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Given the promising results from phase 1/2 clinical trials of therapy involving regulatory T cells (Tregs), it is critical to develop Treg manufacturing methods that use well-defined reagents.Seeking to maximize expansion of human thymic Tregs activated with anti-CD3/CD28 antibody-coated beads and cultured in serum-free medium, the authors investigated the effect of adjusting process parameters including cell density and cell concentration, and feeding strategy on Treg yield and quality.The authors found that levels of expansion and viability varied with cell density on the day of restimulation. Tregs restimulated at low cell densities (1 × 10These data show the previously undescribed consequences of adjusting cell density on Treg expansion and establish a Good Manufacturing Practice-relevant protocol using non-cell-based activation reagents and serum-free media that supports sustained expansion without loss of viability or phenotype.

Chronic graft-versus-host disease (cGvHD) is a major cause of morbidity after hematopoietic stem cell transplantation (HSCT). In large patient populations, we have shown a CD56bright natural killer (NK) population to strongly associate with a lack of cGvHD and we hypothesize that these cells function to suppress cGvHD. We aimed to isolate and define the characteristics of regulatory NK (NKreg) cells associated with suppression of cGvHD. Immunophenotypic evaluation of a large pediatric population found the CD56bright NK population associated with a lack of cGvHD to be perforin-, Granzyme B-, and CD335+. Transcriptome analysis of a small patient cohort of CD56bright compared to CD56dim NK cells found the NKreg cells to also overexpress Granzyme K, IL-7R, GPR183, RANK, GM-CSFR, TCF7, and IL23A. Further analysis of this CD56bright NKreg population found a subpopulation that overexpressed IRF1, and TNF. We also found that viable NKreg cells may be isolated by sorting on CD56+ and CD16- NK cells, and this population can suppress allogeneic CD4+ T cells, but not Treg cells or CD8+ T cells through a non-cytolytic, cell-cell contact dependent mechanism. Suppression was not reliant upon the NKp44, NKp46, or GPR183 receptors. Additionally, NKreg cells do not kill leukemic cells. Moreover, this is the first paper to clearly establish that a CD56brightCD3-CD16-perforin- NKreg population associates with a lack of cGvHD and has several unique characteristics, including the suppression of helper T-cell function in vitro. With further investigation we may decipher the mechanism of NKreg suppression and operationalize expansion of NKreg cells associated with cGvHD suppression.

Fertilizer boron (B) and molybdenum (Mo) were provided to contrasting cultivars of subirrigated pot chrysanthemums at approximately 6–100% of current industry standards in an otherwise balanced nutrient solution during vegetative growth, and then all nutrients were removed during reproductive growth. Two experiments were conducted for each nutrient in a naturally lit greenhouse using a randomized complete block split-plot design. Boron (0.313–5.00 µmol L−1) or Mo (0.031–0.500 µmol L−1) was the main plot, and cultivar was the sub-plot. Petal quilling was observed with leaf-B of 11.3–19.4 mg kg−1 dry mass (DM), whereas Mo deficiency was not observed with leaf-Mo of 1.0–3.7 mg kg−1 DM. Optimized supplies resulted in leaf tissue levels of 48.8–72.5 mg B kg−1 DM and 1.9–4.8 mg Mo kg−1 DM. Boron uptake efficiency was more important than B utilization efficiency in sustaining plant/inflorescence growth with decreasing B supply, whereas Mo uptake and utilization efficiencies appeared to have similar importance in sustaining plant/inflorescence growth with decreasing Mo supply. This research contributes to the development of a sustainable low-input nutrient delivery strategy for floricultural operations, wherein nutrient supply is interrupted during reproductive growth and optimized during vegetative growth.

Regulatory T-cell (Treg) therapy has shown promise in treating autoimmune diseases, transplant rejection, or graft-versus-host disease in early clinical trials. These trials have demonstrated that cell therapy using polyclonal Tregs is feasible and safe, however, the field has been limited by the lack of polyclonal cell specificity and consequent large cell numbers required, and the difficulty in generating autologous products for some patients. Thus, the field is moving toward 'next generation' Treg cell therapies that include genetic modification strategies to engineer specificity and/or modify function, as well as methods to generate Tregs in vitro. In this review, we describe how genetic modification of Tregs using viral transduction or gene editing may be incorporated into Treg manufacturing protocols. We also describe how Tregs may be generated via FOXP3 gene editing or overexpression, or by differentiation from pluripotent stem cells. The application of these various types of engineered Tregs is discussed.

Moderate levels of calcium or magnesium were applied to two cultivars of subirrigated, potted, pinched chrysanthemums during vegetative growth. Market-quality plants were produced with sufficient leaf-calcium or leaf-magnesium even though the delivery of the respective nutrient could be reduced by approximately 87.5%, compared with industry standards. This practice could contribute to low-input production of floricultural crops.

Background Cardiopulmonary exercise-testing (CPET) and the (Portsmouth) Physiological and Operative Severity Score for the enUmeration of Mortality and Morbidity ((P)-POSSUM) are used as pre-operative risk stratification and audit tools in general surgery, however, both have been demonstrated to have limitations in major hepatopancreatobiliary (HPB) surgery. Materials and methods The aim of this review is to determine if CPET and (P)-POSSUM scoring systems accurately predict morbidity and mortality. Eligible articles were identified with an electronic database search. Analysis according to surgery type and tool used was performed. Results Twenty-five studies were included in the final review. POSSUM predicted morbidity demonstrated weighted O/E ratios of 0.75(95%CI0.57–0.97) in hepatic surgery and 0.85(95%CI0.8–0.9) in pancreatic surgery. P-POSSUM predicted mortality in pancreatic surgery demonstrated an O/E ratio of 0.75(95%CI0.27–2.13) and 0.94(95%CI0.57–1.55) in hepatic surgery. In both pancreatic and hepatic surgery an anaerobic threshold(AT) of between 9 0.5–11.5 ml/kg/min was predictive of post-operative complications, and in pancreatic surgery ventilatory equivalence of carbon dioxide(˙VE/˙VCO2) was predictive of 30-day mortality. Conclusion POSSUM demonstrates an overall lack of predictive fit for morbidity, whilst CPET variables provide some predictive power for post-operative outcomes. Development of a new HPB specific risk prediction tool would be beneficial; the combination of parameters from POSSUM and CPET, alongside HPB specific markers could overcome current limitations., Highlights • Current pre-operative scoring for pancreatic and liver surgery is inaccurate. • In pancreatic and liver surgery anaerobic threshold scores were predictive of complications. • In pancreatic surgery ventilatory equivalence of carbon dioxide was predictive of mortality. • P-POSSUM is inaccurate for predicting mortality and morbidity in pancreatic surgery.

Regulatory T cells (Tregs) have a critical role in maintaining self-tolerance and immune homeostasis. There is much interest in using Tregs as a cell therapy to re-establish tolerance in conditions such as inflammatory bowel disease and type 1 diabetes, with many ongoing clinical studies testing the safety and efficacy of this approach. Manufacturing of Tregs for therapy typically involves ex vivo expansion to obtain sufficient cell numbers for infusion and comes with the risk of altering the activity of key biological processes. However, this process also offers an opportunity to tailor Treg function to maximize in vivo activity. In this review, we focus on the roles of antigen-presenting cells (APCs) in the generation and function of Tregs in humans. In addition to stimulating the development of Tregs, APCs activate Tregs and provide signals that induce specialized functional and homing marker expression. Cross talk between Tregs and APCs is a critical, often under-appreciated, aspect of Treg biology, with APCs mediating the key properties of infectious tolerance and bystander suppression. Understanding the biology of human Treg-APC interactions will reveal new ways to optimize Treg-based therapeutic approaches.

The word ‘intercultural’ has been in use in research and practice in different parts of the world for many decades. In daily life, it is less used compared to ‘competitors’ such as multicultural or...

Chronic graft-versus-host disease (cGvHD) is a major cause of morbidity after Hematopoietic Stem Cell Transplantation (HSCT). In large patient populations, we have shown a CD56bright Natural Killer population to strongly associate with a lack of cGvHD and we hypothesize that these cells function to suppress cGvHD. We aimed to isolate and define the characteristics of NKreg cells associated with suppression of cGvHD. Immunophenotypic evaluation of a large pediatric population found the CD56bright NK population associated with a lack of cGvHD to be perforin-, Granzyme B-, and CD335+. Transcriptome analysis of a small patient cohort of CD56bright compared to CD56dim NK cells found the NKreg cells to also overexpress Granzyme K, IL-7R, GPR183, RANK, GM-CSFR, TCF7, and IL23A. Further analysis of this CD56bright NKreg population found a subpopulation that overexpressed IRF1, and TNF. We also found that viable NKreg cells may be isolated by sorting on CD56+ and CD16- NK cells, and this population can suppress allogeneic CD4+ T cells, but not Treg cells or CD8+ T cells through a non-cytolytic, cell-cell contact dependent mechanism. Suppression was not reliant upon the NKp44, NKp46, or GPR183 receptors. Additionally, NKreg cells do not kill leukemic cells. Moreover, this is the first paper to clearly establish that a CD56brightCD3-CD16-perforin- NKreg population associates with a lack of cGvHD and has several unique characteristics, including the suppression of helper T cell function in vitro. With further investigation we may decipher the mechanism of NKreg suppression and operationalize expansion of NKreg cells associated with cGvHD suppression.

There is a long-standing tradition to assess hearing-aid benefits using lab-based speech intelligibility tests. Towards a more everyday-like scenario, the current study investigated the effects of hearing-aid amplification and noise on face-to-face communication between two conversational partners. Eleven pairs, consisting of a younger normal-hearing (NH) and an older hearing-impaired (HI) participant, solved spot-the-difference tasks while their conversations were recorded. In a two-block randomized design, the tasks were solved in quiet or noise, both with and without the HI participant receiving hearing-aid amplification with active occlusion cancellation. In the presence of 70 dB SPL babble noise, participants had fewer, slower, and less well-timed turn-starts, while speaking louder with longer inter-pausal units (IPUs, stretches of continuous speech surrounded by silence) and reducing their articulation rates. All these changes are indicative of increased communication effort. The timing of turn-starts by the HI participants exhibited more variability than that of their NH conversational partners. In the presence of background noise, the timing of turn-starts by the HI participants became even more variable, and their NH partners spoke louder. When the HI participants were provided with hearing-aid amplification, their timing of turn-starts became faster, they increased their articulation rate, and they produced shorter IPUs, all indicating reduced communication effort. In conclusion, measures of the conversational dynamics showed that background noise increased the communication effort, especially for the HI participants, and that providing hearing-aid amplification caused the HI participant to behave more like their NH conversational partner, especially in quiet situations.

Regulatory T cells (Tregs) are a promising therapy for several immune-mediated conditions but manufacturing a homogeneous and consistent product, especially one that includes cryopreservation, has been challenging. Discarded pediatric thymuses are an excellent source of therapeutic Tregs with advantages including cell quantity, homogeneity and stability. Here we report systematic testing of activation reagents, cell culture media, restimulation timing, and cryopreservation to develop a good-manufacturing-practice (GMP)-compatible method to expand and cryopreserve Tregs. By comparing activation reagents, including soluble antibody tetramers, antibody-conjugated beads and artificial antigen-presenting cells (aAPCs), and different media, we found that the combination of Dynabeads Treg Xpander and ImmunoCult-XF medium preserved FOXP3 expression and suppressive function, and resulted in expansion that was comparable to a single stimulation with aAPCs. Cryopreservation tests revealed a critical timing effect: only cells cryopreserved 1–3 days, but not >3 days, after restimulation maintained high viability and FOXP3 expression upon thawing. Restimulation timing was a less critical process parameter than the time between restimulation and cryopreservation. This systematic testing of key variables provides increased certainty regarding methods for in vitro expansion and cryopreservation of Tregs. The ability to cryopreserve expanded Tregs will have broad-ranging applications including enabling centralized manufacturing and long-term storage of cell products.

Background & Aims. T-regulatory (Treg) cells suppress immune responses to maintain homeostasis. There are two main subsets of Treg cells, FOXP3-positive Treg cells that do not produce high levels of effector cytokines and Type 1 Treg (Tr1) cells that are FOXP3-negative and secrete interleukin 10 (IL10). IL10 is an important anti-inflammatory cytokine, therefore Tr1 cells might be uniquely suited for development as a treatment for inflammatory bowel diseases. We aimed to develop methods to isolate and expand human Tr1 cells and define their functions. Methods. We obtained blood samples and colon biopsies from patients with Crohn’s disease or ulcerative colitis or healthy individuals (controls). CD4+ T cells were isolated from blood samples, stimulated with anti-CD3/CD28 beads and Tr1 cells purified using an IL10 cytokine capture assay and cell sorting. FOXP3-positive Treg cells were sorted as CD4+CD25highCD127low cells from unstimulated cells. Tr1 and FOXP3-positive Treg cells were expanded, and phenotypes and gene expression profiles were compared. T cells in peripheral blood mononuclear cells from healthy donors were stimulated with anti-CD3/CD28 beads and the suppressive abilities of Tr1 and FOXP3-positive Treg cells were measured. Human colonic organoid cultures were established then cultured with Tr1 or FOXP3-positive Treg cell culture supernatants and analyzed by immunofluorescence and flow cytometry. T84 cells (human colonic adenocarcinoma epithelial cells) were incubated with Tr1 or FOXP3-positive Treg cell culture supernatants and trans-epithelial electrical resistance was measured to determine epithelial cell barrier function. Results. Phenotypes of Tr1 cells isolated from healthy subjects or patients with Crohn’s disease or ulcerative colitis did not differ significantly following expansion. Tr1 cells and FOXP3-positive Treg cells suppressed proliferation of effector T cells, but only Tr1 cells suppressed secretion of IL1beta (IL1B) and TNF from myeloid cells. Tr1 cells, but not FOXP3-positive Treg cells, isolated from healthy subjects and IBD patients secreted IL22, which regulated repair of the epithelium and promoted barrier function of human intestinal epithelial cells. Tr1 cell culture supernatants promoted differentiation of mucin-producing goblet cells in intestinal organoid cultures. Conclusion. Human Tr1 cells suppress proliferation of effector T cells (adaptive immune response) and production of IL1B and TNF by myeloid cells (innate immune response). They also secrete IL22 to regulate repair of the epithelium and promote barrier function. They might be developed as a cell-based therapy for intestinal inflammatory disorders.

Introduction/Background* Malignant ovarian germ cell tumours (MOGCTs) are rare with a yearly-adjusted incidence of 3.7 per million [1] and account for 1-2% of all ovarian malignancies in Europe. There is a clinical imperative to clarify the optimal surgical approach and establish surgical radicality since this is a predominantly young population and minimising treatment morbidity and optimising future fertility is of real importance. Here we aim to describe the current surgical management of ovarian germ cell tumours and relate this to clinical outcome. Specifically, we aimed to compare outcomes of open versus laparoscopic surgery, the use of fertility-sparing approaches, surgical staging, and the potential utility of cystectomy alone in the management of patients with stage 1 immature teratoma. Methodology A retrospective cohort study of all consecutive patients with primary ovarian germ cell tumours treated in four major UK gynaecology oncology centres over 12 years. Result(s)* One hundred and thirty-seven patients were followed-up for a median of 4.6 years with a 5 year survival of 91% (95% CI 86-97%). Open procedures were likely to be performed for larger (p Assessing the use of surgical staging in our cohort, peritoneal or omental biopsies were infrequently taken (29%, n=40) and were largely negative, returning positive results in 0% of peritoneal and 4% of omental biopsies. In patients with stage one immature teratoma, outcomes of unilateral cystectomy only (n=9) and unilateral salpingo-oophorectomy (n=29) were compared, with no significant difference in death, recurrence rates or residual disease prevalence between the groups. The majority (88%, n=120) of patients had fertility-sparing surgery. This was not associated with higher rates of recurrence or death than non fertility-sparing approaches. Conclusion* Laparoscopic surgery was safe and since routine staging biopsies did not alter outcome, we suggest that their use should be limited. Ovarian cystectomy may be acceptable for early-stage immature teratoma and warrants replication in other cohorts.

Industry standards for nutrient delivery to greenhouse-grown ornamentals are typically in excess of the plant’s needs and can be reduced without causing adverse effects. Previous studies have reduced the level of specific nutrients or suite of nutrients over the entire crop cycle or at the onset of reproductive growth. Here, two split-plot experiments (four blocks each) were conducted with subirrigated, potted, disbudded chrysanthemums (Chrysanthemum morifolium Ramat.) grown under greenhouse conditions with sulphate treatment (2.25 mmol L−1 S supplied continuously over the crop cycle in experiment 2 only and 2.25, 1.125, or 0.5625 mmol L−1 S interrupted at inflorescence emergence) as the main plot and cultivar (‘Olympia’ and ‘Covington’) as the sub-plot. Morphological characteristics of plants with fully-expanded inflorescences were unaffected by decreasing S delivery over the crop cycle. Dry mass (DM) yields and S budgets revealed that supply-based S use and S uptake efficiencies increased markedly in both cultivars with decreasing S delivery. Minor amounts of reduced-S, rather than sulphate, were lost from leaves of ‘Covington’ during inflorescence development. High quality chrysanthemums had sufficient leaf-S (0.17%–0.23% DM) at inflorescence emergence even with the lowest S supply, which would deliver an approximate 87.5% reduction in S over the crop cycle compared with industry standards. The primary mechanism to obtain sufficient S for the growth of chrysanthemums in these studies was increased uptake efficiency.

Summary Regulatory T cell (Treg) therapy has shown promise in early clinical trials for treating graft-versus-host disease, transplant rejection and autoimmune disorders. A challenge has been to isolate sufficiently pure Tregs and expand them to a clinical dose. However, there has been considerable progress in the development and optimization of these methods, resulting in a variety of manufacturing protocols being tested in clinical trials. In this review, we summarize methods that have been used to manufacture Tregs for clinical trials, including the choice of cell source and protocols for cell isolation and expansion. We also discuss alternative culture or genome editing methods for modulating Treg specificity, function or stability that could be applied to future clinical manufacturing protocols to increase the efficacy of Treg therapy.

Regulatory T cell (Treg) therapy is a potential curative approach for a variety of immune-mediated conditions, including autoimmunity and transplantation, in which there is pathological tissue damage. In mice, IL-33R (ST2)–expressing Tregs mediate tissue repair by producing the growth factor amphiregulin, but whether similar tissue-reparative Tregs exist in humans remains unclear. We show that human Tregs in blood and multiple tissue types produced amphiregulin, but this was neither a unique feature of Tregs nor selectively upregulated in tissues. Human Tregs in blood, tonsil, synovial fluid, colon, and lung tissues did not express ST2, so ST2+ Tregs were engineered via lentiviral-mediated overexpression, and their therapeutic potential for cell therapy was examined. Engineered ST2+ Tregs exhibited TCR-independent, IL-33–stimulated amphiregulin expression and a heightened ability to induce M2-like macrophages. The finding that amphiregulin-producing Tregs have a noneffector phenotype and are progressively lost upon TCR-induced proliferation and differentiation suggests that the tissue repair capacity of human Tregs may be an innate function that operates independently from their classical suppressive function.

Aims. Hot accretion flows are thought to be able to power the relativistic jets observed in Active Galactic Nuclei. They can present themselves as SANE (Standard And Normal Evolution) disks or MAD (Magnetically Arrested Disks), two states implying profound differences in the physical properties of the disks themselves and of the outflows they produce. Methods. In this paper we use a multi-frequency and multi-epoch data set to study the giant radio galaxy NGC 315, with the goal to explore the properties of its accretion disk and sub-parsec jet. We analyze the source maps with a pixel-based analysis and we use theoretical models to link the observational properties of the jet to the physical state of the accretion disk. Results. We propose that the bulk flow in NGC 315 accelerates on sub-pc scales, concurrently with the parabolic expansion. We show that this fast acceleration can be theoretically reconciled with a magnetically driven acceleration. Along the acceleration and collimation zone, we observe an unexpected spectral behavior, with very steep spectral index values $\alpha \sim -1.5$ ($S_\nu \propto \nu^\alpha$) between 22 GHz and 43 GHz. Based on the properties of this region, we predict the black hole of NGC 315 to be fast rotating and the magnetic flux threading the accretion disk to be in excellent agreement with that expected in the case of a MAD. Using a new formalism based on the core-shift effect, we model the magnetic field downstream a quasi-parabolic accelerating jet and we reconstruct it up to the event horizon radius. In the MAD scenario, we compare it with the expected magnetic saturation strengths in the disk, finding a good agreement., Comment: 12 pages plus appendix, 8 figures. Accepted for publication in A&A

Pressure to optimise land use and to maximise the economic viability of land has had a detrimental impact on wetlands worldwide. Rehabilitating wetlands has been identified by resource managers as increasingly important to enhance environmental values and restore ecosystem functions that may have been lost through developments effecting wetlands. This paper investigates rehabilitating a geothermally influenced wetland that had been drained and used for grazing stock. The Waikite Wetland (New Zealand) is a relatively unique wetland because the primary water source to the wetland has a significant geothermal water component. This results in the area hosting populations of rare flora and fauna that are significant to New Zealand. A range of management actions that included diverting a geothermal stream back into the wetland, blocking drains, pest control, weed control, native plantings, fencing and building a weir to increase water levels were used to rehabilitate the wetland. This was done to promote thermotolerant vegetation growth, restore wetland water levels and minimise pest plant species re-establishing while minimising the effects on geothermal surface features and allowing indigenous wetland vegetation to re-establish. Physical, chemical and vegetation monitoring show that management actions have increased thermotolerant vegetation growth in the wetland while having a small potential impact on geothermal discharges into the wetland. Increasing the water level in the wetland appears to be helping control plant pest species close to the weir, but has also made sensitive vegetation growing close to the waterways more susceptible to flooding caused by high-intensity rainfall events.