Search

Interstitial meiofauna, organisms smaller than 500 μm that live between sediment grains, are the most abundant animals on Earth. They play crucial roles in biogeochemical cycles, but their responses to microplastics (MPs) remain understudied. Due to their size, meiofauna may be particularly vulnerable to MPs. We quantified how realistic levels of MP contamination affect bioturbation, oxygen penetration depth (OPD), and diffusive oxygen uptake (DOU) in sediment mesocosms over thirteen days. Bioturbation depth and OPD increased, while DOU decreased across all treatments. However, sediments containing MPs had lower bioturbation depth and slightly higher OPD compared to controls. The reduction in bioturbation was likely due to meiofauna stress, while the highest MP contamination caused increased bioturbation depth, likely due to evasion responses. Increased OPD over time was likely due to reduced labile organic matter. This study highlights how bioturbation, OPD, and DOU shift with MP pollution, confirming MPs' impacts on ecosystem functions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

In vitro cultures remain crucial for studying the fundamental mechanisms of human T-cell development. Here, we introduce a novel in vitro cultivation system based on ThymoSpheres (TS): dense spheroids consisting of DLL4-expressing stromal cells and human hematopoietic precursor cells, in the absence of thymic epithelial cells. These spheroids are subsequently cultured at the air-liquid interphase. TS generate large numbers of mature T cells, are easy to manipulate, scalable, and can be repeatably sampled to monitor T-cell differentiation. The mature T cells generated from primary human hematopoietic precursor cells were extensively characterized using single-cell RNA and combined T-cell receptor (TCR) sequencing. These predominantly CD8α T cells exhibit transcriptional and TCR CDR3 characteristics similar to the recently described human polyclonal αβ unconventional T cell (UTC) lineage. This includes the expression of hallmark genes associated with agonist selection, such as IKZF2 (Helios), and the expression of various natural killer receptors. The TCR repertoire of these UTCs is polyclonal and enriched for CDR3-associated autoreactive features and early rearrangements of the TCR-α chain. In conclusion, TS cultures offer an intriguing platform to study the development of this human polyclonal UTC lineage and its inducing selection mechanisms., (© 2024 The Author(s). European Journal of Immunology published by Wiley‐VCH GmbH.)

Objectives: Oral immune tolerance (OT) is a complex process with unknown genetic regulation. Our aim is to explore possible genetic control of OT in patients with rheumatoid arthritis (RA)., Methods: RA patients with increased interferon γ production invitro when their isolated peripheral blood mononuclear cells (PBMC) were cultured with type II bovine collagen α1 chain [α1 (II)] were enrolled in this study and were randomly assigned to the "Low dose" type II collagen (CII) group (30 µg/day for 10 weeks, followed by 50 µg/day for 10 weeks, followed by 70 µg/day for 10 weeks) or "High dose" CII group (90 µg/day for 10 weeks, followed by 110 µg/day for 10 weeks, followed by 130 µg/day for 10 weeks). Heparinized blood was obtained at baseline and after each of the 10 weeks treatment for analysis of the invitro production of IFNγ by their PBMC stimulated by α1(II) . Single nucleotide polymorphism (SNP) analysis of the responders and non-responders to oral CII was conducted using GeneChip Mapping 10 K 2.0 Array., Results: The SNP A-15,737 was found to associate with the ability of CII to suppress IFNγ production by α1(CII)-stimulated RA PBMC. The potential for SNP A-15,737 to associate with the OT response for patients with another autoimmune disease [OT induced by oral type I bovine collagen (CI) in patients with diffuse cutaneous systemic sclersodid (dsSSc)] was also explored., Conclusions: The ROT1 region plays a role in the control of IFNγ production after oral dosing of auto-antigens, thereby determining if oral tolerance to that antigen will develop., Competing Interests: Declaration of competing interests Drs. Arnold E. Postlethwaite and Weikuan Gu are listed on a US Patent #10,450,610B2 owned by University of Tennessee Research Foundation. This work was supported by grant AR45252 (AEP) and AR9–2242 (AEP from NIAMS/NIH and grants from Department of Veterans Affairs (AEP, WG). The authors thank Ms. Angela Cody for excellent help in drafting this manuscript. This publication is dedicated to honoring Dr. Arnold E. Postlethwaite for his more 50 years of dedicated research and contribution on the immune disease and the field of biomedical sciences., (Copyright © 2024 Southern Society for Clinical Investigation. Published by Elsevier Inc. All rights reserved.)

Many school nurses experienced increased work burden and stress during the COVID-19 pandemic. This analysis examined data from a Centers for Disease Control and Prevention cross-sectional, nationwide survey of school nurses in March 2022 to examine associations between school nurses' ability to conduct their core responsibilities and selected nurse and school factors among school nurses during the 2021-2022 school year and COVID-19 pandemic. Perceived adequate staffing and financial compensation reduced the odds of reported difficulties across all core school nursing tasks. Nurses without a registered nurse license and with higher caseloads were more likely to report difficulty in implementing specific tasks. The impact of these factors varied, with inadequate financial compensation having the largest association with school nurses' difficulty implementing all the core responsibilities. The study results improve our understanding of school nurses' challenges in implementing core school nursing responsibilities during the COVID-19 pandemic in the 2021-2022 school year., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

CD70 is an attractive target for chimeric antigen receptor (CAR) T-cell therapy for the treatment of both solid and liquid malignancies. However, the functionality of CD70-specific CAR T cells is modest. We optimized a CD70-specific VHH-based CAR (nanoCAR). We evaluated the nanoCARs in clinically relevant models in vitro, using co-cultures of CD70-specific nanoCAR T cells with malignant rhabdoid tumor organoids, and in vivo, using a diffuse large B-cell lymphoma patient-derived xenograft (PDX) model. Although the nanoCAR T cells were highly efficient in organoid co-cultures, they showed only modest efficacy in the PDX model. We determined that fratricide was not causing this loss in efficacy but rather CD70 interaction in cis with the nanoCAR-induced exhaustion. Knocking out CD70 in nanoCAR T cells using CRISPR/Cas9 resulted in dramatically enhanced functionality in the diffuse large B-cell lymphoma PDX model. Through single-cell transcriptomics, we obtained evidence that CD70 knockout CD70-specific nanoCAR T cells were protected from antigen-induced exhaustion. In addition, we demonstrated that wild-type CD70-specific nanoCAR T cells already exhibited signs of exhaustion shortly after production. Their gene signature strongly overlapped with gene signatures of exhausted CAR T cells. Conversely, the gene signature of knockout CD70-specific nanoCAR T cells overlapped with the gene signature of CAR T-cell infusion products leading to complete responses in chronic lymphatic leukemia patients. Our data show that CARs targeting endogenous T-cell antigens negatively affect CAR T-cell functionality by inducing an exhausted state, which can be overcome by knocking out the specific target., (©2024 American Association for Cancer Research.)

Non-small cell lung cancer (NSCLC) is known for high relapse rates despite resection in early stages. Here, we present the results of a phase I clinical trial in which a dendritic cell (DC) vaccine targeting patient-individual neoantigens is evaluated in patients with resected NSCLC. Vaccine manufacturing is feasible in six of 10 enrolled patients. Toxicity is limited to grade 1-2 adverse events. Systemic T cell responses are observed in five out of six vaccinated patients, with T cell responses remaining detectable up to 19 months post vaccination. Single-cell analysis indicates that the responsive T cell population is polyclonal and exhibits the near-entire spectrum of T cell differentiation states, including a naive-like state, but excluding exhausted cell states. Three of six vaccinated patients experience disease recurrence during the follow-up period of 2 years. Collectively, these data support the feasibility, safety, and immunogenicity of this treatment in resected NSCLC., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Objective: We examine role of ACEs and pathways to risk of opioid misuse among young adults. Participants and Methods: A cross-sectional survey of validated measures of ACEs, risk of opioid misuse, and health conditions with a sample of 1,402 students from a large public university followed by multivariate logistic regression and pathway analysis. Results: Majority (61%) of participants reported at least one ACE. A dose-response relationship between numbers of ACEs with risk for opioid misuse was present. Compared to participants with no ACEs, participants with ≥4 ACEs and 0-3 ACEs were 2.93 (95% CI: 1.95, 4.39; p  < 0.001) and 1.96 (95% CI: 1.46, 2.65; p  < 0.001) times more likely to be at risk for opioid misuse, respectively. Having at least one existing or past health condition significantly mediated the association. Conclusions: Our findings suggest need to include assessment of ACEs as a screening criterion for opioid prescription and administration among college-aged individuals.

Fauna across many taxa and trophic levels have been shown to consume microplastics (MPs) in experiments, providing evidence that supports field-based gut content assessments. Multiple explanations exist regarding why fauna consume MPs, one of which posits that microbial growth on MPs may facilitate faunal ingestion. However, laboratory assessments on the reasons why MPs are consumed remain limited. Here, we assessed if the presence of microbes on MPs altered marine nematode feeding behaviors across current and potential future concentrations of MPs in a local system. We used a microcosm experiment in which field-collected sediment was spiked with bacterially treated or untreated fluorescent plastic microbeads (1.0-5.0 μm) in concentrations of 10 2 , 10 4 , and 10 6 per microcosm, representing local and potential future concentrations of MPs. Ingestion by the dominant interstitial fauna was investigated after 0, 3, and 7 days using bright field microscopy. Nematodes were the only fauna across microcosms that consumed MPs, but this consumption was variable and there were no apparent trends across exposure time, bacterial treatment, or MP concentration. There were also no genera- or feeding-type-specific trends in the number of MPs consumed, though four of the top five nematode genera that consumed MPs were pollution-tolerant genera. Our study demonstrates that microbe-MP associations do not drive marine nematodes to eat MPs, especially at local field concentrations. While there were no trends across any of the nematode genera in our study, we recognize that unrealistic MP concentrations in other studies may provide alternative explanations for nematode consumption of MPs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Wastewater treatment plants (WWTPs) represent major point sources of pollution in coastal systems, affecting benthic ecosystems. In the present study, we assessed the potential role that WWTPs have in shaping nematode communities and established baseline knowledge of free-living nematode community structures in St. Andrew Bay, Florida. Sediment samples were collected from four sites representing areas of WWTP outflow and areas with no apparent outflow, during the winter and summer. Nematode communities across sites were significantly different, and the differences were strongly associated with the distance to the nearest WWTP. While the communities were not different along transects at each site, nor across seasons, community dissimilarity across sites was high, implying strong contrasts throughout the bay system. Dominance of tolerant, opportunistic genera and Ecological Quality Status assessments suggest that the system is stressed by organic enrichment, possibly linked to the WWTPs. Our results suggest that knowledge on the life-history of dominant genera is imperative to assess the ecological quality of a benthic system, in addition to taxonomic and functional metrics. Considering the value of marine nematodes as bioindicators, more work should be done to monitor temporal variability in nematode communities in this system as future infrastructure changes alter its dynamics., (© 2023. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

The Wiskott-Aldrich syndrome (WAS) is an X-linked primary immune deficiency caused by a mutation in the WAS gene. This leads to altered or absent WAS protein (WASp) expression and function resulting in thrombocytopenia, eczema, recurrent infections, and autoimmunity. In T cells, WASp is required for immune synapse formation. Patients with WAS show reduced numbers of peripheral blood T lymphocytes and an altered T-cell receptor repertoire. In vitro , their peripheral T cells show decreased proliferation and cytokine production upon aCD3/aCD28 stimulation. It is unclear whether these T-cell defects are acquired during peripheral activation or are, in part, generated during thymic development. Here, we assessed the role of WASp during T-cell differentiation using artificial thymic organoid cultures and in the thymus of humanized mice. Although CRISPR/Cas9 WAS knockout hematopoietic stem and progenitor cells (HSPCs) rearranged the T-cell receptor and differentiated to T-cell receptor (TCR) + CD4 + CD8 + double-positive (DP) cells similar to wild-type HSPCs, a partial defect in the generation of CD8 single-positive (SP) cells was observed, suggesting that WASp is involved in their positive selection. TCR repertoire analysis of the DP and CD8 + SP population, however, showed a polyclonal repertoire with no bias toward autoreactivity. To our knowledge, this is the first study of the role of WASp in human T-cell differentiation and on TCR repertoire generation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Pille, Avila, Sanchez, Goetgeluk, De Munter, Jansen, Billiet, Weening, Xue, Bonte, Ingels, De Cock, Pascal, Deseins, Kerre, Taghon, Leclercq, Vermijlen, Davis and Vandekerckhove.)

In the human thymus, a CD10+ PD-1+ TCRαβ+ differentiation pathway diverges from the conventional single positive T cell lineages at the early double-positive stage. Here, we identify the progeny of this unconventional lineage in antigen-inexperienced blood. These unconventional T cells (UTCs) in thymus and blood share a transcriptomic profile, characterized by hallmark transcription factors (i.e., ZNF683 and IKZF2), and a polyclonal TCR repertoire with autoreactive features, exhibiting a bias toward early TCRα chain rearrangements. Single-cell RNA sequencing confirms a common developmental trajectory between the thymic and blood UTCs and clearly delineates this unconventional lineage in blood. Besides MME+ recent thymic emigrants, effector-like clusters are identified in this heterogeneous lineage. Expression of Helios and KIR and a decreased CD8β expression are characteristics of this lineage. This UTC lineage could be identified in adult blood and intestinal tissues. In summary, our data provide a comprehensive characterization of the polyclonal unconventional lineage in antigen-inexperienced blood and identify the adult progeny., (© 2023 Billiet et al.)

Ocean manipulation to mitigate climate change may harm deep-sea ecosystems.

Microplastic (MP) pollution is an ongoing problem in coastal systems, where wastewater treatment plants (WWTPs) deposit particles daily. This study examined MP characteristics at WWTP outflow and control sites in St. Andrew Bay in Northwestern Florida, USA. WWTP sites contained mostly polypropylene fragments (180.1 μm avg. size), while reference sites contained polypropylene fragments, and polyethylene and polyester fibers (315.3 μm avg. size). MP sizes were strongly linked to distance from the nearest WWTP, while shape and polymer compositions were more closely related to dissolved oxygen concentrations and distance to the nearest water input source. The prevalence of polypropylene fragments at WWTP sites suggests that extreme weather events during the study flushed land-based debris into the system, where it was buried in the sediments. Increased abundances of polyester and polyethylene terephthalate in the winter at WWTP sites are indicative of the role that laundering synthetic textiles plays in coastal MP pollution., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Aaron Ridall reports financial support was provided by PADI Foundation. Aaron Ridall reports financial support was provided by Friends of Gumbo Limbo. Aaron Ridall reports financial support was provided by Southern Association of Marine Laboratories., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Wastewater treatment plants (WWTPs) cause approximately 25 % of microplastics (MPs) in the marine environment. While research on MPs in WWTP effluent has demonstrated that an abundance of particles enter the marine environment, little effort has gone to assessing MP abundances in coastal sediments to determine their seasonal and spatial variability. Here, we assessed MP abundances in sediments at sites of WWTP outflow and at non-polluted sites over six consecutive seasons within the St. Andrew Bay system in Northwestern Florida. We showed that MP abundances were highest at one of the WWTP sites, where they increased with increasing distance away from the input source (3.16 ± 1.59 MP/kg to 34.03 ± 11.69 MP/kg sediment dry weight). We also found that mean MP abundances were highest in the winter (12.41 ± 3.56 MPs/kg sediment dry weight) and lowest in the spring (2.17 ± 0.63 MPs/kg sediment dry weight). Therefore, while WWTPs differentially retain MPs in their removal processes, MP pollution in the St. Andrew Bay system shows seasonal dynamics like other studies. Although average MP abundance in surface sediments (0-5 cm) was higher than in subsurface sediments (5-10 cm) at all sites, this difference was not as substantial as has been found in other studies. Based on mean MP abundance in surface sediments, we estimate that there are 30 billion MPs within the surface layer of sediment in the St. Andrew Bay system, and that the particles export to the Gulf of Mexico because of seasonal flushing between the winter and spring. The distributions of MPs in the system were also likely driven by extreme weather events that occurred in the bay system during 2018 and 2020, which acts as a cautionary tale for coastal urban ecosystems in the face of sea level rise and climate change., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Introduction: In the United States, physical activity (PA) among adolescents is declining; 75% of high school students do not meet daily PA guidelines. Low rates of PA are more prevalent among high school girls. Schools provide an optimal environment to target and promote PA. However, school climate has not yet been studied for its importance in promoting PA among high school students, particularly girls., Methods: A cross-sectional analysis was conducted using the Georgia Student Health Survey (GSHS) 2.0 data on perceptions of different school climate measures and self-reported weekly PA levels to study gender differences in the association of PA with school climate., Results: Data from a total of 362,926 students (48% males and 52% females) were analyzed. For both genders, the odds of being physically active increased with a more positive report of supportive school environments, school connectedness, peer social support, school physical environments, cultural acceptance, school safety, and adult social support. Peer victimization was associated with increased odds of PA among females but lower odds for males., Conclusion: Results suggest that improving school climate can increase PA among adolescents. As new or existing school-based interventions and policies are considered by states and local governments, improving the school climate should be part of the overall strategy. Future research is needed on peer victimization among physically active females., Implications and Contributions: This study evaluated gender differences in the association between measures of school climate and PA among high school students. School climate or policies fostering positive environments including feelings of safety, connectedness, and peer support may increase adolescent PA; addressing peer victimization and fights may reduce gender disparities in PA., (© 2022 The Foundation for Professionals in Services for Adolescents.)

Messenger RNA (mRNA) has become a promising tool in therapeutic cancer vaccine strategies. Owing to its flexible design and rapid production, mRNA is an attractive antigen delivery format for cancer vaccines targeting mutated peptides expressed in a tumor-the so-called neoantigens. These neoantigens are rarely shared between patients, and inclusion of these antigens in a vaccine requires the production of individual batches of patient-tailored mRNA. The authors have developed MIDRIX NEO , a personalized mRNA-loaded dendritic cell vaccine targeting tumor neoantigens, which is currently being evaluated in a phase 1 clinical study in lung cancer patients. To facilitate this study, the authors set up a Good Manufacturing Practice (GMP)-compliant production process for the manufacture of small batches of personalized neoantigen-encoding mRNA. In this article, the authors describe the complete mRNA production process and the extensive quality assessment to which the mRNA is subjected. Validation runs have shown that the process delivers mRNA of reproducible, high quality. This process is now successfully applied for the production of neoantigen-encoding mRNA for the clinical evaluation of MIDRIX NEO . To the authors' knowledge, this is the first time that a GMP-based production process of patient-tailored neoantigen mRNA has been described., Competing Interests: Declaration of Competing Interest The authors have no commercial, proprietary or financial interest in the products or companies described in this article., (Copyright © 2021 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

We profiled the liver transcriptome, proteome, and metabolome in 347 individuals from 58 isogenic strains of the BXD mouse population across age (7 to 24 months) and diet (low or high fat) to link molecular variations to metabolic traits. Several hundred genes are affected by diet and/or age at the transcript and protein levels. Orthologs of two aging-associated genes, St7 and Ctsd, were knocked down in C. elegans, reducing longevity in wild-type and mutant long-lived strains. The multiomics data were analyzed as segregating gene networks according to each independent variable, providing causal insight into dietary and aging effects. Candidates were cross-examined in an independent diversity outbred mouse liver dataset segregating for similar diets, with ∼80%-90% of diet-related candidate genes found in common across datasets. Together, we have developed a large multiomics resource for multivariate analysis of complex traits and demonstrate a methodology for moving from observational associations to causal connections., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Objectives: Cytomegalovirus (CMV) infection is one of the most common complications in allogeneic hematopoietic stem cell transplant (allo-HSCT) recipients. The classic antiviral treatments have shown clinical efficacy but are often associated with drug resistance. Reconstitution of CMV-specific cellular immunity is essential in controlling CMV infection; therefore, adoptive transfer of CMV-specific T cells is a promising treatment option. We treated a patient with a multidrug resistant CMV infection after haploidentical HSCT with CMV-specific T cells. Methods: The T cells were derived from the HSCT donor who was CMV seropositive. We generated the T cells by a short-term Good Manufacturing Practice (GMP) grade protocol in which a leukapheresis product of the HSCT donor was stimulated with the immunodominant antigen pp65 and interferon-γ secreting cells were isolated. A total of 5 × 10 5  T cells were administered to the patient within 30 hours after leukapheresis. Results: The patient was closely monitored for reconstitution of antiviral T cell immunity and viral replication after adoptive T cell transfer. We observed an in vivo expansion of both CD4 + and CD8 + CMV-specific T cells associated with a significant decrease in viral burden and clinical improvement. Conclusion: This case report further supports the feasibility and effectiveness of adoptive donor T cell transfer for the treatment of drug resistant CMV infections after allo-HSCT.

T cell receptor (TCR)-redirected T cells target intracellular antigens such as Wilms' tumor 1 (WT1), a tumor-associated antigen overexpressed in several malignancies, including acute myeloid leukemia (AML). For both chimeric antigen receptor (CAR)- and TCR-redirected T cells, several clinical studies indicate that T cell subsets with a less-differentiated phenotype (e.g. stem cell memory T cells, T SCM ) survive longer and mediate superior anti-tumor effects in vivo as opposed to more terminally differentiated T cells. Cytokines added during in vitro and ex vivo culture of T cells play an important role in driving the phenotype of T cells for adoptive transfer. Using the OP9-DL1 co-culture system, we have shown previously that we are able to generate in vitro , starting from clinically relevant stem cell sources, T cells with a single tumor antigen (TA)-specific TCR. This method circumvents possible TCR chain mispairing and unwanted toxicities that might occur when introducing a TA-specific TCR in peripheral blood lymphocytes. We now show that we are able to optimize our in vitro culture protocol, by adding IL-21 during maturation, resulting in generation of TA-specific T cells with a less-differentiated phenotype and enhanced in vitro anti-tumor effects. We believe the favorable T SCM -like phenotype of these in vitro generated T cells preludes superior in vivo persistence and anti-tumor efficacy. Therefore, these TA-specific T cells could be of use as a valuable new form of patient-tailored T cell immunotherapy for malignancies for which finding a suitable CAR-T target antigen is challenging, such as AML., Competing Interests: The authors declare no conflict of interest., (© 2021 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Lung cancer remains the leading cause of cancer-related death worldwide. The advent of immune checkpoint inhibitors has led to a paradigm shift in the treatment of metastatic non-small cell and small cell lung cancer. However, despite prolonged overall survival, only a minority of the patients derive clinical benefit from these treatments suggesting that the full anti-tumoral potential of the immune system is not being harnessed yet. One way to overcome this problem is to combine immune checkpoint blockade with different strategies aimed at inducing or restoring cellular immunity in a tumor-specific, robust, and durable way. Owing to their unique capacity to initiate and regulate T cell responses, dendritic cells have been extensively explored as tools for immunotherapy in many tumors, including lung cancer. In this review, we provide an update on the nearly twenty years of experience with dendritic cell-based immunotherapy in lung cancer. We summarize the main results from the early phase trials and give an overview of the future perspectives within this field., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Stevens, Ingels, Van Lint, Vandekerckhove and Vermaelen.)

Human thymic CD8αα + CD10 + PD-1 + αβ T cells selected through early agonist selection have been proposed as the putative thymic precursors of the human CD8αα + intestinal intraepithelial lymphocytes (IELs). However, the progeny of these thymic precursor cells in human blood or tissues has not yet been characterized. Here, we studied the phenotypical and transcriptional differentiation of the thymic IEL precursor (IELp) lineage upon in vitro exposure to cytokines prominent in the peripheral tissues such as interleukin-15 (IL-15) and the inflammatory cytokines interleukin-12 (IL-12) and interleukin-18 (IL-18). We showed that only the CD1a - fraction of the CD10 + PD-1 + IELp population was able to proliferate with IL-15, suggesting that this subset had acquired functionality. These cells downregulated PD-1 expression and completely lost CD10 expression, whereas other surface markers such as CD95 and CXCR3 remained highly expressed. RNA-seq analysis of the IL-15-cultured cells clearly showed induction of innate-like and effector genes. Induction of the cytotoxic machinery by the CD10 + PD-1 + population was acquired in the presence of IL-15 and was further augmented by inflammatory cytokines. Our data suggest that only the CD1a - CD10 + PD-1 + population exits the thymus and survives in the periphery. Furthermore, PD-1 and CD10 expression is not an intrinsic property of this lineage, but rather characterizes a transient stage in differentiation. CD95 and CXCR3 expression combined with the absence of CD28, CCR7, and CD6 expression might be more powerful markers to define this lineage in the periphery.

Objectives. To evaluate the statewide implementation of childhood fitness assessment and reporting in Georgia. Methods. We collected survey data from 1683 (919 valid responses from a random-digit-dialed survey and 764 valid responses from a Qualtrics panel) parents of public school students in Georgia in 2018. Results. Most parents reported that their child participated in fitness assessments at school, yet only 31% reported receiving results. If a child was identified as needing improvement, parents were significantly more likely to change the diet and exercise of both the child and the family. Conclusions. A state-level mandatory fitness assessment for children may be successful in state-level surveillance of fitness levels; parental awareness of the policy, receipt of the fitness assessment information, and action on receiving the screening information require more efforts in implementation.

Chimeric antigen receptor (CAR) T-cells have shown great promise in the treatment of B-cell malignancies. For acute myeloid leukemia (AML), however, the optimal target surface antigen has yet to be discovered. Alternatively, T-cell receptor (TCR)-redirected T-cells target intracellular antigens, marking a broader territory of available target antigens. Currently, adoptive TCR T-cell therapy uses peripheral blood lymphocytes for the introduction of a transgenic TCR. However, this can cause graft-versus-host disease, due to mispairing of introduced and endogenous TCR chains. Therefore, we started from hematopoietic stem and progenitor cells (HSPC), that do not express a TCR yet, isolated from healthy donors, patients in remission after chemotherapy and AML patients at diagnosis. Using the OP9-DL1 in vitro co-culture system and agonist selection, TCR-transduced HSPC develop into mature tumor antigen-specific T-cells with only one TCR. We show here that this approach is feasible with adult HSPC from clinically relevant sources, albeit with slower maturation and lower cell yield compared to cord blood HSPC. Moreover, cryopreservation of HSPC does not have an effect on cell numbers or functionality of the generated T-cells. In conclusion, we show here that it is feasible to generate TA-specific T-cells from HSPC from adult healthy donors and patients and we believe these T-cells could be of use as a very valuable form of patient-tailored T-cell immunotherapy., (© 2020 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Background: Even as many states adopt physical activity policies to promote physical activity and prevent childhood obesity, little is known about differences in policy implementation based on school characteristics. We studied association of school characteristics and changes in physical activity opportunities at the school level during the implementation of a statewide physical activity policy in the state of Georgia., Methods: A web-based school survey was administered to elementary schools at two time points (before and during policy execution). Matched respondents (289 classroom teachers, 234 administrators) reported the frequency and duration of recess and integrated physical activity time. We used paired t-test to assess changes in physical activity opportunities and chi-square tests to assess the association of change in physical activity opportunities with school characteristics. We then constructed a multiple linear regression model following a change score method to identify school-level factors that predict the magnitude of change in physical activity opportunities., Results: There was an overall significant increase in total physical activity opportunities across time; however, schools with higher poverty showed a decrease in physical activity time by 5.3 minutes per day (95% CI: -9.2, -1.3). Further, the changes in physical activity time for schools in suburban Georgia were smaller (-5.7, 95% CI: -9.5, -1.9) compared to schools located in towns., Conclusions: The change in physical activity opportunities was not the same across schools and school characteristics predicted the magnitude of change. Additional efforts at the local level might be needed for equitable policy implementation based on schools' geographical location and poverty level of the student population., Competing Interests: The authors have declared that no competing interests exist.

Recent approval of chimeric antigen receptor (CAR) T cell therapy by the European Medicines Agency (EMA)/Federal and Drug Administration (FDA) and the remarkable results of CAR T clinical trials illustrate the curative potential of this therapy. While CARs against a multitude of different antigens are being developed and tested (pre)clinically, there is still a need for optimization. The use of single-chain variable fragments (scFvs) as targeting moieties hampers the quick generation of functional CARs and could potentially limit the efficacy. Instead, nanobodies may largely circumvent these difficulties. We used an available nanobody library generated after immunization of llamas against Cluster of Differentiation (CD) 20 through DNA vaccination or against the ectodomain of CD33 using soluble protein. The nanobody specific sequences were amplified by PCR and cloned by Gibson Assembly into a retroviral vector containing two different second-generation CAR constructs. After transduction in T cells, we observed high cell membrane nanoCAR expression in all cases. Following stimulation of nanoCAR-expressing T cells with antigen-positive cell lines, robust T cell activation, cytokine production and tumor cell lysis both in vitro and in vivo was observed. The use of nanobody technology in combination with PCR and Gibson Assembly allows for the rapid and effective generation of compact CARs., Competing Interests: Jan Tavernier is affiliated with Orionis Biosciences (as a scientific advisor and/or an employee) and holds equity interests in Orionis Bioscience. Jan Tavernier received financial research support from Orionis Biosciences NV. All the other authors declare no conflict of interest.

GABA type-A (GABA-A) receptors containing the α2 subunit (GABRA2) are expressed in most brain regions and are critical in modulating inhibitory synaptic function. Genetic variation at the GABRA2 locus has been implicated in epilepsy, affective and psychiatric disorders, alcoholism and drug abuse. Gabra2 expression varies as a function of genotype and is modulated by sequence variants in several brain structures and populations, including F2 crosses originating from C57BL/6J (B6J) and the BXD recombinant inbred family derived from B6J and DBA/2J. Here we demonstrate a global reduction of GABRA2 brain protein and mRNA in the B6J strain relative to other inbred strains, and identify and validate the causal mutation in B6J. The mutation is a single base pair deletion located in an intron adjacent to a splice acceptor site that only occurs in the B6J reference genome. The deletion became fixed in B6J between 1976 and 1991 and is now pervasive in many engineered lines, BXD strains generated after 1991, the Collaborative Cross, and the majority of consomic lines. Repair of the deletion using CRISPR- Cas9 -mediated gene editing on a B6J genetic background completely restored brain levels of GABRA2 protein and mRNA. Comparison of transcript expression in hippocampus, cortex, and striatum between B6J and repaired genotypes revealed alterations in GABA-A receptor subunit expression, especially in striatum. These results suggest that naturally occurring variation in GABRA2 levels between B6J and other substrains or inbred strains may also explain strain differences in anxiety-like or alcohol and drug response traits related to striatal function. Characterization of the B6J private mutation in the Gabra2 gene is of critical importance to molecular genetic studies in neurobiological research because this strain is widely used to generate genetically engineered mice and murine genetic populations, and is the most widely utilized strain for evaluation of anxiety-like, depression-like, pain, epilepsy, and drug response traits that may be partly modulated by GABRA2 function.

Recent clinical trials have shown that adoptive chimeric antigen receptor (CAR) T cell therapy is a very potent and possibly curative option in the treatment of B cell leukemias and lymphomas. However, targeting a single antigen may not be sufficient, and relapse due to the emergence of antigen negative leukemic cells may occur. A potential strategy to counter the outgrowth of antigen escape variants is to broaden the specificity of the CAR by incorporation of multiple antigen recognition domains in tandem. As a proof of concept, we here describe a bispecific CAR in which the single chain variable fragment (scFv) is replaced by a tandem of two single-antibody domains or nanobodies (nanoCAR). High membrane nanoCAR expression levels are observed in retrovirally transduced T cells. NanoCARs specific for CD20 and HER2 induce T cell activation, cytokine production and tumor lysis upon incubation with transgenic Jurkat cells expressing either antigen or both antigens simultaneously. The use of nanobody technology allows for the production of compact CARs with dual specificity and predefined affinity., Competing Interests: The authors declare no conflict of interest.

Genetic factors that influence the transition from initial drinking to dependence remain enigmatic. Recent studies have leveraged chronic intermittent ethanol (CIE) paradigms to measure changes in brain gene expression in a single strain at 0, 8, 72 h, and even 7 days following CIE. We extend these findings using LCM RNA-seq to profile expression in 11 brain regions in two inbred strains - C57BL/6J (B6) and DBA/2J (D2) - 72 h following multiple cycles of ethanol self-administration and CIE. Linear models identified differential expression based on treatment, region, strain, or interactions with treatment. Nearly 40% of genes showed a robust effect (FDR < 0.01) of region, and hippocampus CA1, cortex, bed nucleus stria terminalis, and nucleus accumbens core had the highest number of differentially expressed genes after treatment. Another 8% of differentially expressed genes demonstrated a robust effect of strain. As expected, based on similar studies in B6, treatment had a much smaller impact on expression; only 72 genes (p < 0.01) are modulated by treatment (independent of region or strain). Strikingly, many more genes (415) show a strain-specific and largely opposite response to treatment and are enriched in processes related to RNA metabolism, transcription factor activity, and mitochondrial function. Over 3 times as many changes in gene expression were detected in D2 compared to B6, and weighted gene co-expression network analysis (WGCNA) module comparison identified more modules enriched for treatment effects in D2. Substantial strain differences exist in the temporal pattern of transcriptional neuroadaptation to CIE, and these may drive individual differences in risk of addiction following excessive alcohol consumption., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Context: There is an established association between the provision of health care services and maternal mortality. In Ecuador, little is known if the societal value is greater than the resources expended in preventive medicine., Aims: The purpose of this research is to investigate Ecuadorians' willingness to pay to prevent maternal death and disabilities due to complications of care during childbirth in the context of universal coverage., Methods and Materials: The study elicited a "contingent" market on morbidity and mortality outcomes, specific to Ecuador's epidemiologic profiles between a hypothetical market that included a 50% reduction in the risk of maternal mortality from 100 to 50 per 100,000, and a market that included a 50% reduction in the risk of maternal morbidity from 4,000 to 2,000 per 100,000., Results: The average amount participants are willing to pay (WTP) to prevent maternal mortality in the context of universal coverage, was $176 a year (95% CI=$172, $179). The unadjusted mean WTP for a reduction in the maternal morbidity risk was $135 (95% CI=$132, $139). Translated into Value of statistical Life, participant´s from this study valued the prevention of one statistical maternal death at USD $352,000., Conclusion: Results suggest that the costs of maternal care do not outweigh the benefit of prevention, and that Ecuadorians are willing to pay a significant amount to reduce the risk of maternal mortality., Global Health Implications: Reduction of maternal mortality will remain an important global developmental goal in the upcoming years. Having a monetary approximation on the value of these losses may have important implications in the allotting financial and technical resources to reduce it., Competing Interests: Compliance with Ethical Standards Conflict of Interest: Authors declare they have no conflicts of interest.

The rapid divergence of male genitalia is a preeminent evolutionary pattern. This rapid divergence is especially striking in the baculum, a bone that occurs in the penis of many mammalian species. Closely related species often display diverse baculum morphology where no other morphological differences can be discerned. While this fundamental pattern of evolution has been appreciated at the level of gross morphology, nearly nothing is known about the genetic basis of size and shape divergence. Quantifying the genetic basis of baculum size and shape variation has been difficult because these structures generally lack obvious landmarks, so comparing them in three dimensions is not straightforward. Here, we develop a novel morphometric approach to quantify size and shape variation from three-dimensional micro-CT scans taken from 369 bacula, representing 75 distinct strains of the BXD family of mice. We identify two quantitative trait loci (QTL) that explain ∼50% of the variance in baculum size, and a third QTL that explains more than 20% of the variance in shape. Together, our study demonstrates that baculum morphology may diverge relatively easily, with mutations at a few loci of large effect that independently modulate size and shape. Based on a combination of bioinformatic investigations and new data on RNA expression, we prioritized these QTL to 16 candidate genes, which have hypothesized roles in bone morphogenesis and may enable future genetic manipulation of baculum morphology., (Copyright © 2016 Schultz et al.)

Setopagis maculosa (Todd, 1920) (Aves: Caprimulgidae) was described based on a single male specimen collected by Samuel Milton Klages in French Guiana, northeastern South America. Since then, no new specimens have been collected nor have any reliable records been made, and the validity of the species has been questioned. A detailed analysis of the holotype reveals that it has unique and distinctive morphological traits that support the validity and taxonomic status of the species, which is provisionally placed in Setopagis. We present new information on the type locality at the time of its collection, which may shed some light on the habitat preferences of the species, and we provide details on its plumage that have been largely overlooked and that will be important for future field identification.

The deep sea is Earth's largest habitat but little is known about the nature of deep-sea parasitism. In contrast to a few characterized cases of bacterial and protistan parasites, the existence and biological significance of deep-sea parasitic fungi is yet to be understood. Here we report the discovery of a fungus-related parasitic microsporidium, Nematocenator marisprofundi n. gen. n. sp. that infects benthic nematodes at methane seeps on the Pacific Ocean floor. This infection is species-specific and has been temporally and spatially stable over 2 years of sampling, indicating an ecologically consistent host-parasite interaction. A high distribution of spores in the reproductive tracts of infected males and females and their absence from host nematodes' intestines suggests a sexual transmission strategy in contrast to the fecal-oral transmission of most microsporidia. N. marisprofundi targets the host's body wall muscles causing cell lysis, and in severe infection even muscle filament degradation. Phylogenetic analyses placed N. marisprofundi in a novel and basal clade not closely related to any described microsporidia clade, suggesting either that microsporidia-nematode parasitism occurred early in microsporidia evolution or that host specialization occurred late in an ancient deep-sea microsporidian lineage. Our findings reveal that methane seeps support complex ecosystems involving interkingdom interactions between bacteria, nematodes, and parasitic fungi and that microsporidia parasitism exists also in the deep-sea biosphere.

Ongoing greenhouse gas emissions can modify climate processes and induce shifts in ocean temperature, pH, oxygen concentration, and productivity, which in turn could alter biological and social systems. Here, we provide a synoptic global assessment of the simultaneous changes in future ocean biogeochemical variables over marine biota and their broader implications for people. We analyzed modern Earth System Models forced by greenhouse gas concentration pathways until 2100 and showed that the entire world's ocean surface will be simultaneously impacted by varying intensities of ocean warming, acidification, oxygen depletion, or shortfalls in productivity. In contrast, only a small fraction of the world's ocean surface, mostly in polar regions, will experience increased oxygenation and productivity, while almost nowhere will there be ocean cooling or pH elevation. We compiled the global distribution of 32 marine habitats and biodiversity hotspots and found that they would all experience simultaneous exposure to changes in multiple biogeochemical variables. This superposition highlights the high risk for synergistic ecosystem responses, the suite of physiological adaptations needed to cope with future climate change, and the potential for reorganization of global biodiversity patterns. If co-occurring biogeochemical changes influence the delivery of ocean goods and services, then they could also have a considerable effect on human welfare. Approximately 470 to 870 million of the poorest people in the world rely heavily on the ocean for food, jobs, and revenues and live in countries that will be most affected by simultaneous changes in ocean biogeochemistry. These results highlight the high risk of degradation of marine ecosystems and associated human hardship expected in a future following current trends in anthropogenic greenhouse gas emissions., Competing Interests: The authors have declared that no competing interests exist.

Because of the unique conditions that exist around the Antarctic continent, Southern Ocean (SO) ecosystems are very susceptible to the growing impact of global climate change and other anthropogenic influences. Consequently, there is an urgent need to understand how SO marine life will cope with expected future changes in the environment. Studies of Antarctic organisms have shown that individual species and higher taxa display different degrees of sensitivity to environmental shifts, making it difficult to predict overall community or ecosystem responses. This emphasizes the need for an improved understanding of the Antarctic benthic ecosystem response to global climate change using a multitaxon approach with consideration of different levels of biological organization. Here, we provide a synthesis of the ability of five important Antarctic benthic taxa (Foraminifera, Nematoda, Amphipoda, Isopoda, and Echinoidea) to cope with changes in the environment (temperature, pH, ice cover, ice scouring, food quantity, and quality) that are linked to climatic changes. Responses from individual to the taxon-specific community level to these drivers will vary with taxon but will include local species extinctions, invasions of warmer-water species, shifts in diversity, dominance, and trophic group composition, all with likely consequences for ecosystem functioning. Limitations in our current knowledge and understanding of climate change effects on the different levels are discussed.

The Gollum Channels and Whittard Canyon (NE Atlantic) are two areas that receive high input of organic matter and phytodetritus from euphotic layers, but they are typified by different trophic and hydrodynamic conditions. Sediment biogeochemistry was analysed in conjunction with structure and diversity of the nematode community and differences were tested between study areas, water depths (700 m vs 1000 m), stations, and sediment layers. The Gollum Channels and Whittard Canyon harboured high meiofauna abundances (1054-1426 ind. 10 cm(-2)) and high nematode diversity (total of 181 genera). Next to enhanced meiofauna abundance and nematode biomass, there were signs of high levels of organic matter deposition leading to reduced sedimentary conditions, which in turn structured the nematode community. Striking in this respect was the presence of large numbers of 'chemosynthetic' Astomonema nematodes (Astomonema southwardorum, Order Monhysterida, Family Siphonolaimidae). This genus lacks a mouth, buccal cavity and pharynx and possesses a rudimentary gut containing internal, symbiotic prokaryotes which have been recognised as sulphur-oxidising bacteria. Dominance of Astomonema may indicate the presence of reduced environments in the study areas, which is partially confirmed by the local biogeochemical environment. The nematode communities were mostly affected by sediment layer differences and concomitant trophic conditions rather than other spatial gradients related to study area, water depth or station differences, pointing to small-scale heterogeneity as the main source of variation in nematode structure and function. Furthermore, the positive relation between nematode standing stocks, and quantity and quality of the organic matter was stronger when hydrodynamic disturbance was greater. Analogically, this study also suggests that structural diversity can be positively correlated with trophic conditions and that this relation is tighter when hydrodynamic disturbance is greater.

Background: Recent climate-induced ice-shelf disintegration in the Larsen A (1995) and B (2002) areas along the Eastern Antarctic Peninsula formed a unique opportunity to assess sub-ice-shelf benthic community structure and led to the discovery of unexplored habitats, including a low-activity methane seep beneath the former Larsen B ice shelf. Since both limited particle sedimentation under previously permanent ice coverage and reduced cold-seep activity are likely to influence benthic meiofauna communities, we characterised the nematode assemblage of this low-activity cold seep and compared it with other, now seasonally ice-free, Larsen A and B stations and other Antarctic shelf areas (Weddell Sea and Drake Passage), as well as cold-seep ecosystems world-wide., Principal Findings: The nematode community at the Larsen B seep site differed significantly from other Antarctic sites in terms of dominant genera, diversity and abundance. Densities in the seep samples were high (>2000 individuals per 10 cm(2)) and showed below-surface maxima at a sediment depth of 2-3 cm in three out of four replicates. All samples were dominated by one species of the family Monhysteridae, which was identified as a Halomonhystera species that comprised between 80 and 86% of the total community. The combination of high densities, deeper density maxima and dominance of one species is shared by many cold-seep ecosystems world-wide and suggested a possible dependence upon a chemosynthetic food source. Yet stable (13)C isotopic signals (ranging between -21.97±0.86‰ and -24.85±1.89‰) were indicative of a phytoplankton-derived food source., Conclusion: The recent ice-shelf collapse and enhanced food input from surface phytoplankton blooms were responsible for the shift from oligotrophic pre-collapse conditions to a phytodetritus-based community with high densities and low diversity. The parthenogenetic reproduction of the highly dominant Halomonhystera species is rather unusual for marine nematodes and may be responsible for the successful colonisation by this single species.

An autoantigenic role for collagen type I (CI) has been suggested previously in diffuse cutaneous systemic sclerosis (dcSSc). Whether CI is indeed capable of affecting the immune system in dcSSc is not known. Patients with early (3 years or less) or late (>3 years) dcSSc and healthy controls donated blood. Peripheral blood mononuclear cells (PBMC) were cultured with or without CI, and expression of genes known for their involvement in autoimmune and inflammatory processes was assessed using cDNA arrays; results were confirmed by real-time polymerase chain reaction and enzyme-linked immunosorbent assay for selected genes. Patients with early and late dcSSc were similarly different from healthy controls in basal gene expression. When cultured with CI, PBMC from patients with early dcSSc differed from healthy controls in expression of 34 genes, whereas PBMC from patients with late dcSSc differed from healthy controls in expression of only 29 genes. Direct comparisons of matched PBMC samples cultured with and without CI revealed differences in expression of eight genes in healthy controls, of five genes in patients with early dcSSc, and no differences in patients with late dcSSc. Thus, PBMC from patients with dcSSc respond differently than do PBMC from healthy controls when cultured with CI. Exposure to CI in culture of PBMC from patients in the early stage of dcSSc in contrast to PBMC from patients with late-stage dcSSc evokes a greater degree of activation of immune-related genes, suggesting that CI is more dominant as an autoantigen in early versus late dcSSc., (© 2010 British Society for Immunology.)

1alpha,25-Dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) is known to inhibit prostate cancer cells in vitro. Its effects on proliferation in the presence of living bone have not been reported, but are especially relevant since much of the morbidity and mortality associated with prostate cancer is due to metastatic bone disease. We investigated the effect of 1alpha,25(OH)(2)D(3) on MatLyLu-beta(2) cells (MatLyLu cells), a rat prostate cancer line, co-cultured in transwells with living rat calvaria. Cultures of MatLyLu cells with living calvaria treated with 1alpha,25(OH)(2)D(3) exhibited a statistically significant increase in proliferation (range 1.4 to 1.7-fold; p<0.05). Cultures of MatLylu cells alone, with spleen cells, muscle tissue, or with living or inactivated calvarial bone showed no differences in proliferation. To investigate the mechanism for enhanced proliferation, Galardin, a matrix metalloproteinase (MMP) inhibitor, or pamidronate, an antiresorptive agent, was added. Enhanced proliferation was prevented by either agent, but not to an equal extent. The presence of 1alpha,25(OH)(2)D(3) may lead to proteolytic release or activation of growth factors from bone. These results may explain the variability in reports on the in vivo effects of Vitamin D and suggest a potential concern in using Vitamin D or its analogs alone in patients with metastatic prostate cancer.