep regulation and measures of memory retention. Thyronamines promptly regulate body temperature. Lastly, there are numerous inactivation mechanisms for the hormones, including decarboxylation, deiodination, oxidative deamination, glucuronidation, sulfation and acetylation. Therefore, at the current state of the research field, thyroid hormones and their derivatives satisfy most, but not all, of the criteria for definition as neurotransmitters.

Height Fluctuations and Surface Gradients in Topographic Measurements.

Topographic maps are composed of pixels associated with coordinates (x, y, z) on a surface. Each pixel location (x, y) is linked with fluctuations in a measured height sample (z). Fluctuations here are uncertainties in heights estimated from multiple topographic measurements at the same position. Height samples (z) are measured at individual locations (x, y) in topographic measurements and compared with gradients on topographies. Here, gradients are slopes on a surface calculated at the scale of the sampling interval from inclination angles of vectors that are normal to triangular facets formed by adjacent height samples (z = z(x, y)). Similarities between maps of gradients logs and height fluctuations are apparent. This shows that the fluctuations are exponentially dependent on local surface gradients. The highest fluctuations correspond to tool/material interactions for turned surfaces and to regions of maximum plastic deformation for sandblasted surfaces. Finally, for abraded, heterogeneous, multilayer surfaces, fluctuations are dependent on both abrasion and light/sub-layer interactions. It appears that the natures of irregular surface topographies govern fluctuation regimes, and that regions which are indicative of surface functionality, or integrity, can have the highest fluctuations.

Tracing Founder Mutations in Circulating and Tissue-Resident Follicular Lymphoma Precursors.

Follicular lymphomas (FL) are characterized by BCL2 translocations, often detectable in blood years before FL diagnosis, but also observed in aging healthy individuals, suggesting additional lesions are required for lymphomagenesis. We directly characterized early cooperating mutations by ultradeep sequencing of prediagnostic blood and tissue specimens from 48 subjects who ultimately developed FL. Strikingly, CREBBP lysine acetyltransferase (KAT) domain mutations were the most commonly observed precursor lesions, and largely distinguished patients developing FL (14/48, 29%) from healthy adults with or without detected BCL2 rearrangements (0/13, P = 0.03 and 0/20, P = 0.007, respectively). CREBBP variants were detectable a median of 5.8 years before FL diagnosis, were clonally selected in FL tumors, and appeared restricted to the committed B-cell lineage. These results suggest that mutations affecting the CREBBP KAT domain are common lesions in FL cancer precursor cells (CPC), with the potential for discriminating subjects at risk of developing FL or monitoring residual disease. SIGNIFICANCE: Our study provides direct evidence for recurrent genetic aberrations preceding FL diagnosis, revealing the combination of BCL2 translocation with CREBBP KAT domain mutations as characteristic committed lesions of FL CPCs. Such prediagnostic mutations are detectable years before clinical diagnosis and may help discriminate individuals at risk for lymphoma development. This article is highlighted in the In This Issue feature, p. 1275.

Investigating and modeling positron emission tomography factors associated with large cell transformation from low-grade lymphomas.

Low-grade lymphomas have a 1%-3% annual risk of transformation to a high-grade histology, and prognostic factors remain undefined. We set to investigate the role of positron emission tomography (PET) metrics in identification of transformation in a retrospective case-control series of patients matched by histology and follow-up time. We measured PET parameters including maximum standard uptake value (SUV-max) and total lesion glycolysis (TLG), and developed a PET feature and lactate dehydrogenase (LDH)-based model to identify transformation status within discovery and validation cohorts. For our discovery cohort, we identified 53 patients with transformation and 53 controls with a similar distribution of follicular lymphoma (FL). Time to transformation and control follow-up time was similar. We observed a significant incremental increase in SUV-max and TLG between control, pretransformation and post-transformation groups (P < 0.05). By multivariable analysis, we identified a significant interaction between SUV-max and TLG such that SUV-max had highest significance for low volume cases (P = 0.04). We developed a scoring model incorporating SUV-max, TLG, and serum LDH with improved identification of transformation (area under the curve [AUC] = 0.91). Our model performed similarly for our validation cohort of 23 patients (AUC = 0.90). With external and prospective validation, our scoring model may provide a specific and noninvasive tool for risk stratification for patients with low-grade lymphoma.

A randomized trial on the effects of heat stress abatement on environmental conditions and growth, feed efficiency, and concentration of metabolites of pre-weaned female Holstein calves.

Heat abatement strategies for pre-weaned calves are seldom adopted. Our objectives were to determine the effects of adding fans to barns on environmental conditions and growth, feed efficiency, concentration of metabolites and health of pre-weaned female Holstein calves. Calves born from July 15th to 30th of 2019 were eligible for enrollment. At birth (d 0), calves were assigned randomly to: SH (n = 125) - hutch under a barn with no cooling, SHF (n = 101) - hutch under a barn cooled through fans. Body weight (BW) and wither-height were measured at birth and d 68. Calves were evaluated thrice weekly (0700-1000 h) using the Calf Health Scoring Chart (UW-Madison). A sub-sample of hutches (SH = 26, SHF = 25) was evaluated for air velocity and temperature at 1000 and 1600 h thrice weekly and calves housed in these hutches were evaluated for rectal temperature (RT) at 1600 h and respiratory rate (RR) at 1000 and 1600 h. Calves were fed a liquid diet twice a day (d 2-18 = 5.56 L/d; d 19-49 = 7.58 L/d; d 50-56 = 3.84 L/d; d 57-63 = 1.64 L/d) and starter ad libitum starting on d 14. A sub-sample of calves (SH = 56, SHF = 44) had intakes of liquid feed and starter measured daily, BW and wither-height measured weekly from birth to d 68, and blood sampled on d 1, 14, 28, 42, 49, 52, 56, 58, 63 and 65 for the measurement of fatty acids, ß-hydroxybutyrate, and glucose concentrations. The SHF treatment increased air velocity by 0.8 m/sec and reduced air temperature by 0.3 ºC. The SHF treatment reduced RT (38.70 ± 0.03 vs. 38.78 ± 0.02 °C) and the percentage of calves with hyperthermia (RT ≥ 39.2 °C; 20.6 ± 1.9 vs. 30.2 ± 2.0%) at 1000 h. Treatment did not affect feed efficiency (SH = 0.53 ± 0.01, SHF = 0.53 ± 0.01 g of BW gained/g of dry matter intake), nor did it affect BW (SH = 81.6 ± 0.7, SHF = 82.9 ± 0.8 kg) and wither-height (SH = 89.5 ± 0.3, SHF = 90.1 ± 0.3 cm) on d 68. Concentrations of metabolites were not affected by treatment. Cooling the environment through fans reduced RT and the risk of hyperthermia at 1000 h but it did not affect performance of pre-weaned Holstein calves.

Top-down Determination of Fluctuations in Topographic Measurements.

A top-down method is presented and studied for quantifying topographic map height (z) fluctuations directly from measurements on surfaces of interest. Contrary to bottom-up methods used in dimensional metrology, this method does not require knowledge of transfer functions and fluctuations of an instrument. Fluctuations are considered here to be indicative of some kinds of uncertainties. Multiple (n), successive topographic measurements (z = z(x,y)) are made at one location without moving the measurand relative to the measurement instrument. The measured heights (z) at each position (x,y) are analyzed statistically. Fluctuation maps are generated from the calculated variances. Three surfaces were measured with two interferometric measuring microscopes (Bruker ContourGT™ and Zygo NewView™ 7300). These surfaces included an anisotropic, turned surface; an isotropic, sandblasted surface; and an abraded, heterogeneous, multilayer surface having different, complex, multiscale morphologies. In demonstrating the method, it was found that few non-measured points persisted for all 100 measurements at any location. The distributions of uncertainties are similar to those of certain features on topographic maps at the same locations, suggesting that topographic features can augment measurement fluctuations. This was especially observed on the abraded ophthalmic lens; a scratch divides the topographic map into two zones with different uncertainty values. The distributions of fluctuations can be non-Gaussian. Additionally, they can vary between regions within some measurements.

Determinants of resistance to engineered T cell therapies targeting CD19 in large B cell lymphomas.

Most relapsed/refractory large B cell lymphoma (r/rLBCL) patients receiving anti-CD19 chimeric antigen receptor (CAR19) T cells relapse. To characterize determinants of resistance, we profiled over 700 longitudinal specimens from two independent cohorts (n = 65 and n = 73) of r/rLBCL patients treated with axicabtagene ciloleucel. A method for simultaneous profiling of circulating tumor DNA (ctDNA), cell-free CAR19 (cfCAR19) retroviral fragments, and cell-free T cell receptor rearrangements (cfTCR) enabled integration of tumor and both engineered and non-engineered T cell effector-mediated factors for assessing treatment failure and predicting outcomes. Alterations in multiple classes of genes are associated with resistance, including B cell identity (PAX5 and IRF8), immune checkpoints (CD274), and those affecting the microenvironment (TMEM30A). Somatic tumor alterations affect CAR19 therapy at multiple levels, including CAR19 T cell expansion, persistence, and tumor microenvironment. Further, CAR19 T cells play a reciprocal role in shaping tumor genotype and phenotype. We envision these findings will facilitate improved chimeric antigen receptor (CAR) T cells and personalized therapeutic approaches.

Molecular Monitoring of Lymphomas.

Molecular monitoring of tumor-derived alterations has an established role in the surveillance of leukemias, and emerging nucleic acid sequencing technologies are likely to similarly transform the clinical management of lymphomas. Lymphomas are well suited for molecular surveillance due to relatively high cell-free DNA and circulating tumor DNA concentrations, high somatic mutational burden, and the existence of stereotyped variants enabling focused interrogation of recurrently altered regions. Here, we review the clinical scenarios and key technologies applicable for the molecular monitoring of lymphomas, summarizing current evidence in the literature regarding molecular subtyping and classification, evaluation of treatment response, the surveillance of active cellular therapies, and emerging clinical trial strategies.

Heat abatement during the pre-weaning period: effects on lying behavior and disbudding-related responses of male Holstein calves.

Strategies to abate heat stress are seldom adopted for pre-weaned dairy calves and little is known about their effects on behavior and pain sensitivity of youngstock. Our objectives were to determine the effects of heat stress abatement on lying behavior and disbudding-related pain sensitivity, wound healing, and change in intake. Male Holstein calves (n = 60; 0 to 68 d of age) were assigned randomly at birth (d 0) to 1 of 3 treatments: hutch outdoors with 50% of its area covered with plywood (control = 20), hutch in a barn with no cooling (SH = 21), and hutch in a barn with ceiling fans (SHF = 19). Calves were fitted with lying-behavior loggers on the hind leg from d 1 to 68. On d 32 ± 8 (±SD), we disbudded calves using hot iron, 30 min after cornual nerves were blocked with lidocaine. Immediately before (0 h), and at 1, 2, and 3 h after disbudding, we evaluated calves for mechanical nociceptive threshold (MNT) and head (ear flick, head shake, head rubbing) and somatic (tail flicking, foot stamping, restlessness) behaviors. On d 1, 3, 7, and 14 after disbudding, we evaluated the MNT and, on d 7 and 14, we evaluated wound healing (1 = crust, 5 = exudate). We calculated the relative change in milk solids and starter intake from d 0 to 6 relative to disbudding compared with the average of the 72 h preceding the procedure. The lying time was 0.6 h/d greater for the SHF treatment compared with the SH treatment. The control treatment resulted in 3.2 and 4.1 more lying bouts per day than the SH and SHF treatments, respectively; consequently, the control treatment resulted in lying-bout duration 7.7 and 10.9 min/event shorter than the SH and SHF treatments, respectively. We did not detect an effect of treatment on the number of disbudding-related head and somatic behaviors and MNT. The odds of calves having abnormal wound was 3.5 and 3.2 times greater for the control treatment compared with the SH and SHF treatments, respectively. We did not detect an effect of treatment on the relative change in intake of milk solids and starter. Heat abatement improves the welfare of pre-weaned dairy calves and may hasten healing.

Heat abatement during the pre-weaning period: effects on growth, feed efficiency, metabolites, and insulin of male Holstein calves.

Heat stress abatement strategies for pre-weaned dairy calves are seldom evaluated. An experiment was conducted to evaluate the effects of housing calves under a barn and provision of fans to calves housed under a barn on calfhood performance. The experiment was conducted in a dairy in southern Georgia, USA. Male Holstein calves (n = 60; 0 to 68 day of age) were assigned randomly at birth (day 0) to 1 of 3 treatments: hutch outdoors with 50% of its area covered with plywood (control = 20), hutch in a barn with no cooling (SH = 21), and hutch in a barn with ceiling fans (SHF = 19). Body weight (BW) was measured at birth, and total serum protein and wither-height were measured 24 to 48 h after birth. A sub-set of hutches was evaluated for air speed and temperature, and rectal temperature (RT) and respiratory frequency (RF) of calves housed in these hutches were measured at 0900 and 1500 h. Intakes of liquid feed (days 14 to 63) and starter (days 14 to 68) were recorded daily, BW and wither-height were measured weekly, and feed efficiency was calculated weekly. Blood was sampled on days 1, 14, 28, 42, 49, 52, 56, 58, 63, and 65 for the measurement of fatty acids, ß-hydroxybutyrate, glucose, and insulin. The SHF treatment resulted in air velocity 0.56 to 0.83 m/s greater (P < 0.01) than the control and SH treatments, respectively, whereas the control treatment resulted in air temperature 1.2 to 3.2 °C greater (P < 0.01) than the SH and SHF treatments, respectively. The RT of calves in the control treatment was 0.1 to 1.1 °C greater (P ≤ 0.03) than the SH and SHF treatments, respectively, and the control treatment resulted in RF 39.4 to 60.2 mov/min greater (P < 0.01) than the SH and SHF treatments, respectively. Treatment did not (P ≥ 0.27) affect feed efficiency and concentrations of metabolites and insulin, but calves in the control treatment were 2.6 cm shorter (P = 0.03) than calves in the SHF treatments at weaning. Provision of fans to calves housed under a barn reduced RT, RF, but only had a minute impact on wither-height.