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Exposure to diesel engine exhaust (DEE) contributes appreciably to the burden of occupational cancer. This study aims to estimate the potential impact of a range of interventions on the future burden of cancer from occupational exposure to DEE in Australia. The future excess fraction method, a novel method based on the lifetime risk approach, was used to model changes in the future burden of cancer among the Australian working age population exposed to DEE at work in 2012 under various intervention strategies. The interventions modeled were based on the widely accepted hierarchy of control model. At baseline, 600 (0.4%) future bladder and 4,450 (0.6%) future lung cancer cases over the lifetime of the cohort were estimated to be attributable to occupational exposure to DEE in those exposed in 2012. Up to 2,000 of these cases were estimated to be avoidable through the use of various interventions. Exhaust hoses (engineering controls) were estimated to be particularly effective. This study provides an indication of which intervention strategies may be most useful in reducing the future burden of cancer associated with occupational DEE exposure. These results show the potential effect of changing current exposure, rather than focusing on past exposures, and thus provide relevant information for policy planning.

Sensitivity Analysis - Bladder

Recent metagenome assembled genome (MAG) analyses have profoundly impacted Rickettsiology systematics. Discovery of basal lineages (Mitibacteraceae and Athabascaceae) with predicted extracellular lifestyles reveals an evolutionary timepoint for the transition to host dependency, which occurred independent of mitochondrial evolution. Notably, these basal rickettsiae carry the Rickettsialesvirhomolog (rvh) type IV secretion system (T4SS) and purportedly uservhto kill congener microbes rather than parasitize host cells as described for derived rickettsial pathogens. MAG analysis also substantially increased diversity for genusRickettsiaand delineated a basal lineage (Tisiphia) that stands to inform on the rise of human pathogens from protist and invertebrate endosymbionts. Herein, we probed Rickettsiales MAG and genomic diversity for the distribution ofRickettsia rvheffectors to ascertain their origins. A sparse distribution of mostRickettsia rvheffectors outside of Rickettsiaceae lineages indicates uniquervhevolution from basal extracellular species and other rickettsial families. Remarkably, nearly every effector was found in multiple divergent forms with variable architectures, illuminating profound roles for gene duplication and recombination in shaping effector repertoires inRickettsiapathogens. Lateral gene transfer plays a prominent role shaping thervheffector landscape, as evinced by the discover of many effectors on plasmids and conjugative transposons, as well as pervasive effector gene exchange betweenRickettsiaandLegionellaspecies. Our study exemplifies how MAGs can provide incredible insight on the origins of pathogen effectors and how their architectural modifications become tailored to eukaryotic host cell biology.

We assessed minimal residual disease (MRD) detection and B-cell aplasia after tisagenlecleucel therapy for acute lymphoblastic leukemia (ALL) to define biomarkers predictive of relapse (N = 143). Next-generation sequencing (NGS) MRD detection >0 in bone marrow (BM) was highly associated with relapse. B-cell recovery [signifying loss of functional chimeric antigen receptor (CAR) T cells] within the first year of treatment was associated with a hazard ratio (HR) for relapse of 4.5 [95% confidence interval (CI), 2.03–9.97; P < 0.001]. Multivariate analysis at day 28 showed independent associations of BMNGS-MRD >0 (HR = 4.87; 95% CI, 2.18–10.8; P < 0.001) and B-cell recovery (HR = 3.33; 95% CI, 1.44–7.69; P = 0.005) with relapse. By 3 months, the BMNGS-MRD HR increased to 12 (95% CI, 2.87–50; P < 0.001), whereas B-cell recovery was not independently predictive (HR = 1.27; 95% CI, 0.33–4.79; P = 0.7). Relapses occurring with persistence of B-cell aplasia were largely CD19− (23/25: 88%). Detectable BMNGS-MRD reliably predicts risk with sufficient time to consider approaches to relapse prevention such as hematopoietic cell transplantation (HCT) or second CAR-T cell infusion. Significance: Detectable disease by BMNGS-MRD with or without B-cell aplasia is highly predictive of relapse after tisagenlecleucel therapy for ALL. Clonotypic rearrangements used to follow NGS-MRD did not change after loss of CD19 or lineage switch. High-risk patients identified by these biomarkers may benefit from HCT or investigational cell therapies. See related commentary by Ghorashian and Bartram, p. 2. This article is highlighted in the In This Issue feature, p. 1

BackgroundThe genus Rickettsia (Alphaproteobacteria: Rickettsiales) encompasses numerous obligate intracellular species with predominantly ciliate and arthropod hosts. Notable species are pathogens transmitted to mammals by blood-feeding arthropods. Mammalian pathogenicity evolved from basal, non-pathogenic host-associations; however, some non-pathogens are closely related to pathogens. One such species, Rickettsia buchneri, is prevalent in the blacklegged tick, Ixodes scapularis. While I. scapularis transmits several pathogens to humans, it does not transmit Rickettsia pathogens. We hypothesize that R. buchneri established a mutualism with I. scapularis, blocking tick superinfection with Rickettsia pathogens.MethodsTo improve estimates for assessing R. buchneri infection frequency in blacklegged tick populations, we used comparative genomics to identify an R. buchneri gene (REIS_1424) not present in other Rickettsia species present throughout the I. scapularis geographic range. Bioinformatic and phylogenomics approaches were employed to propose a function for the hypothetical protein (263 aa) encoded by REIS_1424.ResultsREIS_1424 has few analogs in other Rickettsiales genomes and greatest similarity to non-Proteobacteria proteins. This cohort of proteins varies greatly in size and domain composition, possessing characteristics of Recombination hotspot (Rhs) and contact dependent growth inhibition (CDI) toxins, with similarity limited to proximal C-termini (~145 aa). This domain was named CDI-like/Rhs-like C-terminal toxin (CRCT). As such proteins are often found as toxin-antidote (TA) modules, we interrogated REIS_1423 (151 aa) as a putative antidote. Indeed, REIS_1423 is similar to proteins encoded upstream of CRCT domain-containing proteins. Accordingly, we named these proteins CDI-like/Rhs-like C-terminal toxin antidotes (CRCA). R. buchneri expressed both REIS_1423 and REIS_1424 in tick cell culture, and PCR assays showed specificity for R. buchneri over other rickettsiae and utility for positive detection in three tick populations. Finally, phylogenomics analyses uncovered divergent CRCT/CRCA modules in varying states of conservation; however, only R. buchneri and related Tamurae/Ixodes Group rickettsiae carry complete TA modules.ConclusionWe hypothesize that Rickettsia CRCT/CRCA modules circulate in the Rickettsia mobile gene pool, arming rickettsiae for battle over arthropod colonization. While its functional significance remains to be tested, R. buchneri CRCT/CRCA serves as a marker to positively identify infection and begin deciphering the role this endosymbiont plays in the biology of the blacklegged tick.

Pseudomonas aeruginosa is a Gram-negative nosocomial pathogen and one of the most prevalent organisms isolated from burn wounds worldwide. Pseudomonas aeruginosa strain M2 (O5 serotype, type B flagella) is utilized for examining the murine model associated with burns. Pseudomonas aeruginosa M2 is similar in lethality to common laboratory P. aeruginosa strains when infecting CD-1 mice. Conversely, we recently showed that, relative to these strains, P. aeruginosa M2-infected mice are more susceptible to sepsis and demonstrate a 6-log reduction in LD50 from subcutaneous infection at the infection site directly after 10% total body surface area burn. To better understand this striking phenotypic difference from other P. aeruginosa strains employed in burn models, we sequenced the P. aeruginosa M2 genome. A total of 4,136,641 read pairs were obtained, providing an average genome coverage of 97.5X; subsequent assembly yielded a draft genome with 187 contigs comprising 6,360,304 bp with a G + C content of 66.45%. Genome-based phylogeny estimation of 92 P. aeruginosa strains placed P. aeruginosa M2 with P. aeruginosa-12-4-4(59), a nonairway clinical strain isolated from the blood culture of a burn patient. Phylogenomic analyses identified genes shared between P. aeruginosa M2 and P. aeruginosa 14, another strain exhibiting increased lethality in thermal tissues, as well as P. aeruginosa M2 unique genes with diverse functions like degradation of toxic aromatic compounds, iron scavenging, swarming motility and biofilm formation, defense against invasive DNA, and host assault. Predicted lateral gene transfers illuminate proteins heretofore uncharacterized for roles in P. aeruginosa biology. Our work yields a rich resource for assessing P. aeruginosa genes required for increased lethality in burn tissue seroma.

Peptidoglycan (PG) is a highly cross-linked peptide-glycan mesh that confers structural rigidity and shape to most bacterial cells. Polymerization of new PG is usually achieved by the concerted activity of two membrane-bound machineries, class-A penicillin binding proteins (aPBPs) and class-B penicillin binding proteins (bPBPs) in complex with shape, elongation, division, and sporulation (SEDS) proteins. Here, we have identified four phylogenetically distinct groups of bacteria that lack any identifiable aPBPs. We performed experiments on a panel of species within one of these groups, the Rickettsiales, and found that bacteria lacking aPBPs build a PG-like cell wall with minimal abundance and rigidity relative to cell walls of aPBP-containing bacteria. This reduced cell wall may have evolved to minimize the activation of host responses to pathogens and endosymbionts while retaining the minimal PG-biosynthesis machinery required for cell elongation and division. We term these “peptidoglycan-intermediate” bacteria, a cohort of host-associated species that includes some human pathogens. IMPORTANCE Peptidoglycan (PG) is a large, cross-linked polymer that forms the cell wall of most bacterial species and confers shape, rigidity, and protection from osmotic shock. It is also a potent stimulator of the immune response in animals. PG is normally polymerized by two groups of enzymes, aPBPs and bPBPs working together with shape, elongation, division, and sporulation (SEDS) proteins. We have identified a diverse set of host-associated bacteria that have selectively lost aPBP genes while retaining bPBP/SEDS and show that some of these build a minimal PG-like structure. It is expected that these minimal cell walls built in the absence of aPBPs improve the evolutionary fitness of host-associated bacteria, potentially through evasion of PG-recognition by the host immune system.

Pediatric neuroblastoma (NB) patients receive multi-modal therapy and may experience care fragmented among multiple institutions with a significant travel burden, which has been associated with poor outcomes for some adult cancers. We hypothesized that fragmented care for pediatric NB patients is associated with inferior outcomes compared to treatment consolidated at one location. We reviewed paper and electronic records for pediatric NB patients who received ≥1 hematopoietic stem cell transplant (HSCT) at Duke from 1990-2017. Fragmented care was defined by treatment at >1 institution and grouped by 2 institutions vs. 3+ institutions. Distances were calculated using Google Maps. To compare all care groups, we used Fisher's Exact and Kruskal-Wallis tests for demographic and treatment characteristics, Kaplan-Meier for unadjusted overall survival (OS), and Cox proportional hazards for factors associated with OS. Of 127 eligible patients, 102 (80.3%) patients experienced fragmented care, with 17 treated at 3+ facilities. Kaplan-Meier analysis did not associate fragmented care with increased mortality (log-rank p = 0.13). With multivariate analysis, only earlier diagnostic decade and greater distance to HSCT remained significantly associated with worsened OS. In this single institutional study, we found fragmented care did not impact overall survival. Worsened overall survival was associated with increased travel distance for HSCT and further research should aim to improve supportive processes for patients undergoing HSCT for high-risk neuroblastoma.

Spikes in rickettsioses occur as deforestation, urbanization, and homelessness increase human exposure to blood-feeding arthropods. Still, effective Rickettsia vaccines remain elusive., Species of Rickettsia (Alphaproteobacteria: Rickettsiales) are obligate intracellular parasites of a wide range of eukaryotes, with recognized arthropod-borne human pathogens belonging to the transitional group (TRG), typhus group (TG), and spotted fever group (SFG) rickettsiae. Growing in the host cytosol, rickettsiae pilfer numerous metabolites to make a typical Gram-negative bacterial cell envelope. The O-antigen of rickettsial lipopolysaccharide (LPS) is immunogenic and has been shown to tether the S-layer to the rickettsial surface; however, little is known about the structure and immunogenicity of the Rickettsia lipid A moiety. The structure of lipid A, the membrane anchor of LPS, affects the ability of this molecule to interact with components of the host innate immune system, specifically the MD-2/TLR4 receptor complex. To dissect the host responses that can occur during Rickettsia in vitro and in vivo infection, structural analysis of Rickettsia lipid A is needed. Lipid A was extracted from four Rickettsia species and structurally analyzed. R. akari (TRG), R. typhi (TG), and R. montanensis (SFG) produced a similar structure, whereas R. rickettsii (SFG) altered the length of a secondary acyl group. While all structures have longer acyl chains than known highly inflammatory hexa-acylated lipid A structures, the R. rickettsii modification should differentially alter interactions with the hydrophobic internal pocket in MD2. The significance of these characteristics toward inflammatory potential as well as membrane dynamics between arthropod and vertebrate cellular environments warrants further investigation. Our work adds lipid A to the secretome and O-antigen as variable factors possibly correlating with phenotypically diverse rickettsioses. IMPORTANCE Spikes in rickettsioses occur as deforestation, urbanization, and homelessness increase human exposure to blood-feeding arthropods. Still, effective Rickettsia vaccines remain elusive. Recent studies have determined that Rickettsia lipopolysaccharide anchors the protective S-layer to the bacterial surface and elicits bactericidal antibodies. Furthermore, growing immunological evidence suggests vertebrate sensors (MD-2/TLR4 and noncanonical inflammasome) typically triggered by the lipid A portion of lipopolysaccharide are activated during Rickettsia infection. However, the immunopotency of Rickettsia lipid A is unknown due to poor appreciation for its structure. We determined lipid A structures for four distinct rickettsiae, revealing longer acyl chains relative to highly inflammatory bacterial lipid A. Surprisingly, lipid A of the Rocky Mountain spotted fever agent deviates in structure from other rickettsiae. Thus, lipid A divergence may contribute to variable disease phenotypes, sounding an alarm for determining its immunopotency and possible utility (i.e., as an adjuvant or anti-inflammatory) for development of more prudent rickettsiacidal therapies.

Infections and graft-versus-host disease (GVHD) have historically resulted in high mortality among children undergoing umbilical cord blood transplantation (UCBT). However, recent advances in clinical practice have likely improved outcomes of these patients. We conducted a retrospective cohort study of children (18years of age) undergoing UCBT at Duke University between January 1, 1995 and December 31, 2014. We compared 2-year all-cause and cause-specific mortality during 3 time periods based on year of transplantation (1995 to 2001, 2002 to 2007, and 2008 to 2014). We used multivariable Cox regression to identify demographic and UCBT characteristics that were associated with all-cause mortality, transplantation-related mortality, and death from invasive aspergillosis after adjustment for time period. During the 20-year study period 824 children underwent UCBT. Two-year all-cause mortality declined from 48% in 1995 to 2001 to 30% in 2008 to 2014 (P = .0002). White race and nonmalignant UCBT indications were associated with lower mortality. Black children tended to have a higher risk of death for which GVHD (18% versus 11%; P = .06) or graft failure (9% versus 3%; P = .01) were contributory than white children. Comparing 2008 to 2014 with 1995 to 2001, more than half (59%) of the reduced mortality was attributable to a reduction in infectious mortality, with 45% specifically related to reduced mortality from invasive aspergillosis. Antifungal prophylaxis with voriconazole was associated with lower mortality from invasive aspergillosis than low-dose amphotericin B lipid complex (hazard ratio, .09; 95% confidence interval, .01 to .76). With the decline in mortality from invasive aspergillosis, adenovirus and cytomegalovirus have become the most frequentinfectious causes of death in children after UCBT. Advances in clinical practice over the past 20years improved survival of children after UCBT. Reduced mortality from infections, particularly invasive aspergillosis, accounted for the largest improvement in survival and was associated with use of voriconazole for antifungal prophylaxis.

Rickettsia buchneri (formerly Rickettsia endosymbiont of Ixodes scapularis, or REIS) is an obligate intracellular endoparasite of the black-legged tick, the primary vector of Lyme disease in North America. It is noteworthy among the rickettsiae for its relatively large genome (1.8 Mb) and extraordinary proliferation of mobile genetic elements (MGEs), which comprise nearly 35% of its genome. Previous analysis of the R. buchneri genome identified several integrative conjugative elements named Rickettsiales amplified genomic elements (RAGEs); the composition of these RAGEs suggests that continued genomic invasions by MGEs facilitated the proliferation of rickettsial genes related to an intracellular lifestyle. In this study, we compare the genomic diversity at RAGE loci among sequenced rickettsiae that infect three related Ixodes spp., including two strains of R. buchneri and Rickettsia endosymbiont of Ixodes pacificus strain Humboldt, as well as a closely related species R. tamurae infecting Amblyomma testudinarium ticks. We further develop a novel multiplex droplet digital PCR assay and use it to quantify copy number ratios of chromosomal R. buchneri RAGE-A and RAGE-B to the single-copy gene gltA within natural populations of I. scapularis. Our results reveal substantial diversity among R. buchneri at these loci, both within individual ticks as well as in the I. scapularis population at large, demonstrating that genomic rearrangement of MGEs is an active process in these intracellular bacteria.

Reduced dexterity is a major problem in cold weather, with a need for a countermeasure that increases hand (Thand) and finger (Tfing) temperatures and improves dexterity. The purpose of this study was to determine whether electric heat (set point, 42°C) applied to the forearm (ARM, 82 W), face (FACE, 9.2 W), or combination of both (COMB, 91.2 W), either at the beginning of cold exposure (COLD; 0.5°C, 120 min; 2 clo insulation, seated, bare-handed) or after Tfing fell to 10.5°C [delayed trials (D)], improves Thand, Tfing, dexterity, and finger key pinch strength (Sfing). Volunteers ( n = 8; 26 ± 9 yr) completed 7 experimental trials in COLD: ARM, ARM-D, FACE, FACE-D, COMB, COMB-D, and no heating (CON). Temperatures were measured before (BASE) and throughout COLD. Tests of dexterity [Purdue Pegboard assembly (PP) and magazine loading (MAGLOAD)] and Sfing were measured at BASE and after 45 and 90 min of COLD. Data presented are at minute 90. Thand was warmer ( P < 0.001) during ARM (18.0 ± 2.6°C) and COMB (18.9 ± 2.0°C) versus CON (15.3 ± 1.5°C) and FACE (15.8 ± 1.5°C) for heating that was initiated at the beginning of COLD. Tfing was higher ( P < 0.04) during COMB (12.7 ± 5.1°C) versus CON (9.7 ± 2.1°C) and FACE (8.9 ± 2.2°C). The change from BASE for PP (no. of pieces) was less ( P < 0.005) in COMB (−4.5 ± 3.3) and ARM (−5.0 ± 6.0) versus CON (−13.0 ± 7.3) and FACE (−10.0 ± 8.3), and for MAGLOAD, it tended ( P = 0.06) to be less in COMB (−8.9 ± 6.2 cartridges) versus CON (−14.8 ± 3.7 cartridges). There was no change in Sfing from BASE (10.5 kg) to minute 90 in ARM or COMB (0.7 ± 1.4 and −0.2 ± 1.7 kg, respectively) but a decrease ( P < 0.01) in CON and FACE (−2.1 ± 2.0 and −1.6 ± 1.9 kg, respectively). There were no differences in Thand, Tfing, dexterity, and Sfing at minute 90 when comparing heating that was initiated at the beginning of COLD versus delayed heating. In conclusion, heating using either COMB or ARM, compared with CON and FACE, improved Thand and Tfing and reduced the decline in dexterity by 20%–50% and Sfing by 90%. Furthermore, delayed heating had no deleterious effect on Thand, Tfing, dexterity, and Sfing compared with heating that started at the beginning of cold exposure. NEW & NOTEWORTHY The present study demonstrated that, during sedentary cold air exposure, localized heating that was applied from the beginning of cold exposure on the forearm increases hand and finger temperatures and finger strength, leading to subsequent improvements in manual dexterity. In addition, localized heating that was delayed until finger temperatures cooled significantly also caused higher peripheral temperatures, leading to better strength and manual dexterity, compared with no heating.

Context: In the ELIANA trial, tisagenlecleucel, a chimeric antigen receptor (CAR)-T cell therapy, demonstrated efficacy (81% overall remission rate [ORR]; 60% complete remission [CR]) and safety in pediatric and young adult patients with relapsed/refractory (r/r) B-cell acute lymphoblastic leukemia (B-ALL) (Maude et al. N Engl J Med. 2018). Sustained remissions were associated with B-cell aplasia. In patients with short CAR-T cell persistence, reinfusion with tisagenlecleucel may restore B-cell aplasia in some patients (Maude et al. J Clin Oncol. 2016). Objective: To determine the efficacy and safety of tisagenlecleucel reinfusion in pediatric and young adult patients with B-ALL experiencing loss of B-cell aplasia. Design: HESTER (NCT04225676) is an ongoing phase II, open-label, multicenter trial being conducted in the United States. Clinical and laboratory assessments will be done at months 1, 3, 6, and 12 post-reinfusion. Patients: Eligible patients must be ≤25 years old, with CD19(+) B-ALL and a previous infusion of commercial tisagenlecleucel with ≥1 additional tisagenlecleucel dose available. Patients must exhibit loss of B-cell aplasia, defined as peripheral blood (PB) absolute B lymphocyte count ≥50/µL or PB B lymphocytes ≥10% of the total, but are not required to be minimal residual disease negative (MRD-). Patients with active central nervous system metastasis or treated with prior gene or other T-cell therapies are excluded. Estimated enrollment is approximately 54 patients. Interventions: Reinfusion with tisagenlecleucel will be given once during the study, with the dose range of 0.2-5.0×106 CAR-positive viable T-cells/kg for patients ≤50 kg body weight or 0.1-2.5×108 CAR-positive viable T-cells for patients >50 kg body weight. Reinfusion must occur within 9 months from manufacture. Main Outcomes Measures: The primary efficacy endpoint is the proportion of patients who reestablish B-cell aplasia (circulating B lymphocytes

The cat flea, Ctenocephalides fells, is widely recognized as a global veterinary pest and a vector of pathogenic bacteria. We recently reported on the C. felis nuclear genome, which is characterized by over 38% protein coding gene duplication, extensive tRNA gene family expansion, and remarkable gene copy number variation (CNV) between individual fleas. Herein, we describe the assembly of the C. felis mitochondrial genome, a novel resource for comparative genomics of fleas and other insects. The order and content of mitochondrial genes is highly consistent with four previously sequenced flea mitochondrial genomes, limiting CNV to siphonapteran nuclear genomes.

The soil microbiome plays an essential role in processing and storage of nitrogen (N) and carbon (C), and is influenced by vegetation above-ground through imparted differences in chemistry, structure, mass of plant litter, root physiology, and dominant mycorrhizal associations. We used shotgun metagenomic sequencing and bioinformatic analysis to quantify the abundance and distribution of gene families involved in soil microbial N and C cycling beneath three deciduous hardwood tree species: ectomycorrhizal (ECM)-associated Quercus rubra (red oak), ECM-associated Castanea dentata (American chestnut), and arbuscular mycorrhizal (AM)-associated Prunus serotina (black cherry). Chestnut exhibited the most distinct soil microbiome of the three species, both functionally and taxonomically, with a general suppression of functional genes in the nitrification, denitrification, and nitrate reduction pathways. These changes were related to low inorganic N availability in chestnut stands as soil was modified by poor, low-N litter quality relative to red oak and black cherry soils.IMPORTANCEPrevious studies have used field biogeochemical process rates, isotopic tracing, and targeted gene abundance measurements to study the influence of tree species on ecosystem N and C dynamics. However, these approaches do not enable a comprehensive systems-level understanding of the relationship between microbial diversity and metabolism of N and C below-ground. We analyzed microbial metagenomes from soils beneath red oak, American chestnut, and black cherry stands and showed that tree species can mediate the abundance of key microbial genes involved in N and (to a lesser extent) C metabolism pathways in soil. Our results highlight the genetic framework underlying tree species’ control over soil microbial communities, and below-ground C and N metabolism, and may enable land managers to select tree species to maximize C and N storage in soils.

Wolbachiae are obligate intracellular bacteria that infect arthropods and certain nematodes. Usually maternally inherited, they may provision nutrients to (mutualism) or alter sexual biology of (reproductive parasitism) their invertebrate hosts. We report the assembly of closed genomes for two novel wolbachiae,wCfeT andwCfeJ, found co-infecting cat fleas (Ctenocephalides felis) of the Elward Laboratory colony (Soquel, CA).wCfeT is basal to nearly all describedWolbachiasupergroups, whilewCfeJ is related to supergroups C, D and F. Both genomes contain laterally transferred genes that inform on the evolution ofWolbachiahost associations.wCfeT carries the Biotin synthesis Operon of Obligate intracellular Microbes (BOOM); our analyses reveal five independent acquisitions of BOOM across theWolbachiatree, indicating parallel evolution towards mutualism. Alternately,wCfeJ harbors a toxin-antidote operon analogous to thewPipcinABoperon recently characterized as an inducer of cytoplasmic incompatibility (CI) in flies.wCfeJcinBand immediate-5’ end genes are syntenic to large modular toxins encoded in CI-like operons of certainWolbachiastrains andRickettsiaspecies, signifying that CI toxins streamline by fission of larger toxins. Remarkably, theC.felisgenome itself contains two CI-like antidote genes, divergent from wCfeJcinA, revealing episodic reproductive parasitism in cat fleas and evidencing mobility of CI loci independent of WO-phage. Additional screening revealed predominant co-infection (wCfeT/wCfeJ) amongstC.feliscolonies, though occasionallywCfeJ singly infects fleas in wild populations. Collectively, genomes ofwCfeT,wCfeJ, and their cat flea host supply instances of lateral gene transfers that could drive transitions between parasitism and mutualism.ImportanceMany arthropod and certain nematode species are infected with wolbachiae which are intracellular bacteria well known for reproductive parasitism (RP). Like other RP strategies,Wolbachia-induced cytoplasmic incompatibility, CI, increases prevalence and frequency in host populations. Mutualism is another strategy employed by wolbachiae to maintain host infection, with some strains synthesizing and supplementing certain B vitamins (particularly biotin) to invertebrate hosts. Curiously, we discovered two novelWolbachiastrains that co-infect cat fleas (Ctenocephalides felis):wCfeT carries biotin synthesis genes, whilewCfeJ carries a CI-inducing toxin-antidote operon. Our analyses of these genes highlight their mobility across theWolbachiaphylogeny and source to other intracellular bacteria. Remarkably, theC.felisgenome also carries two CI-like antidote genes divergent from thewCfeJ antidote gene, indicating episodic RP in cat fleas. Collectively,wCfeT andwCfeJ inform on the rampant dissemination of diverse factors that mediateWolbachiastrategies for persisting in invertebrate host populations.

BackgroundFleas (Insecta: Siphonaptera) are small flightless parasites of birds and mammals; their blood-feeding can transmit many serious pathogens (i.e. the etiological agents of bubonic plague, endemic and murine typhus). The lack of flea genome assemblies has hindered research, especially comparisons to other disease vectors. Accordingly, we sequenced the genome of the cat flea,Ctenocephalides felis, an insect with substantial human health and veterinary importance across the globe.ResultsBy combining Illumina and PacBio sequencing with Hi-C scaffolding techniques, we generated a chromosome-level genome assembly forC.felis. Unexpectedly, our assembly revealed extensive gene duplication across the entire genome, exemplified by ∼38% of protein-coding genes with two or more copies and over 4,000 tRNA genes. A broad range of genome size determinations (433-551 Mb) for individual fleas sampled across different populations supports the widespread presence of fluctuating copy number variation (CNV) inC. felis. Similarly broad genome sizes were also calculated for individuals ofXenopsylla cheopis(Oriental rat flea), indicating that this remarkable “genome-in-flux” phenomenon could be a siphonapteran-wide trait. Finally, from theC. felissequence reads we also generated closed genomes for two novel strains ofWolbachia, one parasitic and one symbiotic, found to co-infect individual fleas.ConclusionRampant CNV inC.felishas dire implications for gene-targeting pest control measures and stands to complicate standard normalization procedures utilized in comparative transcriptomics analysis. Coupled with co-infection by novelWolbachiaendosymbionts – potential tools for blocking pathogen transmission – these oddities highlight a unique and underappreciated disease vector.

Background: Tisagenlecleucel is an anti-CD19 chimeric antigen receptor (CAR)-T cell therapy approved for pediatric and young adult patients with relapsed/refractory (r/r) B-cell acute lymphoblastic leukemia (B-ALL) and adult patients with r/r diffuse large B-cell lymphoma. The ELIANA trial showed efficacy (81% overall remission rate [ORR]; 60% complete remission [CR]) and safety of tisagenlecleucel in r/r B-ALL (Maude et al. N Engl J Med. 2018). In the ELIANA trial, sustained remissions were associated with B-cell aplasia, an expected on-target effect of tisagenlecleucel and a pharmacodynamic marker for tisagenlecleucel persistence. In some patients who demonstrated short CAR-T cell persistence, reinfusion with 1 or more additional doses of tisagenlecleucel has restored B-cell aplasia and produced a 60% CR rate in patients who were reinfused with humanized CD19-targeted CAR-T cell therapy (Maude et al. J Clin Oncol. 2016). This prolongs the period of tisagenlecleucel activity and immunosurveillance and may therefore prolong durable remission. We introduce a trial in progress investigating the efficacy and safety of tisagenlecleucel reinfusion in pediatric and young adult patients with B-ALL experiencing a loss of B-cell aplasia. Study Design and Methods: HESTER (NCT04225676) is a phase II, open-label, multicenter trial to determine the efficacy and safety of tisagenlecleucel reinfusion in pediatric and young adult patients with B-ALL experiencing loss of B-cell aplasia. Eligible patients must be ≤25 years of age with a confirmed diagnosis of CD19(+) leukemia. Patients must have been previously infused with commercial tisagenlecleucel and have at least 1 additional dose of commercial tisagenlecleucel prescribed to them in the course of medical practice. Commercial tisagenlecleucel must be given for reinfusion within 9 months of the initial manufacturing date. Patients must have loss of B-cell aplasia defined as peripheral blood (PB) absolute B lymphocyte count ≥50/μL or PB B lymphocyte ≥10% of the total lymphocytes; patients are not required to be minimal residual disease negative (MRD)(-). Karnofsky (age ≥16 years) or Lansky (age The primary efficacy endpoint is the proportion of patients who reestablish B-cell aplasia within 12 months of reinfusion as measured by circulating B lymphocytes ( For the primary analysis, a minimum of 10% of patients reestablishing B-cell aplasia within 12 months after reinfusion is expected with an estimated true rate of 25%. All secondary and exploratory variables will be summarized descriptively. Estimated enrollment is about 54 patients in the United States. Clinical Trial Information: NCT04225676 Disclosures Boyer: Thunder Biotech Inc: Consultancy. Grupp:Servier: Research Funding; Cellectis: Other; Roche: Consultancy; Adaptimmune: Other: SAB; Jazz: Other: SSC; TCR2: Other: SAB; GlaxoSmithKline: Consultancy; CRISPR Therapeutics/Vertex Pharmaceuticals: Other; Juno/BMS: Other; Janssen/JnJ: Consultancy; Humanigen: Consultancy; CBMG: Consultancy; Kite/Gilead: Research Funding; Allogene: Other; Novartis: Consultancy, Other: SSC, Research Funding. Hermiston:Novartis: Membership on an entity's Board of Directors or advisory committees; Sobi: Membership on an entity's Board of Directors or advisory committees. Kovacs:Novartis: Current Employment. Magley:Novartis: Current Employment. Myers:Novartis: Consultancy, Honoraria, Other: ELIANA trial Steering Committee, Speakers Bureau. Phillips:Novartis: Membership on an entity's Board of Directors or advisory committees. Pulsipher:Bellicum: Honoraria; Mesoblast: Honoraria; Miltenyi: Honoraria, Research Funding; Adaptive: Research Funding; Novartis: Honoraria; Jasper: Honoraria. Purkayastha:Novartis: Current Employment. Willert:Novartis: Current Employment.

The soil microbiome plays an essential role in processing and storage of nitrogen (N) and carbon (C) and is influenced by vegetation above-ground through imparted differences in chemistry, structure, mass of plant litter, root physiology, and dominant mycorrhizal associations. We used shotgun metagenomic sequencing to quantify the abundance and distribution of gene families involved in soil microbial N and C cycling beneath three deciduous hardwood tree species: ectomycorrhizal (ECM)-associated Quercus rubra (red oak), ECM-associated Castanea dentata (American chestnut), and arbuscular mycorrhizal (AM)-associated Prunus serotina (black cherry). Chestnut exhibited the most distinct soil microbiome of the three species, both functionally and taxonomically, with a general suppression of functional genes in the nitrification, denitrification, and nitrate reduction pathways. These changes were related to low inorganic N availability in chestnut stands as soil was modified by poor, low-N litter quality relative to red oak and black cherry soils. Previous studies have used field biogeochemical process rates, isotopic tracing, and targeted gene abundance measurements to study the influence of tree species on ecosystem N and C dynamics. However, these approaches do not enable a comprehensive systems-level understanding of the relationship between microbial diversity and dynamics of N and C below-ground. We analyzed microbial metagenomes from soils beneath red oak, American chestnut, and black cherry stands and showed that tree species can mediate the abundance of key microbial genes involved in N and (to a lesser extent) C metabolism pathways in soil. We compare gene abundance values to measures of N and C flux from incubated soil. Our results highlight the genetic framework underlying tree species’ control over soil microbial communities, and below-ground C and N metabolism, and may enable land managers to select tree species to maximize C and N storage in soils.

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening disorder of immune dysregulation characterized by fever, hepatosplenomegaly, cytopenias, central nervous system disease, increased inflammatory markers, and hemophagocytosis. Currently, allogeneic hematopoietic stem cell transplantation is the only curative approach for patients with HLH, with reported survival ranging from 50% to 70% with myeloablative conditioning (MAC) regimens. However, donor availability and transplantation-related mortality associated with conventional MAC are major barriers to success. Unrelated umbilical cord blood transplantation (UCBT) provides a readily available alternative donor source for patients lacking matched related donors. Accordingly, we report the results of UCBT in 14 children treated between 1998 and 2016. All children received standard HLH chemotherapy before UCBT. The median age at diagnosis was 2.7 months (range, .8 to 10.4) and at transplantation was 7.5 months (range, 3.8 to 17). Ten patients received MAC with busulfan/cyclophosphamide/etoposide /antithymocyte globulin (ATG) (n = 5), busulfan/cyclophosphamide /ATG (n = 4), or busulfan /melphalan/ATG (n = 1). Four patients received reduced-toxicity conditioning (RTC) with alemtuzumab/fludarabine/melphalan/hydroxyurea ± thiotepa. Cord blood units were mismatched at either 1 (n = 9) or 2 (n = 5) loci and delivered a median total nucleated cell dose of 11.9 × 107/kg (range, 4.6 to 27.9) and CD34+ dose of 3.1 × 105/kg (range, 1.1 to 6.8). The cumulative incidence of neutrophil engraftment by day 42 was 78.6% (95% confidence interval [CI], 42.9% to 93.4%) with a median of 19 days (range, 13 to 27), and that for platelet (50,000) engraftment by day 100 was 64.3% (95% CI, 28.2% to 85.7%) with a median of 51 days (range, 31 to 94). Six patients developed either grade II (n = 5) or grade IV (n = 1) acute graft-versus-host disease (GVHD); no extensive chronic GVHD was seen. Ten patients (71.4%) are alive and well at a median of 11.2 years after transplantation (range, .85 to 18.25), 9 of whom maintain sustained full donor chimerism after a single UCBT, whereas 1 patient with autologous recovery after first UCBT with RTC has achieved full donor chimerism after a second UCBT with MAC. This series demonstrates that, in combination with standard HLH therapy, UCBT after MAC or RTC conditioning can provide long-term survival with durable complete donor chimerism comparable to that of conventional donors. UCBT should be considered for patients with HLH lacking a fully matched related or unrelated adult donor.

At our institution, a high proportion of children with onychocryptosis (ingrown toenail) requiring surgical intervention were noted to have a history of hematopoietic stem cell transplantation (HSCT). We analyzed the characteristics of patients who underwent surgical intervention for onychocryptosis and examined our institutional HSCT database to determine if an association exists between onychocryptosis and HSCT.Surgical cases for onychocryptosis performed from 2000 to 2012 were identified. Nine demographic, clinical, and perioperative variables for both patients with and without prior HSCT were assessed. In a separate analysis, the institutional HSCT database was then queried to identify the prevalence and clinical characteristics associated with onychocryptosis after HSCT.We identified 17 children who had undergone surgical management of onychocryptosis, of which 8 (47.1%) had previous HSCT. Children who had undergone HSCT had an aggressive form of onychocryptosis with 50.0% having bilateral great toe and nail edge involvement and 37.5% having a recurrence. In HSCT cohort analysis of 1069 children, 91 (8.5%) had onychocryptosis. Male sex, non-black race, acute graft versus host disease, and increasing age at transplantation were independently associated with onychocryptosis.HSCT is strongly associated with onychocryptosis requiring surgical intervention. Children with a history of HSCT may also have more aggressive toenail disease, with higher rates of surgical intervention, bilateral ingrown toenails, recurrence, and need for return to the operating room. Clinicians should perform careful screening and early treatment in these patients.

Background Fleas (Insecta: Siphonaptera) are small flightless parasites of birds and mammals; their blood-feeding can transmit many serious pathogens (i.e., the etiological agents of bubonic plague, endemic and murine typhus). The lack of flea genome assemblies has hindered research, especially comparisons to other disease vectors. Accordingly, we sequenced the genome of the cat flea, Ctenocephalides felis, an insect with substantial human health and veterinary importance across the globe. Results By combining Illumina and PacBio sequencing of DNA derived from multiple inbred female fleas with Hi-C scaffolding techniques, we generated a chromosome-level genome assembly for C. felis. Unexpectedly, our assembly revealed extensive gene duplication across the entire genome, exemplified by ~ 38% of protein-coding genes with two or more copies and over 4000 tRNA genes. A broad range of genome size determinations (433–551 Mb) for individual fleas sampled across different populations supports the widespread presence of fluctuating copy number variation (CNV) in C. felis. Similarly, broad genome sizes were also calculated for individuals of Xenopsylla cheopis (Oriental rat flea), indicating that this remarkable “genome-in-flux” phenomenon could be a siphonapteran-wide trait. Finally, from the C. felis sequence reads, we also generated closed genomes for two novel strains of Wolbachia, one parasitic and one symbiotic, found to co-infect individual fleas. Conclusion Rampant CNV in C. felis has dire implications for gene-targeting pest control measures and stands to complicate standard normalization procedures utilized in comparative transcriptomics analysis. Coupled with co-infection by novel Wolbachia endosymbionts—potential tools for blocking pathogen transmission—these oddities highlight a unique and underappreciated disease vector.

Summary Background The ELIANA trial showed that 61 (81%) of 75 paediatric and young adult patients with relapsed or refractory B-cell acute lymphoblastic leukaemia achieved overall remission after treatment with tisagenlecleucel, a chimeric antigen receptor targeted against the CD19 antigen. We aimed to evaluate patient-reported quality of life in these patients before and after tisagenlecleucel infusion. Methods ELIANA, a global, single-arm, open-label, phase 2 trial, was done in 25 hospitals across Australia, Austria, Belgium, Canada, France, Germany, Italy, Japan, Norway, Spain, and the USA. Patients with B-cell acute lymphoblastic leukaemia aged at least 3 years at the time of screening and 21 years or younger at the time of initial diagnosis who were in second or greater bone marrow relapse, chemorefractory, relapsed after allogeneic stem-cell transplantation, or were otherwise ineligible for allogeneic stem-cell transplantation were enrolled. Patients received a single intravenous administration of a target dose of 0·2–5 × 106 transduced viable T cells per kg for patients weighing 50 kg or less or 0·1–2·5 × 108 transduced viable T cells for patients weighing more than 50 kg. The primary outcome, reported previously, was the proportion of patients who achieved remission. A prespecified secondary endpoint, reported here, was patient-reported quality of life measured with the Pediatric Quality of Life Inventory (PedsQL) and European Quality of Life-5 Dimensions questionnaire (EQ-5D). Patients completed the questionnaires at baseline, day 28, and months 3, 6, 9, and 12 after treatment. The data collected were summarised using descriptive statistics and post-hoc mixed models for repeated measures. Change from baseline response profiles were illustrated with cumulative distribution function plots. The proportion of patients achieving the minimal clinically important difference and normative mean value were reported. Analysis was per protocol. This study is registered with ClinicalTrials.gov , NCT02435849 . Findings Between April 8, 2015, and April 25, 2017, 107 patients were screened, 92 were enrolled, and 75 received tisagenlecleucel. 58 patients aged 8–23 years were included in the analysis of quality of life. At baseline, 50 (86%) patients had completed the PedsQL questionnaire and 48 (83%) had completed the EQ-5D VAS. Improvements in patient-reported quality-of-life scores were observed for all measures at month 3 after tisagenlecleucel infusion (mean change from baseline to month 3 was 13·3 [95% CI 8·9–17·6] for the PedsQL total score and 16·8 [9·4–24·3] for the EQ-5D visual analogue scale). 30 (81%) of 37 patients achieved the minimal clinically important difference at month 3 for the PedsQL total score and 24 (67%) of 36 patients achieved this for the EQ-5D visual analogue scale. Interpretation These findings, along with the activity and safety results of ELIANA, suggest a favourable benefit–risk profile of tisagenlecleucel in the treatment of paediatric and young adult patients with relapsed or refractory B-cell acute lymphoblastic leukaemia. Funding Novartis.

Extensive clonal T cell expansion is associated with response to immunotherapy., In both human and murine systems, we have developed an adoptive cellular therapy platform against medulloblastoma and glioblastoma that uses dendritic cells pulsed with a tumor RNA transcriptome to expand polyclonal tumor-reactive T cells against a plurality of antigens within heterogeneous brain tumors. We demonstrate that peripheral TCR Vβ repertoire analysis after adoptive cellular therapy reveals that effective response to adoptive cellular therapy is concordant with massive in vivo expansion and persistence of tumor-specific T cell clones within the peripheral blood. In preclinical models of medulloblastoma and glioblastoma, and in a patient with relapsed medulloblastoma receiving adoptive cellular therapy, an early and massive expansion of tumor-reactive lymphocytes, coupled with prolonged persistence in the peripheral blood, is observed during effective therapeutic response to immunotherapy treatment.

BACKGROUND Recurrent medulloblastoma and malignant glioma are lethal tumors that are virtually incurable. The cytomegalovirus (CMV) antigen pp65 is ubiquitously expressed on medulloblastoma and malignant glioma but not on healthy brain. We evaluated autologous CMV pp65 RNA-pulsed dendritic cell (DC) vaccines in children and young adults in a phase I trial. METHODS Circulating monocytes were harvested using leukapheresis, differentiated into DCs, matured, and pulsed with pp65 RNA using electroporation. DCs were packaged into vaccines (2x107DC/vaccine) and administered intradermally following tetanus-diphtheria toxoid site preconditioning every 2 weeks x3, then monthly. The primary objectives of the study were to establish the feasibility of generating at least 3 vaccines and safety. An exploratory objective was to evaluate the ability of vaccination to create and enhance patient pp65-specific T cell responses. RESULTS Eleven patients were enrolled with medulloblastoma (n=3) or glioblastoma (n=8). Ages ranged from 9–30 years old (mean 15.5y). Ten of 11 patients (91%) generated at least 3 vaccines (mean 6.2). Eight patients received at least 3 vaccines. To date, 4 patients have received all generated vaccines without progression, 4 patients have progressed, and 2 patients are still receiving vaccines. There have not been any severe adverse events probably or definitely related to vaccines. More mature data will be presented at ISPNO. CONCLUSIONS Leukapheresis and monocyte differentiation is a feasible strategy for generating adequate DCs for active immunization in children with malignant brain tumors. CMV pp65 RNA-pulsed DCs are well-tolerated and immunogenic. Efficacy endpoints will be evaluated in a subsequent phase II trial.

Plumajes Aberrantes en Accipiter cooperii Documentamos dos tipos de plumajes melanicos aberrantes, palido y oscuro, en tres individuos de Accipiter cooperii en estado silvestre. Capturamos y fotografiamos dos individuos de A. cooperii con plumaje palido en Dakota del Norte y un individuo de la misma especie en Virginia. Este es el primer estudio de muda de un individuo de A. cooperii de un plumaje aberrante juvenil palido a un segundo plumaje basico palido. Ademas, una de las dos crias macho presenta un plumaje palido como su madre, lo que sugiere que la aberracion es heredable. Tambien describimos el primer plumaje melanico oscuro para A. cooperii. [Traduccion del equipo editorial]

BACKGROUND Recurrent medulloblastoma and malignant glioma are virtually incurable. In children, these tumors are often disseminated or occur in deep midline structures that preclude surgery and are not amenable to highly inflammatory treatments. Active immunization is an attractive immunotherapeutic approach. We are conducting a phase I trial evaluating autologous CMV pp65 RNA-pulsed dendritic cell (DC) vaccines in children with recurrent malignant glioma or medulloblastoma. A primary objective is establishing the feasibility of using leukapheresis to harvest sufficient monocytes in order to generate at least 3 autologous DC vaccines. METHODS For children unable to receive two 16-guage IVs, a temporary pheresis catheter is placed in the jugular vein under anesthesia. Leukapheresis is performed with the goal of processing 3 total blood volumes. Circulating monocytes are collected and cultured for 7 days in media supplemented with GM-CSF and IL-4, then matured into dendritic cells and loaded with antigen. The target dose for a single vaccine is 2 x 107 dendritic cells. RESULTS To date, five patients (ages 9, 13, 16, 17, 30) have undergone leukapheresis. All patients tolerated leukapheresis without complications, with run times between 3 to 6 hours depending on achievable pheresis flow rate. Patients generated 2, 3, 7, 8, 8, and 8 vaccines. The medulloblastoma patient who generated 2 vaccines underwent a second leukapheresis that generated 8 vaccines. In this patient, although the initial leukapheresis collected a high number of monocytes, the conversion rate to mature DCs was low. Thusfar, there has not been a strong correlation between the number of monocytes collected and the eventual number of mature dendritic cells. CONCLUSIONS Leukapheresis is tolerable for younger children and appears to be a viable strategy for harvesting sufficient monocytes for creating DC vaccines. Conversion rates between harvested monocytes and the final number of mature dendritic cells vary.

While typically a flea parasite and opportunistic human pathogen, the presence of Rickettsia felis (strain LSU-Lb) in the non-blood-feeding, parthenogenetically reproducing booklouse, Liposcelis bostrychophila, provides a system to ascertain factors governing not only host transitions but also obligate reproductive parasitism (RP). Analysis of plasmid pLbAR, unique to R. felis str. LSU-Lb, revealed a toxin–antitoxin module with similar features to prophage-encoded toxin–antitoxin modules utilized by parasitic Wolbachia strains to induce another form of RP, cytoplasmic incompatibility, in their arthropod hosts. Curiously, multiple deubiquitinase and nuclease domains of the large (3,841 aa) pLbAR toxin, as well the entire antitoxin, facilitated the detection of an assortment of related proteins from diverse intracellular bacteria, including other reproductive parasites. Our description of these remarkable components of the intracellular mobilome, including their presence in certain arthropod genomes, lends insight on the evolution of RP, while invigorating research on parasite-mediated biocontrol of arthropod-borne viral and bacterial pathogens.

We performed a phase I study to assess feasibility, safety, and efficacy of ALT+DCV following HDC +ASCR (group A) or NMAC (group B) in pts with r-PNET. METHODS: Eligible pts underwent surgery to confirm diagnosis and obtain tumor for vaccine preparation. Pts with local (group A) or metastatic (group B) recurrence received 4 cycles of cytoreductive chemotherapy prior to either MAC (carboplatin + thiotepa + etoposide) (group A) or NMAC (cyclophosphamide + Fludarabine) (group B). Two dose levels of ALT were evaluated [3 x 10(6) or 3 x 10(7) cells/kg] with 3 intradermal biweekly DCVs (10(7)cells each). Safety evaluation for DLT assessment was 2 weeks past third DCV with monthly vaccines thereafter as available. Correlative studies for immune response included TCR sequencing and measurement of serum cytokines RESULTS: Ten pts (M5; F5) were treated on protocol (group A 1, group B 9). Nine evaluable pts received immunotherapy; DCV [median 3 doses (3-9)] + ALT [3 x10(6)/kg (n=4) or 3 x 10(7)/kg (n=5)]. There were no DLTs observed. At median of 21 months follow-up, 3 pts are alive with (n=2) or without (n=1) disease 33-42 months following treatment. TCR RNA sequencing demonstrates massive clonal expansion of T cells following ALT+DCV and positive correlation with clinical outcomes. Persistence of tumor-reactive (IFN-gamma secreting) T cell clones in the peripheral blood was observed 4 months after treatment in a patient with long-term disease control. CONCLUSIONS: ALT+DCV is feasible, safe, and shows signs of biologic and clinical activity in some pts with r-PNET.