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Background: After introducing IL-1/IL-6 inhibitors, some patients with Still and Still-like disease developed unusual, often fatal, pulmonary disease. This complication was associated with scoring as DReSS (drug reaction with eosinophilia and systemic symptoms) implicating these inhibitors, although DReSS can be difficult to recognize in the setting of systemic inflammatory disease., Objective: To facilitate recognition of IL-1/IL-6 inhibitor-DReSS in systemic inflammatory illnesses (Still/Still-like) by looking at timing and reaction-associated features. We evaluated outcomes of stopping or not stopping IL-1/IL-6 inhibitors after DReSS reaction began., Methods: In an international study collaborating primarily with pediatric specialists, we characterized features of 89 drug-reaction cases versus 773 drug-exposed controls and compared outcomes of 52 cases stopping IL-1/IL-6 inhibitors with 37 cases not stopping these drugs., Results: Before the reaction began, drug-reaction cases and controls were clinically comparable, except for younger disease-onset age for reaction cases with preexisting cardiothoracic comorbidities. After the reaction began, increased rates of pulmonary complications and macrophage activation syndrome differentiated drug-reaction cases from drug-tolerant controls (P = 4.7 × 10 -35 and P = 1.1 × 10 -24 , respectively). The initial DReSS feature was typically reported 2 to 8 weeks after initiating IL-1/IL-6 inhibition. In drug-reaction cases stopping versus not stopping IL-1/IL-6-inhibitor treatment, reaction-related features were indistinguishable, including pulmonary complication rates (75% [39 of 52] vs 76% [28 of 37]). Those stopping subsequently required fewer medications for treatment of systemic inflammation, had decreased rates of macrophage activation syndrome, and improved survival (P = .005, multivariate regression). Resolution of pulmonary complications occurred in 67% (26 of 39) of drug-reaction cases who stopped and in none who continued inhibitors., Conclusions: In systemic inflammatory illnesses, recognition of IL-1/IL-6-inhibitor-associated reactions followed by avoidance of IL-1/IL-6 inhibitors significantly improved outcomes., (Copyright © 2024 American Academy of Allergy, Asthma & Immunology. All rights reserved.)

Objective: To use a standardised reporting tool to identify potential eligible candidates for cochlear implant (CI) referral and quantify the proportion of adults who had a CI referral discussion after presenting with an audiogram within United Kingdom (UK) audiometric criteria., Design: Retrospective multicentre 6-month audit of Audiology clinic databases., Study Sample: A total of 810 adults from five geographically diverse UK Audiology sites., Results: Data were collected in late 2019 after UK CI audiometric candidacy criteria changed; one site collected only 3 months of data. The proportion of potential eligible adults (based only on audiometry) considered for CI referral was 64% (521 out of 810) and varied by site (from 50% to 83%). About 24% of patients (123 out of 521) declined CI referral; this also varied across sites (12-45%). The median age of patients where CI referral was not considered was 80 years - significantly higher than the group where CI referral was considered (73 years)., Conclusions: CI referral is dependent on where adults live, and how old they are. Older adults are significantly less likely to be considered for CI referral by Audiologists. Audiology clinics need more support to empower staff to talk to patients about CI referral.

Alternative 3-dimensional (3D) skin models that replicate in vivo human skin are required to investigate important events during wound healing, such as collective cell migration, epidermal layer formation, dermal substrate formation, re-epithelialisation and collagen production. In this study, a matched human 3D skin equivalent model (3D-SEM) was developed from human skin cells (fibroblast and keratinocytes), characterised using haematoxylin and eosin, immunofluorescence staining and microRNA profiling. The 3D-SEM was then functionally tested for its use in wound healing studies. Mesenchymal stem cells (MSCs) were isolated and characterised according to the criteria stipulated by the International Society for Cell Therapy. Cytokine and growth factor secretions were analysed by enzyme-linked immunosorbent assay. MSC-conditioned medium (MSC-CM) was then tested for wound healing capacity using the developed 3D-SEM at different timepoints i.e., at one, two and four weeks. The constructed 3D-SEM showed consistent development of skin-like structures composed of dermal layers and epidermal layers, with the ability to express epidermal differentiation markers and full stratification. They also showed prolonged longevity in culture media, retaining full differentiation and stratification within the four weeks. MicroRNA profiling revealed a strong correlation in microRNA expression between the developed 3D-SEM and the original native skin (p<0.001; R=0.64). Additionally, MSC-CM significantly enhanced migration, proliferation and differentiation of epidermal cells in the wounded models compared to control models at the different timepoints. In conclusion, in this study, the developed 3D-SEM mimicked native skin at the cellular and molecular levels, and clearly showed the important stages of skin regeneration during the healing process. MSC secretome contains growth factors that play a pivotal role in the healing process and could be used as a therapeutic option to accelerate skin healing.

Biologics, including monoclonal antibodies (mAb), have proved to be effective and successful therapeutic agents, particularly in the treatment of cancer and immune-inflammatory conditions, as well as allergies and infections. However, their use carries an inherent risk of an immune-mediated adverse drug reaction. In this study, we describe the use of a novel pre-clinical human in vitro skin explant test for predicting skin sensitization and adverse immune reactions. The skin explant test was used to investigate the effects of therapeutic antibodies, which are known to cause a limited reaction in a small number of patients or more severe reactions., Material and Methods: Immune responses were determined by T cell proliferation and multiplex cytokine analysis, as well as histopathological analysis of skin damage (grades I-IV in increasing severity), predicting a negative (grade I) or positive (grade ≥ II) response for an adverse skin sensitization effect., Results: T cell proliferation responses were significantly increased in the positive group ( p < 0.004). Multiplex cytokine analysis showed significantly increased levels of IFNγ, TNFα, IL-10, IL-12, IL-13, IL-1β, and IL-4 in the positive response group compared with the negative response group ( p < 0.0001, p < 0.0001, p < 0.002, p < 0.01, p < 0.04, p < 0.006, and p < 0.004, respectively)., Conclusions: Overall, the skin explant test correctly predicted the clinical outcome of 13 out of 16 therapeutic monoclonal antibodies with a correlation coefficient of 0.770 ( p = 0.0001). This assay therefore provides a valuable pre-clinical test for predicting adverse immune reactions, including T cell proliferation and cytokine release, both associated with skin sensitization to monoclonal antibodies.

Introduction: Monoclonal antibodies (mAbs) are important therapeutics. However, the enhanced potential for aggregation has become a critical quality parameter during the production of mAbs. Furthermore, mAb aggregation may also present a potential health risk in a clinical setting during the administration of mAb therapeutics to patients. While the extent of immunotoxicity in patient populations is uncertain, reports show it can lead to immune responses via cell activation and cytokine release. In this study, an autologous in vitro skin test designed to predict adverse immune events, including skin sensitization, was used as a novel assay for the assessment of immunotoxicity caused by mAb aggregation. Material and Methods : Aggregation of mAbs was induced by a heat stress protocol, followed by characterization of protein content by analytical ultra-centrifugation and transmission electron microscopy, revealing a 4% aggregation level of total protein content. Immunotoxicity and potential skin sensitization caused by the aggregates, were then tested in a skin explant assay. Results: Aggregated Herceptin and Rituximab caused skin sensitization, as shown by histopathological damage (grade II-III positive response) together with positive staining for Heat Shock Protein 70 (HSP70). Changes in T cell proliferation were not observed. Cytokine analysis revealed a significant increase of IL-10 for the most extreme condition of aggregation (65 °C at pH3) and a trend for an overall increase of IFN-γ, especially in response to Rituximab. Conclusions: The skin explant assay demonstrated that aggregated mAbs showed adverse immune reactions, as demonstrated as skin sensitization, with histopathological grades II-III. The assay may, therefore, be a novel tool for assessing immunotoxicity and skin sensitization caused by mAb aggregation.

Osteoarthritis (OA) is a degenerative joint disease, causing impaired mobility. There are currently no effective therapies other than palliative treatment. Mesenchymal stromal cells (MSCs) and their secreted extracellular vesicles (MSC-EVs) have shown promise in attenuating OA progression, promoting chondral regeneration, and modulating joint inflammation. However, the precise molecular mechanism of action driving their beneficial effects has not been fully elucidated. In this study, we analyzed MSC-EV-treated human OA chondrocytes (OACs) to assess viability, proliferation, migration, cytokine and catabolic protein expression, and microRNA and mRNA profiles. We observed that MSC-EV-treated OACs displayed increased metabolic activity, proliferation, and migration compared to the controls. They produced decreased proinflammatory (Il-8 and IFN-γ) and increased anti-inflammatory (IL-13) cytokines, and lower levels of MMP13 protein coupled with reduced expression of MMP13 mRNA, as well as negative microRNA regulators of chondrogenesis (miR-145-5p and miR-21-5p). In 3D models, MSC-EV-treated OACs exhibited enhanced chondrogenesis-promoting features (elevated sGAG, ACAN, and aggrecan). MSC-EV treatment also reversed the pathological impact of IL-1β on chondrogenic gene expression and extracellular matrix component (ECM) production. Finally, MSC-EV-treated OACs demonstrated the enhanced expression of genes associated with cartilage function, collagen biosynthesis, and ECM organization and exhibited a signature of 24 differentially expressed microRNAs, associated with chondrogenesis-associated pathways and ECM interactions. In conclusion, our data provide new insights on the potential mechanism of action of MSC-EVs as a treatment option for early-stage OA, including transcriptomic analysis of MSC-EV-treated OA, which may pave the way for more targeted novel therapeutics.

Background: Chronic graft-versus-host disease (cGvHD) is a complex disorder that typically manifests after allogeneic hematopoietic stem cell transplantation (HSCT). It is a major cause of non-relapse mortality, which makes finding biomarkers associated with its occurrence a priority. Recent studies increasingly indicate that microRNAs (miRNAs, short regulatory RNA molecules) can be used as biomarkers of various disorders. They can circulate in patients' bodies encapsulated within extracellular vesicles (EVs)., Objectives: To identify miRNAs associated with the occurrence of cGvHD in EVs isolated from the plasma of patients after allogeneic HSCT., Material and Methods: We performed global miRNA expression profiling in a pilot cohort of 3 cGvHD cases and 4 non-cGvHD patients without disease symptoms 90 days after the transplantation (control group)., Results: The 2 groups were naturally clustered according to their miRNA profiles using unsupervised hierarchical clustering analysis. We identified 3 miRNAs that were differentially expressed in the cGvHD patients compared to the non-cGvHD patients. The levels of hsa-miR-630 and hsa-miR-374b-5p were lower in the cGvHD patients: 4.1-fold (p = 0.002) and 2.7-fold (p = 0.044), respectively. In contrast, the levels of hsa-miR-29c-3p were 5.8-fold higher (p = 0.004)., Conclusions: Our results suggest that miRNA profiles from plasma EVs may act as markers of cGvHD onset.

Extracellular vesicles (EV) and the microRNAs that they contain are increasingly recognised as a rich source of informative biomarkers, reflecting pathological processes and fundamental biological pathways and responses. Their presence in biofluids makes them particularly attractive for biomarker identification. However, a frequent caveat in relation to clinical studies is low abundance of EV RNA content. In this study, we used NanoString nCounter technology to assess the microRNA profiles of n  = 64 EV low concentration RNA samples (180-49125 pg), isolated from serum and cell culture media using precipitation reagent or sequential ultracentrifugation. Data was subjected to robust quality control parameters based on three levels of limit of detection stringency, and differential microRNA expression analysis was performed between biological subgroups. We report that RNA concentrations > 100 times lower than the current NanoString recommendations can be successfully profiled using nCounter microRNA assays, demonstrating acceptable output ranges for imaging parameters, binding density, positive/negative controls, ligation controls and normalisation quality control. Furthermore, despite low levels of input RNA, high-level differential expression analysis between biological subgroups identified microRNAs of biological relevance. Our results demonstrate that NanoString nCounter technology offers a sensitive approach for the detection and profiling of low abundance EV-derived microRNA, and may provide a solution for research studies that focus on limited sample material., Competing Interests: The authors declare no conflicts of interest., (© 2022 The Authors. Journal of Extracellular Biology published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)

Hematopoietic allogeneic stem cell transplantation (allo-SCT) is a curative option for patients with hematological malignancies. However, due to disparities in major and minor histocompatibility antigens between donor and recipient, severe inflammatory complications can occur, among which chronic graft-versus-host disease (cGVHD) can be life-threatening. A classical therapeutic approach to the prevention and treatment of cGVHD has been broad immunosuppression, but more recently adjuvant immunotherapies have been tested. This review summarizes and discusses immunomodulatory approaches with T cells, including chimeric antigen receptor (CAR) and regulatory T cells, with natural killer (NK) cells and innate lymphoid cells (ILCs), and finally with mesenchymal stromal cells (MSC) and extracellular vesicles thereof. Clinical studies and pre-clinical research results are presented likewise., Competing Interests: AD was employed by Alcyomics Ltd. The remaining 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 © 2022 Doglio, Crossland, Alho, Penack, Dickinson, Stary, Lacerda, Eissner and Inngjerdingen.)

Tolerogenic dendritic cell (tolDC) therapies aim to restore self-tolerance in patients suffering from autoimmune diseases. Phase 1 clinical trials with tolDC have shown the feasibility and safety of this approach, but have also highlighted a lack of understanding of their distribution in vivo . Fluorine-19 magnetic resonance imaging ( 19 F-MRI) promises an attractive cell tracking method because it allows for detection of 19 F-labelled cells in a non-invasive and longitudinal manner. Here, we tested the suitability of nanoparticles containing 19 F ( 19 F-NP) for labelling of therapeutic human tolDC for detection by 19 F-MRI. We found that tolDC readily endocytosed 19 F-NP with acceptable effects on cell viability and yield. The MRI signal-to-noise ratios obtained are more than sufficient for detection of the administered tolDC dose (10 million cells) at the injection site in vivo , depending on the tissue depth and the rate of cell dispersal. Importantly, 19 F-NP labelling did not revert tolDC into immunogenic DC, as confirmed by their low expression of typical mature DC surface markers (CD83, CD86), low secretion of pro-inflammatory IL-12p70, and low capacity to induce IFN-γ in allogeneic CD4 + T cells. In addition, the capacity of tolDC to secrete anti-inflammatory IL-10 was not diminished by 19 F-NP labelling. We conclude that 19 F-NP is a suitable imaging agent for tolDC. With currently available technologies, this imaging approach does not yet approach the sensitivity required to detect small numbers of migrating cells, but could have important utility for determining the accuracy of injecting tolDC into the desired target tissue and their efflux rate., 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. The reviewer PR declared participation with the authors in the INSTRuCT Consortium to the handling editor., (Copyright © 2022 Cooke, Neal, Wood, de Vries, Anderson, Diboll, Pratt, Stanway, Nicorescu, Moyse, Hiles, Caulfield, Dickinson, Blamire, Thelwall, Isaacs and Hilkens.)

Acute myeloid leukemia (AML) is a hematological malignancy with an undefined heritable risk. Here we perform a meta-analysis of three genome-wide association studies, with replication in a fourth study, incorporating a total of 4018 AML cases and 10488 controls. We identify a genome-wide significant risk locus for AML at 11q13.2 (rs4930561; P = 2.15 × 10 -8 ; KMT5B). We also identify a genome-wide significant risk locus for the cytogenetically normal AML sub-group (N = 1287) at 6p21.32 (rs3916765; P = 1.51 × 10 -10 ; HLA). Our results inform on AML etiology and identify putative functional genes operating in histone methylation (KMT5B) and immune function (HLA)., (© 2021. The Author(s).)

Rheumatoid arthritis (RA) and ankylosing spondylitis (AS) belong to the most common inflammatory rheumatic diseases. MicroRNAs (miRNAs) are small 18-22 RNA molecules that function as posttranscriptional regulators. They are abundantly present within extracellular vesicles (EVs), small intercellular communication vesicles that can be found in bodily fluids and that have key functions in pathological and physiological pathways. Recently, EVs have gained much interest because of their diagnostic and therapeutic potential. Using NanoString profiling technology, the miRNA repertoire of serum EVs was determined and compared in RA and AS patients before and after anti-TNF therapy to assess its potential use as a diagnostic and prognostic biomarker. Furthermore, possible functional effects of those miRNAs that were characterized by the most significant expression changes were evaluated using in silico prediction algorithms. The analysis revealed a unique profile of differentially expressed miRNAs in RA and AS patient serum EVs. We identified 12 miRNAs whose expression profiles enabled differentiation between RA and AS patients before induction of anti-TNF treatment, as well as 4 and 14 miRNAs whose repertoires were significantly changed during the treatment in RA and AS patients, respectively. In conclusion, our findings suggest that extracellular vesicle miRNAs could be used as potential biomarkers associated with RA and AS response to biological treatment., Competing Interests: The authors declare no conflict of interest., (Copyright © 2021 Joanna Wielińska et al.)

Introduction: Acute graft vs. host disease (aGvHD) is a frequent complication following allogeneic haematopoeitic transplantation (HSCT). Despite recent advances, there are no universally accepted biomarkers to determine development of aGvHD. MicroRNAs miR-146a and miR-155 have been previously associated with aGvHD and show promise as clinically translatable biomarkers. In this study, we performed comprehensive expression profiling of miR-146a, miR-155, and miR-155 * expression in aGvHD target tissue and biofluids and relate expression to post-HSCT outcomes. Materials and Methods: MicroRNA expression was assessed by qRT-PCR in gastrointestinal ( n = 31) and skin ( n = 31) biopsies as well as serum (exploratory cohort n = 34, verification cohort n = 81, diagnostic cohort n = 65) and urine (exploratory cohort n = 30, verification cohort n = 56, diagnostic cohort n = 20) biofluids, including extracellular vesicle (EV) cohorts (serum EV n = 15, urine EV n = 30). Expression was related to aGvHD incidence, severity and overall survival. Results: In GI samples, expression of miR-155 ( p = 0.03) and miR-146a ( p = 0.03) was higher at aGvHD onset compared to patients with no GvHD. In skin biopsies, expression of miR-155 ( p = 0.004) was upregulated in aGvHD patients compared to normal control skin. Expression of miR-146a was higher in aGvHD compared to no aGvHD biopsies ( p = 0.002). In serum, miR-155 ( p = 0.03) and miR-146a ( p = 0.02) expression was higher at day 14 (D14), while in urine expression was elevated at D7 post-HSCT in patients who developed aGvHD compared to those disease-free. This was verified in an independent serum (miR-155 p = 0.005, miR-146a p = 0.003) and urine (miR-155 p = 0.02, miR-146a p = 0.04) cohort, where both microRNAs were also associated with aGvHD by ROC analysis. In serum and urine samples taken at the time of aGvHD symptoms, expression of miR-155 and miR-146a was also elevated (serum miR-155 p = 0.03, miR-146a p < 0.001; urine miR-155 p = 0.02, miR-146a p = 0.02). In contrast, miR-146a and miR-155 were downregulated at D14 in serum EVs and at D7 in urine EVs in patients who developed aGvHD compared to those that remained disease-free, in both an exploratory (serum miR-155 p = 0.02, miR-146a p = 0.06; urine miR-155 p = 0.02, miR-146a p = 0.07) and an independent cohort (serum miR-155 p = 0.01, miR-146a p = 0.02). Conclusions: These results further support a role for miR-155 and miR-146a as non-invasive, clinically relevant biomarkers for aGvHD. However, the link between their involvement in generalized inflammation and in specific pathophysiology requires further investigation at a systemic level., 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 Crossland, Norden, Ghimire, Juric, Pearce, Lendrem, Collin, Mischak-Weissinger, Holler, Greinix and Dickinson.)

With the reduction or elimination of animal testing, manufacturers are left with limited options, as few robust in vitro tests are available and human studies are costly. Recently, concerns have been raised regarding potential adverse health effects associated with use of WEN by Chaz Dean (WCD) cleansing conditioners. The purpose of this study was to evaluate the immunogenic potential of a WCD hair cleansing conditioner by utilizing a novel in vitro human skin explant test. Peripheral blood mononuclear cells (PBMCs) and human skin biopsies were obtained from healthy volunteers. Monocyte derived dendritic cells (MoDCs) were generated, primed by 0.01% WCD cleansing conditioner exposure for 24 h, co-cultured with autologous lymphocytes for 4 days, and then cultured with skin biopsies for 3 days. The skin biopsies then underwent histopathological evaluation, and T cell proliferation and IFNγ levels were determined. Overall, this study showed that treatment with 0.01% WCD cleansing conditioner resulted in a negative prediction for in vivo immune response. Further, this analysis shows that the skin explant test is a viable alternative to animal testing for complex mixtures or commercially available products.

Prognostic, diagnostic or predictive biomarkers are urgently needed for assessment of chronic graft-versus-host disease (cGvHD), a major risk for patients undergoing allogeneic hematopoietic stem cell transplantation. The main goal of this review generated within the COST Action EUROGRAFT "Integrated European Network on Chronic Graft Versus Host Disease" was to identify potential novel biomarkers for cGvHD besides the widely accepted molecular and cellular biomarkers. Thus, the focus was on cellular biomarkers, alloantibodies, glycomics, endothelial derived particles, extracellular vesicles, microbiome, epigenetic and neurologic changes in cGvHD patients. Both host-reactive antibodies in general, and particularly alloantibodies have been associated with cGvHD and require further consideration. Glycans attached to IgG modulate its activity and represent a promising predictive and/or stratification biomarker for cGVHD. Furthermore, epigenetic changes such as microRNAs and DNA methylation represent potential biomarkers for monitoring cGvHD patients and novel targets for developing new treatment approaches. Finally, the microbiome likely affects the pathophysiology of cGvHD; bacterial strains as well as microbial metabolites could display potential biomarkers for dysbiosis and risk for the development of cGvHD. In summary, although there are no validated biomarkers currently available for clinical use to better inform on the diagnosis, prognosis or prediction of outcome for cGvHD, many novel sources of potential markers have shown promise and warrant further investigation using well characterized, multi-center patient cohorts., 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. The reviewer BB declared a shared affiliation, with no collaboration, with one of the authors, FP, to the handling editor at the time of review., (Copyright © 2020 Crossland, Perutelli, Bogunia-Kubik, Mooney, Milutin Gašperov, Pučić-Baković, Greinix, Weber, Holler, Pulanić, Wolff, Dickinson, Inngjerdingen and Grce.)

AbstractBird nests represent an extended phenotype that is variable among and within species in terms of nest location and construction materials. It has also been suggested that nests indicate niche construction, although empirical evidence to support this is lacking. The nest wall is often considered to confer some insulation because this would help minimize the energetic expenditure by the incubating adult. However, it has been previously suggested that nests are constructed primarily for their structural role rather than for insulation, but to date, studies have used a variety of techniques to investigate the insulation of nest walls but only for relatively few species. This study used temperature loggers to determine insulatory values of nests for 16 previously undescribed species and to replicate values for eight previously described species. In addition, data for nest wall insulation that were determined using temperature loggers have been collated for a total of 32 different passerine species. The effects of nest dimensions, mass, and composition on these values were examined. Base thickness, but not wall thickness, and nest mass significantly positively affected insulatory values. This study found that the proportions of feathers and moss in the nest wall significantly positively correlated with insulatory values. This suggests that there is a key role for nest materials in determining insulation, which provides empirical evidence that nest building constitutes niche construction. The data will also help us interpret data from future studies of the thermal properties of nests. Ultimately, we need to develop our understanding of the role of insulatory values in the evolution of nest function.

Fms-like tyrosine kinase 3 (Flt3) is expressed on progenitor cells and acute myeloid leukemia (AML) blasts. Fms-like tyrosine kinase 3 ligand (Flt3L) is detectable during homeostasis and increases in hypoplasia due to genetic defects or treatment with cytoreductive agents. Conversely, Flt3+ AML is associated with depletion of Flt3L to undetectable levels. After induction chemotherapy, Flt3L is restored in patients entering complete remission (CR) but remains depressed in those with refractory disease. Weekly sampling reveals marked differences in the kinetics of Flt3L response during the first 6 weeks of treatment, proportionate to the clearance of blasts and cellularity of the bone marrow. In the UK NCRI AML17 trial, Flt3L was measured at day 26 in a subgroup of 140 patients with Flt3 mutation randomized to the tyrosine kinase inhibitor lestaurtinib or placebo. In these patients, attainment of CR was associated with higher Flt3L at day 26 (Mann-Whitney UP < .0001). Day 26 Flt3L was also associated with survival; Flt3L ≤291 pg/mL was associated with inferior event-free survival (EFS), and Flt3L >1185 pg/mL was associated with higher overall survival (OS; P = .0119). The separation of EFS and OS curves increased when minimal residual disease (MRD) status was combined with Flt3L measurement, and Flt3L retained a near-significant association with survival after adjusting for MRD in a proportional hazards model. Serial measurement of Flt3L in patients who had received a hematopoietic stem cell transplant for AML illustrates the potential value of monitoring Flt3L to identify relapse. Measurement of Flt3L is a noninvasive test with the potential to inform clinical decisions in patients with AML., (© 2019 by The American Society of Hematology.)

The occurrence of drug hypersensitivity reactions (DHRs) following administration of low molecular weight (LMW) drugs is an important health concern. However, in vivo animal models which could be used as tools for the prediction of DHRs are lacking. As a result, research has focused on development of in vitro tools for predicting DHRs. In this study a novel human in vitro pre-clinical skin explant test was used to predict T cell-mediated hypersensitivity responses induced by LMW drugs. Responses in the skin explant test for 12 LMW drugs associated with T cell-mediated hypersensitivity in the clinic (abacavir, amoxicillin, carbamazepine, diclofenac, lamotrigine, lapatinib, lumiracoxib, nevirapine, ofloxacin, phenytoin, propranolol, sulfamethoxazole) were compared with responses for 5 drugs with few/no reports of T cell-mediated hypersensitivity reactions (acetaminophen, cimetidine, flecainide, metformin, verapamil). Changes in skin histology following in vitro exposure to the drugs as well as T cell proliferation and interferon gamma (IFNγ) production were studied. The results of the skin explant assays showed a good positive correlation (r = 0.77, p < .001) between the test outcome (prediction of positive or negative) and the clinical classification of the tested drugs. The T cell proliferation assay showed a correlation of r = 0.60 (p < .01) and the IFNγ assay r = 0.51 (p < .04). The data suggest that the skin explant model could be a useful tool to predict the potential of LMW drugs to induce DHRs., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Avian nests provide a location for incubation, and in many cases rearing of nestlings. These energetically demanding activities have meant that nest insulation has been the subject of many studies but few to date have dealt with how variation among species in nest construction materials could affect nest insulation. This study investigated the insulation of nests constructed by three species of Old Word warblers (Sylviidae), which vary in size and composition. Insulatory values, i.e. difference in cooling rate of temperature loggers placed inside and outside a nest, and internal cooling rates within the nest cup were determined using temperature loggers under still-air and moving-air conditions. Insulatory values determined in still-air conditions of a laboratory were significantly different among nests of the different species but not when the values were determined within the smaller volume of a wind tunnel. Moving-air increased insulatory values by an order of magnitude but also increased internal cooling rates in all species. Insulatory values were positively correlated with the nest base thickness. Moving-air increased the cooling rate of the external temperature logger much more than the internal logger, which inflated the insulatory value of a nest wall in moving-air. Reasons for these results may reflect the thermal properties of the materials individually or in combination. Future testing of nest insulation should be under standard conditions that limit air movement but the role of the nest location in situ should be investigated in future research., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Mesenchymal stromal cells (MSCs) are potent regulators of immune responses largely through paracrine signaling. MSC secreted extracellular vesicles (MSC-EVs) are increasingly recognized as the key paracrine factors responsible for the biological and therapeutic function of MSCs. We report the first comprehensive study demonstrating the immunomodulatory effect of MSC-EVs on dendritic cell (DC) maturation and function. MSC-EVs were isolated from MSC conditioned media using differential ultracentrifugation. Human monocyte-derived DCs were generated in the absence or presence of MSC-EVs (20 ug/ml) then subjected to phenotypic and functional analysis in vitro . MSC-EV treatment impaired antigen uptake by immature DCs and halted DC maturation resulting in reduced expression of the maturation and activation markers CD83, CD38, and CD80, decreased secretion of pro-inflammatory cytokines IL-6 and IL-12p70 and increased production of anti-inflammatory cytokine TGF-β. MSC-EV treated DCs also demonstrated a diminished CCR 7 expression after LPS stimulation, coupled with a significantly reduced ability to migrate toward the CCR7-ligand CCL21, although they were still able to stimulate allogeneic T cell proliferation in vitro . Through microRNA profiling we have identified 49 microRNAs, which were significantly enriched in MSC-EVs compared to their parent MSCs. MicroRNAs with known effect on DC maturation and functions, including miR-21-5p, miR-142-3p, miR-223-3p, and miR-126-3p, were detected within the top 10 most enriched miRNAs in MSC-EVs, with MiR-21-5p as the third highest expressed miRNA in MSC-EVs. In silico analysis revealed that miR-21-5p targets the CCR7 gene for degradation. To verify these observations, DCs were transfected with miR-21-5p mimics and analyzed for their ability to migrate toward the CCR7-ligand CCL21 in vitro . MiR-21-5p mimic transfected DCs showed a clear trend of reduced CCR7 expression and a significantly decreased migratory ability toward the CCL21. Our findings suggest that MSC-EVs are able to recapitulate MSC mediated DC modulation and MSC-EV enclosed microRNAs may represent a novel mechanism through which MSCs modulate DC functions. As MSCs are currently used in clinical trials to treat numerous diseases associated with immune dysregulation, such as graft-versus-host disease and inflammatory bowel disease, our data provide novel evidence to inform potential future application of MSC-EVs as a cell-free therapeutic agent.

Haematopoietic stem cell transplantation (HSCT) remains the only cure for most haematological malignancies, however, the mortality rate remains high. Complications after HSCT include relapse, graft versus host disease (GvHD), graft rejection and infection. Over the last few years several groups, have demonstrated that non-HLA gene polymorphisms can be predictive of outcome after HSCT. Since the glucocorticoid cortisol is pivotal in the regulation of the immune system, we decided to examine single nucleotide polymorphisms (SNPs; rs6198, rs33388 and rs33389) within the glucocorticoid receptor (GR) and correlate with HSCT outcome. The training set consisted of patients (n = 458) who underwent HSCT for acute leukaemia between 1983 and 2005. In the recipients, the absence of the ACT haplotype and absence of the T allele of rs33388 were associated with decreased OS and the absence of the ACT haplotype, the absence of the T allele of rs33388 and the presence of the ATA haplotype were associated with increased risk of relapse. In addition, the presence of the ACT haplotype in the recipient showed a trend to be associated with increased risk of chronic graft versus host disease (cGvHD). The patients in this cohort received mainly myeloablative conditioning (n = 327). The SNPs in the glucocorticoid receptor were then investigated in a validation set (n = 251) of HSCT patients transplanted for acute leukaemia from 2006. This cohort contained significantly more patients that had received reduced intensity conditioning (RIC). Some of the results could be validated in these patients. However, contrary to the training set, the absence of the haplotype ACT in the donor in this cohort was associated with increased risk of cGvHD. Differences in the conditioning were shown to influence the results. These results are the first to associate GR SNPs with HSCT outcome and demonstrate the inherent problems of replicating SNP association studies in HSCT, due to different pre-transplant regimens., (© 2018 John Wiley & Sons Ltd.)

Allogeneic hematopoietic stem cell transplantation is a curative treatment for numerous hematological malignancies. However, acute graft-versus-host disease (aGvHD) is a major complication affecting 40-70% of all transplant patients, whereby the earliest and most frequent presentation is in the skin. MicroRNAs play a role in varied biological process and have been reported as potential biomarkers for aGvHD. More recently, microRNAs have received added attention as circulatory biomarkers that can be detected in biofluids. In this study, we performed global microRNA expression profiling using a discovery cohort of diagnostic cutaneous aGvHD biopsies ( n  = 5, stages 1-3) and healthy volunteers ( n  = 4), in order to identify a signature list of microRNAs that could be used as diagnostic biomarkers for cutaneous aGvHD. Candidate microRNAs ( n  = 8) were then further investigated in a validation cohort of post-HSCT skin biopsies ( n  = 17), pre-HSCT skin biopsies ( n  = 6) and normal controls ( n  = 6) for their association with aGvHD. Expression of let-7c ( p  = 0.014), miR-503-5p ( p  = 0.003), miR-365a-3p ( p  = 0.02), miR-34a-5p ( p  < 0.001) and miR-34a-3p ( p  = 0.006) were significantly differentially expressed between groups and significantly associated with survival outcome in post-HSCT patients (miR-503-5p ROC AUC = 0.83 p  = 0.021, Log Rank p  = 0.003; miR-34a-3p ROC AUC = 0.93, p  = 0.003, Log Rank p  = 0.004). There was no association with relapse. A statistical interaction between miR-34a-3p and miR-503-5p ( p  = 0.016) was diagnostic for aGvHD. Expression levels of the miR-34a-5p protein target p53 were assessed in the epidermis of the skin, and an inverse correlation was identified ( r 2  = 0.44, p  = 0.039). Expression of the validated candidate microRNAs was also assessed at day 28 post-HSCT in the sera of transplant recipients, in order to investigate their potential as circulatory microRNA biomarkers. Expression of miR-503-5p ( p  = 0.001), miR-34a-5p ( p  = 0.005), and miR-34a-3p ( p  = 0.004) was significantly elevated in the sera of patients who developed aGvHD versus no-aGvHD ( n  = 30) and miR-503-5p was associated with overall survival (OS) (ROC AUC = 0.80, p  = 0.04, Log Rank p  = 0.041). In conclusion, this investigation reports that microRNA expression levels in clinical skin biopsies, obtained at the time of cutaneous aGvHD onset, show potential as diagnostic biomarkers for aGvHD and as predictive biomarkers for OS. In addition, the same microRNAs can be detected in the circulation and show predictive association with post-HSCT outcomes.

Objective: To conduct a systematic review of the benefits of non-linear frequency compression (NLFC) in adults and children., Design: Ten databases were searched for studies comparing the effects of NLFC and conventional processing (CP) for the period January 2008 to September 2017., Study Sample: Twelve articles were included in this review: four adults and school-aged only, one pre-school only and three with both adults and school-aged children., Results: A two-stage process was implemented to grade the evidence. The individual studies were graded based on their study type (from 1 = highest quality of evidence to 5 = the lowest quality) and then sub-graded based on their quality ("a" for "good quality" or "b" for "lesser quality"). All studies were awarded 4a, except the single pre-school study, which was awarded 2a. The overall evidence for each population was graded based on the quality, quantity and consistency of the studies. The body of evidence was rated as very low for both adults and school-aged children, but high for pre-school children., Conclusion: The low number (and quality) of studies means that evidence supporting the benefit from NLFC is inconclusive. Further high-quality RCTs are required to provide a conclusive answer to this question.

Mesenchymal stromal cells (MSCs) are a promising cell source to develop cell therapy for many diseases. Human platelet lysate (PLT) is increasingly used as an alternative to foetal calf serum (FCS) for clinical-scale MSC production. To date, the global surface protein expression of PLT-expended MSCs (MSC-PLT) is not known. To investigate this, paired MSC-PLT and MSC-FCS were analysed in parallel using high-throughput flow cytometry for the expression of 356 cell surface proteins. MSC-PLT showed differential surface protein expression compared to their MSC-FCS counterpart. Higher percentage of positive cells was observed in MSC-PLT for 48 surface proteins, of which 13 were significantly enriched on MSC-PLT. This finding was validated using multiparameter flow cytometry and further confirmed by quantitative staining intensity analysis. The enriched surface proteins are relevant to increased proliferation and migration capacity, as well as enhanced chondrogenic and osteogenic differentiation properties. In silico network analysis revealed that these enriched surface proteins are involved in three distinct networks that are associated with inflammatory responses, carbohydrate metabolism and cellular motility. This is the first study reporting differential cell surface protein expression between MSC-PLT and MSC-FSC. Further studies are required to uncover the impact of those enriched proteins on biological functions of MSC-PLT.

Acute graft-versus-host disease (aGvHD) is a major cause of adverse outcome in hematopoietic stem cell transplantation (HSCT), with a high incidence (20-50%). A novel, non-invasive diagnostic test to predict for prevalence and severity would enable improved prophylaxis and reduce morbidity. Circulatory microRNAs (miRNAs) miR-423, miR-199, miR-93*, and miR-377 have previously been associated with aGvHD in post-HSCT patient plasma, but validation is lacking and their expression within extracellular vesicles (EVs) has not been explored. This study replicated elevated serum expression of miR-423 ( p  < 0.001), miR-199 ( p  = 0.04), miR-93* ( p  < 0.001), and miR-377 ( p  = 0.03) in aGvHD, using a prognostic cohort of day 14 (D14) post-HSCT patient samples ( n  = 81). Expression also associated with disease severity. Further analysis at aGvHD diagnosis in an independent cohort ( n  = 65) confirmed high miR-423 ( p  = 0.02), miR-199 ( p  = 0.007), and miR-93* ( p  = 0.004) expression at disease onset. Investigation of expression patterns during early HSCT sequential timepoints (pre-HSCT to D28) identified elevated miRNAs at D7 post-HSCT in all transplant patients. In a novel investigation of miRNA expression in serum EVs ( n  = 15), miR-423 ( p  = 0.09), miR-199 ( p  = 0.008), and miR-93* ( p  = 0.001) levels were lower at D14 in patients who later developed aGvHD, and this was replicated for miR-423 ( p  = 0.02) and miR-199 ( p  = 0.04) ( n  = 47). Comparing serum to circulating EVs, at D14 patients remaining aGvHD-free had higher expression of miR-423 ( p  = 0.03), miR-199 ( p  = 0.009), and miR-93* ( p  = 0.002) in the EV fraction. Results verify the capacity for circulating miR-423, miR-199, and miR-93* as diagnostic and prognostic aGvHD biomarkers. The novel finding of their differential expression in EVs suggests a potential role in aGvHD etiology.

The success of hematopoietic stem cell transplantation (HSCT) lies with the ability of the engrafting immune system to remove residual leukemia cells via a graft-versus-leukemia effect (GvL), caused either spontaneously post-HSCT or via donor lymphocyte infusion. GvL effects can also be initiated by allogenic mismatched natural killer cells, antigen-specific T cells, and activated dendritic cells of leukemic origin. The history and further application of this GvL effect and the main mechanisms will be discussed and reviewed in this chapter.

Alemtuzumab conditioning is highly effective at reducing the incidence of acute and chronic graft-versus-host disease (GVHD) in reduced-intensity fludarabine and melphalan transplantation with cyclosporine monotherapy. Less frequent and lower dose scheduling may be used with sibling donors, but an optimal regimen for matched unrelated donors has not been defined. In this retrospective observational study of 313 patients, the incidence and severity of GVHD was compared in patients receiving 3 different dose schedules: the standard 100-mg regimen (20 mg on days -7 to -3), 60 mg (30 mg on days -4 and -2), or 50 mg (10 mg on days -7 to -3). Patients treated with 100 mg, 60 mg, or 50 mg developed acute GVHD grades I to IV with an incidence of 74%, 65%, and 64%, respectively, whereas 36%, 32%, and 41% developed chronic GHVD. An excess of severe acute grades III/IV GVHD was observed in the 50-mg cohort (15% versus 2% to 6%; P = .016). The relative risk of severe acute grade GVHD remained more than 3-fold higher in the 50-mg cohort compared with the 100-mg cohort after adjustment for differences in HLA match, age, gender mismatch, cytomegalovirus risk, and diagnosis (P = .030). The findings indicate that the 60-mg alemtuzumab schedule was comparable with the 100-mg schedule, but more attenuated schedules may increase the risk of severe grade GVHD., (Copyright © 2017 The American Society for Blood and Marrow Transplantation. All rights reserved.)

The outcome of hematopoietic stem cell transplantation (HSCT) is controlled by genetic factors among which the leukocyte antigen human leukocyte antigen (HLA) matching is most important. In addition, minor histocompatibility antigens and non-HLA gene polymorphisms in genes controlling immune responses are known to contribute to the risks associated with HSCT. Besides single-nucleotide polymorphisms (SNPs) in protein coding genes, SNPs in regulatory elements such as microRNAs (miRNAs) contribute to these genetic risks. However, genetic risks require for their realization the expression of the respective gene or miRNA. Thus, gene and miRNA expression studies may help to identify genes and SNPs that indeed affect the outcome of HSCT. In this review, we summarize gene expression profiling studies that were performed in recent years in both patients and animal models to identify genes regulated during HSCT. We discuss SNP-mRNA-miRNA regulatory networks and their contribution to the risks associated with HSCT in specific examples, including forkheadbox protein 3 and regulatory T cells, the role of the miR-155 and miR-146a regulatory network for graft-versus-host disease, and the function of MICA and its receptor NKG2D for the outcome of HSCT. These examples demonstrate how SNPs affect expression or function of proteins that modulate the alloimmune response and influence the outcome of HSCT. Specific miRNAs targeting these genes and directly affecting expression of mRNAs are identified. It might be valuable in the future to determine SNPs and to analyze miRNA and mRNA expression in parallel in cohorts of HSCT patients to further elucidate genetic risks of HSCT.

Acute graft-versus-host disease (aGvHD) is the most frequent and serious complication following hematopoietic stem cell transplantation (HSCT), with a high mortality rate. A clearer understanding of the molecular pathogenesis may allow for improved therapeutic options or guide personalized prophylactic protocols. Circulating microRNAs are expressed in body fluids and have recently been associated with the etiology of aGvHD, but global expression profiling in a HSCT setting is lacking. This study profiled expression of n  = 799 mature microRNAs in patient serum, using the NanoString platform, to identify microRNAs that showed altered expression at aGvHD diagnosis. Selected microRNAs ( n  = 10) were replicated in independent cohorts of serum samples taken at aGvHD diagnosis ( n  = 42) and prior to disease onset (day 14 post-HSCT, n  = 47) to assess their prognostic potential. Sera from patients without aGvHD were used as controls. Differential microRNAs were investigated in silico for predicted networks and mRNA targets. Expression analysis identified 61 microRNAs that were differentially expressed at aGvHD diagnosis. miR-146a ( p  = 0.03), miR-30b-5p ( p  = 0.007), miR-374-5p ( p  = 0.02), miR-181a ( p  = 0.03), miR-20a ( p  = 0.03), and miR-15a ( p  = 0.03) were significantly verified in an independent cohort ( n  = 42). miR-146a ( p  = 0.01), miR-20a ( p  = 0.03), miR-18 ( p  = 0.03), miR-19a ( p  = 0.03), miR-19b ( p  = 0.01), and miR-451 ( p  = 0.01) were differentially expressed 14 days post-HSCT in patients who later developed aGvHD ( n  = 47). High miR-19b expression was associated with improved overall survival (OS) ( p  = 0.008), whereas high miR-20a and miR-30b-5p were associated with lower rates of non-relapse mortality ( p  = 0.05 and p  = 0.008) and improved OS ( p  = 0.016 and p  = 0.021). Pathway analysis associated the candidate microRNAs with hematological and inflammatory disease. Circulating biofluid microRNAs show altered expression at aGvHD onset and have the capacity to act as prognostic and diagnostic biomarkers. Their differential expression in serum suggests a role for circulatory microRNAs in aGvHD pathology, which warrants further investigation.

For over 60 years, hematopoietic stem cell transplantation has been the major curative therapy for several hematological and genetic disorders, but its efficacy is limited by the secondary disease called graft versus host disease (GvHD). Huge advances have been made in successful transplantation in order to improve patient quality of life, and yet, complete success is hard to achieve. This review assimilates recent updates on pathophysiology of GvHD, prophylaxis and treatment of GvHD-related complications, and advances in the potential treatment of GvHD.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) may be curative, but is associated with significant morbidity and mortality. Chronic graft-versus-host disease (cGvHD), characterized by inflammation and fibrosis of multiple target organs, considerably contributes to the morbidity and mortality even years after allo-HSCT. Diagnosis of cGvHD is based on clinical features and histology of biopsies. Here, we report the generation of a urinary cGvHD-specific proteome-pattern (cGvHD&#95;MS14) established by capillary electrophoresis-mass spectrometry to predict onset and severity of cGvHD as an unbiased laboratory test. cGvHD&#95;MS14 was evaluated on samples from 412 patients collected prospectively in four transplant centers. Sensitivity and specificity was 84 and 76% by cGvHD&#95;MS14 classification. Sensitivity further increased to 93% by combination of cGvHD&#95;MS14 with relevant clinical variables to a logistic regression model. cGvHD was predicted up to 55 days prior to clinical diagnosis. Acute GvHD is not recognized by cGvHD&#95;MS14. cGvHD&#95;MS14 consists of 14 differentially excreted peptides, six of those have been sequenced to date and are fragments from thymosin β-4, eukaryotic translation initiation factor 4γ2, fibrinogen β-chain or collagens. In conclusion, the cGvHD&#95;MS14-pattern allows early, highly sensitive and specific prediction of cGvHD as an independent diagnostic criterion of clinical diagnosis potentially allowing early therapeutic intervention.

NK cells have shown promise in therapy of hematological cancers, in particular against acute myeloid leukemia. In contrast, the more NK cell-resistant acute lymphoblastic leukemia (ALL) is difficult to treat with NK-cell-based therapies, and we hypothesized that pre-activation of NK cells could overcome this resistance. We show in pediatric and adult patients with T-cell ALL (T-ALL) perturbed NK cell effector functions at diagnosis. Using an in vivo rat model for T-ALL, Roser leukemia (RL), suppressed NK cell effector functions were observed. NK cells from T-ALL patients had reduced expression of the activating receptors NKp46 and DNAM-1, but not NKG2D. In contrast to T-ALL patients, NKG2D but not NKp46 was downregulated on NK cells during rat RL. Decreased frequencies of terminally differentiated NKG2A + CD57 - CD56 dim NK cells in human T-ALL was paralleled in the rat by reduced frequencies of bone marrow NK cells expressing the maturation marker CD11b, possibly indicating impairment of differentiation during leukemia. RL was highly resistant to autologous NK cells, but this resistance was overcome upon pre-activation of NK cells with IL-12, IL-15, and IL-18, with concomitant upregulation of activation markers and activating receptors. Importantly, adoptive transfers of IL-12, IL-15, and IL-18 pre-activated NK cells significantly slowed progression of RL in vivo . The data thus shows that T-ALL blasts normally resistant to NK cells may be targeted by cytokine pre-activated autologous NK cells, and this approach could have potential implications for immunotherapeutic protocols using NK cells to more efficiently target leukemia.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the main curative therapy for hematological malignancy such as leukemias, lymphomas, or multiple myelomas and some other hematological disorders. In this therapy, cure of hematological diseases relies on graft-versus-malignancy effects by allogenic immune cells. However, severe posttransplant treatment-associated complications such as acute graft-versus-host disease (aGvHD) and chronic graft-versus-host disease (cGvHD) limit this approach. Most research into GvHD has concentrated on the aGvHD, while the more complex and multifaceted chronic form has been largely poorly investigated. cGvHD is a multi-organ autoimmune disorder and is the major cause of non-relapse morbidity and mortality following allo-HSCT, occurring in about 50% of patients, or 13,000-15,000 patients per year worldwide. Therefore, there is a high medical need for an early prediction of these therapy-associated toxicities. Biomarkers have gained importance over the last decade in diagnosis, in prognosis, and in prediction of pending diseases or side effects. Biomarkers can be cells, factors isolated from target tissues, or soluble factors that can be detected in body fluids. In this review, we aim to summarize some of the recent developments of biomarkers in the field of allo-HSCT. We will focus on cell-based biomarkers (B-cell subsets) for cGvHD and soluble factors including microRNA (miRNA), which are excreted into serum/plasma and urine. We also discuss the potential role of cytosolic and extracellular 70 kDa heat shock proteins (HSP70) as potential biomarkers for aGvHD and their role in preclinical models. Proteomic biomarkers in the blood have been used as predictors of treatment responses in patients with aGvHD for many years. More recently, miRNAs have been found to serve as a biomarker to diagnose aGvHD in the plasma. Another development relates to urine-based biomarkers that are usually detected by capillary electrophoresis and mass spectrometry. These biomarkers have the potential to predict the development of severe aGvHD (grades III-IV), overall mortality, and the pending development of cGvHD in patients posttransplant.

Objectives: To assess the safety of intra-articular (IA) autologous tolerogenic dendritic cells (tolDC) in patients with inflammatory arthritis and an inflamed knee; to assess the feasibility and acceptability of the approach and to assess potential effects on local and systemic disease activities., Methods: An unblinded, randomised, controlled, dose escalation Phase I trial. TolDC were differentiated from CD14+ monocytes and loaded with autologous synovial fluid as a source of autoantigens. Cohorts of three participants received 1×10 6 , 3×10 6 or 10×10 6 tolDC arthroscopically following saline irrigation of an inflamed (target) knee. Control participants received saline irrigation only. Primary outcome was flare of disease in the target knee within 5 days of treatment. Feasibility was assessed by successful tolDC manufacture and acceptability via patient questionnaire. Potential effects on disease activity were assessed by arthroscopic synovitis score, disease activity score (DAS)28 and Health Assessment Questionnaire (HAQ). Immunomodulatory effects were sought in peripheral blood., Results: There were no target knee flares within 5 days of treatment. At day 14, arthroscopic synovitis was present in all participants except for one who received 10×10 6 tolDC; a further participant in this cohort declined day 14 arthroscopy because symptoms had remitted; both remained stable throughout 91 days of observation. There were no trends in DAS28 or HAQ score or consistent immunomodulatory effects in peripheral blood. 9 of 10 manufactured products met quality control release criteria; acceptability of the protocol by participants was high., Conclusion: IA tolDC therapy appears safe, feasible and acceptable. Knee symptoms stabilised in two patients who received 10×10 6 tolDC but no systemic clinical or immunomodulatory effects were detectable., Trial Registration Number: NCT01352858., Competing Interests: Conflicts of Interest: None declared., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

The EBMT risk score is an established tool successfully used in the prognosis of survival post-HSCT and is applicable for a range of haematological disorders. One of its main advantages is that score generation involves summation of clinical parameters that are available pretransplant. However, the EBMT risk score is recognized as not being optimal. Previous analyses, involving patients with various diagnoses, have shown that non-HLA gene polymorphisms influence outcome after allogeneic HSCT. This study is novel as it focuses only on patients having acute leukaemia (N = 458) and attempts to demonstrate how non-HLA gene polymorphisms can be added to the EBMT risk score in a Cox regression model to improve prognostic ability for overall survival. The results of the study found that three genetic factors improved EBMT risk score. The presence of MAL (rs8177374) allele T in the patient, absence of glucocorticoid receptor haplotype (consisting of rs6198, rs33389 and rs33388) ACT in the patient and absence of heat-shock protein 70-hom (+2437) (rs2227956) allele C in the patient were associated with decreased survival time. When compared to the EBMT risk score, the scores combining EBMT risk score with the genetic factors had an improved correlation with clinical outcome and better separation of risk groups. A bootstrapping technique, involving repeated testing of a model using multiple validation sets, also revealed that the newly proposed model had improved predictive value when compared to the EBMT risk score alone. Results support the view that non-HLA polymorphisms could be useful for pretransplant clinical assessment and provide evidence that polymorphisms in the recipient genotype may influence incoming donor cells, suppressing the initiation of the graft versus leukaemia effect and reducing survival., (© 2016 John Wiley & Sons Ltd.)

Allogeneic hematopoietic stem cell transplantation is associated with serious complications, and improvement of the overall clinical outcome of patients with hematological malignancies is necessary. During the last decades, posttransplant donor-derived adoptive cellular immunotherapeutic strategies have been progressively developed for the treatment of graft-versus-host disease (GvHD), infectious complications, and tumor relapses. To date, the common challenge of all these cell-based approaches is their implementation for clinical application. Establishing an appropriate manufacturing process, to guarantee safe and effective therapeutics with simultaneous consideration of economic requirements is one of the most critical hurdles. In this review, we will discuss the recent scientific findings, clinical experiences, and technological advances for cell processing toward the application of mesenchymal stromal cells as a therapy for treatment of severe GvHD, virus-specific T cells for targeting life-threating infections, and of chimeric antigen receptors-engineered T cells to treat relapsed leukemia.

Background: Graft-versus-host (GvH) disease (GvHD) remains a serious concern for patients undergoing antiviral cellular therapy. Despite the major improvements in cellular immunotherapy, the immunogenicity of virus-specific T cells has not yet been fully defined. This present study aims to examine how cytomegalovirus (CMV)-specific cytotoxic T lymphocytes (CTLs) respond to allogeneic antigen stimulation and whether they give rise to GvHD target tissue damage., Methods: Cytomegalovirus-specific CTLs were isolated by the IFN-γ secretion assay (gamma-catch) from healthy seropositive volunteers and expanded in vitro. The levels of intracellular IFN-γ, cytotoxic activity, IFN-γ and granzyme B secretion, and CD25 expression were measured using flow cytometry (fluorescence-activated cell sorting). The ability of CMV-CTLs to induce GvHD target tissue damage was evaluated using the human in vitro skin explant assay (skin explant assay)., Results: Cytomegalovirus-specific CTLs responded specifically to CMV-phosphoprotein 65 stimulation by secreting IFN-γ and killing virus peptide loaded autologous phytohemagglutinin (PHA) blasts. Compared with unselected peripheral blood mononuclear cells, CMV-CTLs induced significantly less severe cutaneous GvH tissue damage. This observation coincided with low levels of CD25 expression, as well as IFN-γ and granzyme B secretion after allogeneic antigen stimulation in both the mixed lymphocyte reaction and in the skin explant assay., Conclusions: Cytomegalovirus-specific CTLs isolated by the IFN-γ secretion assay from HLA-unmatched healthy donors exhibited a high level of anti-CMV potency without inducing significant cutaneous GvH tissue damage in vitro. This finding provides novel evidence supporting the safe use of in vitro expanded CMV-CTLs as an antiviral therapy in transplant patients with refractory CMV infections.

MicroRNAs (miRNA) have emerged as central regulators of diverse biological processes and contribute to driving pathology in several diseases. Acute graft-versus-host disease (aGvHD) represents a major complication after allogeneic hematopoietic stem cell transplantation, caused by alloreactive donor T cells attacking host tissues leading to inflammation and tissue destruction. Changes in miRNA expression patterns occur during aGvHD, and we hypothesized that we could identify miRNA signatures in target tissues of aGvHD that may potentially help understand the underlying molecular pathology of the disease. We utilized a rat model of aGvHD with transplantation of fully MHC-mismatched T cell depleted bone marrow, followed by infusion of donor T cells. The expression pattern of 423 rat miRNAs was investigated in skin, gut, and lung tissues and intestinal T cells with the NanoString hybridization platform, in combination with validation by quantitative PCR. MHC-matched transplanted rats were included as controls. In the skin, upregulation of miR-34b and downregulation of miR-326 was observed, while in the intestines, we detected downregulation of miR-743b and a trend toward downregulation of miR-345-5p. Thus, tissue-specific expression patterns of miRNAs were observed. Neither miR-326 nor miR-743b has previously been associated with aGvHD. Moreover, we identified upregulation of miR-146a and miR-155 in skin tissue of rats suffering from aGvHD. Analysis of intestinal T cells indicated 23 miRNAs differentially regulated between aGvHD and controls. Two of these miRNAs were differentially expressed either in skin (miR-326) or in intestinal (miR-345-5p) tissue. Comparison of intestinal and peripheral blood T cells indicated common dysregulated expression of miR-99a, miR-223, miR-326, and miR-345-5p. Analysis of predicted gene targets for these miRNAs indicated potential targeting of an inflammatory network both in skin and in the intestines that may further regulate inflammatory cytokine production. In conclusion, comprehensive miRNA profiling in rats suffering from aGvHD demonstrate tissue-specific differences in the expression patterns of miRNA that may not be detected by profiling of peripheral blood T cells alone. These tissue-specific miRNAs may contribute to distinct pathologic mechanisms and could represent potential targets for therapy.

Sensitization to chemicals resulting in an allergy is an important health issue. The current gold-standard method for identification and characterization of skin-sensitizing chemicals was the mouse local lymph node assay (LLNA). However, for a number of reasons there has been an increasing imperative to develop alternative approaches to hazard identification that do not require the use of animals. Here we describe a human in-vitro skin explant test for identification of sensitization hazards and the assessment of relative skin sensitizing potency. This method measures histological damage in human skin as a readout of the immune response induced by the test material. Using this approach we have measured responses to 44 chemicals including skin sensitizers, pre/pro-haptens, respiratory sensitizers, non-sensitizing chemicals (including skin-irritants) and previously misclassified compounds. Based on comparisons with the LLNA, the skin explant test gave 95% specificity, 95% sensitivity, 95% concordance with a correlation coefficient of 0.9. The same specificity and sensitivity were achieved for comparison of results with published human sensitization data with a correlation coefficient of 0.91. The test also successfully identified nickel sulphate as a human skin sensitizer, which was misclassified as negative in the LLNA. In addition, sensitizers and non-sensitizers identified as positive or negative by the skin explant test have induced high/low T cell proliferation and IFNγ production, respectively. Collectively, the data suggests the human in-vitro skin explant test could provide the basis for a novel approach for characterization of the sensitizing activity as a first step in the risk assessment process., (Copyright © 2015 John Wiley & Sons, Ltd.)

Previous studies from Multhoff and colleagues reported that plasma membrane Hsp70 acts as a tumour-specific recognition structure for activated NK cells, and that the incubation of NK cells with Hsp70 and/or a 14-mer peptide derived from the N-terminal sequence of Hsp70 (TKDNNLLGRFELSG, TKD, aa 450-463) plus a low dose of IL-2 triggers NK cell proliferation and migration, and their capacity to kill cancer cells expressing membrane Hsp70. Herein, we have used flow cytometry to determine the influence of in vitro stimulation of peripheral blood mononuclear cells from healthy individuals with IL-2 or IL-15, either alone or in combination with TKD peptide on the cell surface expression of CD94, NK cell activatory receptors (CD16, NK2D, NKG2C, NKp30, NKp44, NKp46, NKp80, KIR2DL4, DNAM-1 and LAMP1) and NK cell inhibitory receptors (NKG2A, KIR2DL2/L3, LIR1/ILT-2 and NKR-P1A) by CD3+CD56+ (NKT), CD3+CD4+, CD3+CD8+ and CD19+ populations. NKG2D, DNAM-1, LAMP1 and NKR-P1A expression was upregulated after the stimulation with IL-2 or IL-15 alone or in combination with TKD in NKT, CD8+ T cells and B cells. CD94 was upregulated in NKT and CD8+ T cells. Concurrently, an increase in a number of CD8+ T cells expressing LIR1/ILT-2 and CD4+ T cells positive for NKR-P1A was observed. The proportion of CD8+ T cells that expressed NKG2D was higher after IL-2/TKD treatment, when compared with IL-2 treatment alone. In comparison with IL-15 alone, IL-15/TKD treatment increased the proportion of NKT cells that were positive for CD94, LAMP1 and NKRP-1A. The more potent effect of IL-15/TKD on cell surface expression of NKG2D, LIR1/ILT-2 and NKRP-1A was observed in B cells compared with IL-15 alone. However, this increase was not of statistical significance. IL-2/TKD induced significant upregulation of LAMP1 in CD8+ T cells compared with IL-2 alone. Besides NK cells, other immunocompetent cells present within the fraction of peripheral blood mononuclear cells were influenced by the treatment with low-dose interleukins themselves or in combination with hsp70 derived (TKD) peptide.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative treatment for numerous hematological malignancies. However, acute graft-versus-host disease (aGVHD) is still the major complication causing mortality. MicroRNAs (miRNAs) play a significant role in inflammation and have potential as prognostic and diagnostic biomarkers. This study investigated the role of two immune-specific miRNAs (miR-146a and miR-155) as biomarkers for aGVHD incidence in the peripheral blood of allo-HSCT patients prior to disease onset. The study showed that miR-146a and its statistical interaction with miR-155 at day +28 were predictive of aGVHD incidence. Interestingly, the expression levels of miR-146a and miR-155 negatively correlated with the transcription factor, SPI1 (PU.1gene) mRNA expression.

MicroRNAs are small regulatory molecules that demonstrate useful biomarker potential. They have been recognised in biofluids, where they are protected from degradation by encapsulation into extracellular vesicles (EVs). A number of commercial products are available for the isolation of EVs and their RNA content; however, extensive protocol comparisons are lacking. Furthermore, robust qRT-PCR assessment of microRNA expression within EVs is problematic, as endogenous controls (ECs) previously used in cellular samples may not be present. This study compares EV isolation and RNA extraction methods (EV precipitation reagents, RNA isolation kits and ultracentrifugation) from serum or urine samples and evaluates suitable ECs for incorporation into qRT-PCR analysis. Results were assessed by electron microscopy, nanoparticle tracking analysis and bioanalyzer concentrations. The stability of 8 ECs was compared for both serum and urine EV RNA and retrospectively validated in independent cohorts (serum n=55, urine n=50). The Life Technologies precipitation reagent gave superior serum EV recovery compared to SBI reagent, as assessed by NTA size distribution, increased RNA concentration, and lower small RNA Ct values. Similarly, the Norgen Biotek Urine Exosome RNA Isolation Kit gave improved results for urine EV isolation compared to ultracentrifugation, when determined by the same parameters. The Qiagen miRNeasy™ RNA isolation kit gave suitable serum EV RNA concentrations compared to other kits, as assessed by Bioanalyzer and small RNA qRT-PCR. Small RNAs HY3 (S.D=1.77, CoV=6.2%) and U6 (S.D=2.14, CoV=8.6%) were selected as optimal ECs for serum EV microRNA expression analysis, while HY3 (S.D=1.67, CoV=6.5%) and RNU48 (S.D=1.85, CoV=5.3%) were identified as suitable for urine studies. In conclusion, this study identifies optimal methods for isolation of serum and urine EV RNA, and suitable ECs for normalisation of qRT-PCR studies. Such reports should aid in the standardisation of EV microRNA data, particularly for biomarker studies., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Graft versus host disease (GVHD) is a major complication of haematopoietic SCT (HSCT). A number of inflammatory cytokines/chemokines are implicated in GVHD and have been identified in numerous single centre studies as potential biomarkers for acute and/or chronic GVHD. In this study, we analysed candidate inflammatory biomarkers (B-cell activating factor (BAFF), interleukin 33 (IL-33), CXCL10 and CXCL11) in a two-centre study. Biomarkers were evaluated pre-transplant and at serial time points post transplant in acute and chronic GVHD patient sera with time-matched control samples from patients without GVHD. Further validation was performed using the human skin explant assay, clinical GVHD biopsies and mRNA expression analysis. BAFF was significantly increased pre-transplant. BAFF, IL-33, CXCL10 and CXCL11 showed increased levels in acute GVHD patient sera and high protein expression in grades II-III of the in vitro skin explant graft versus host reaction (GVHR) group. BAFF, CXCL10 and CXCL11 also showed increased mRNA expression levels in clinical biopsies compared with the no/low-grade GVHD group. BAFF, CXCL10 and CXCL11 levels were increased in chronic GVHD patient sera. The results identify BAFF and CXCL10 as predictive biomarkers for acute GVHD and BAFF, CXCL10 and CXCL11 as useful diagnostic biomarkers for acute GVHD and chronic GVHD.

Haematopoietic stem cell transplantation (HSCT) remains the only cure for many haematological neoplasms; however, the mortality rate remains high, at around 30-80%. Complications after HSCT include relapse, graft-versus-host disease, graft rejection and infection. High-resolution HLA matching has improved survival in HSCT over recent years; however, GVHD still remains a serious complication. Single nucleotide polymorphisms (SNPS) within genes that are involved with an individual's capability to mount an immune response to infectious pathogens, residual leukaemia, alloantigens or genes involved in drug metabolism have been studied for their association with HSCT outcome. Indeed, over the last 15 years, several groups, including ourselves, have demonstrated that non-HLA gene polymorphisms can be predictive of HSCT outcome. Can genetic characteristics of the patient and donor be used in the future to tailor HSCT protocols and determine GVHD prophylaxis? This review summarizes some of the recent SNP association studies in HSCT and highlights some of the disparities therein, discussing the integral problems of performing genetic association studies on diseases with complex outcomes using heterogeneous cohorts. The review will comment on recent genomewide association studies (GWAS) and discuss their relevance in this field, and it will also comment on recent meta-analysis combining GWAS studies with other studies such as gene expression micro array data in the field of autoimmune disease and solid organ transplantation. It will mention possible novel candidate gene polymorphisms, for example SNPS in microRNAs. In addition, it will discuss some of the inherent problems associated with gene association studies including the GRIPs (genetic risk prediction studies) recommendations. In summary, this review will assess the usefulness of non-HLA genomic studies in HSCT with regard to predicting outcome and modifying therapy., (© 2015 John Wiley & Sons Ltd.)

Graft-versus-host disease (GVHD) can be a fatal complication of allogeneic stem cell transplantation (allo-HSCT). GVHD can be classified as acute (aGVHD: up to 100 days) or chronic (cGVHD: after 100 days) based on the time-point of disease occurrence. At present there are a limited number of biomarkers available for use in the clinic. Thus, the aim of this research was to evaluate the biomarker potential of the extensively studied Heat Shock Protein 70 family members (HSPA1A/HSPA1B and HSPA1L) at the messenger RNA (mRNA) level in acute and cGVHD patient cohorts. In the skin biopsies, HSPA1L mRNA expression was lower in patients with severe aGVHD (grades II-III) when compared to those with none or low grade aGVHD (grades 0-I) and normal controls. In whole blood, HSPA1L mRNA expression level was significantly (p = 0.008) up-regulated at 28 days post-transplant in cGVHD patients with a significant area under the curve (AUC = 0.773). In addition, HSPA1B expression in whole blood was significantly higher at 3 months post-transplant in both the aGVHD grade II-III (p = 0.012) and cGVHD (p = 0.027) patients. Our initial results in this small cohort show that quantifying HSPA1L mRNA expression in the whole blood of allo-HSCT patients at day 28 post-allo-HSCT may be a useful predictive biomarker for cGVHD., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Food stored, prepared, cooked and eaten at home contributes to foodborne disease which, globally, presents a significant public health burden. The aim of the study reported here was to investigate, analyse and interpret domestic kitchen practices in order to provide fresh insight about how the domestic setting might influence food safety. Using current theories of practice meant the research, which drew on qualitative and ethnographic methods, could investigate people and material things in the domestic kitchen setting whilst taking account of people's actions, values, experiences and beliefs. Data from 20 UK households revealed the extent to which kitchens are used for a range of non-food related activities and the ways that foodwork extends beyond the boundaries of the kitchen. The youngest children, the oldest adults and the family pets all had agency in the kitchen, which has implications for preventing foodborne disease. What was observed, filmed and photographed was not a single practice but a series of entangled encounters and actions embedded and repeated, often inconsistently, by the individuals involved. Households derived logics and principles about foodwork that represented rules of thumb about 'how things are done' that included using the senses and experiential knowledge when judging whether food is safe to eat. Overall, food safety was subsumed within the practice of 'being' a household and living everyday life in the kitchen. Current theories of practice are an effective way of understanding foodborne disease and offer a novel approach to exploring food safety in the home., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Aim: Aberrant DNA methylation patterns are a hallmark of cancer, although the extent to which they underlie cancer development is unknown. In this study, we aimed to determine whether acute lymphoblastic leukemia (ALL) patients in clinical remission retained abnormal DNA methylation patters and whether these were associated with patient outcome., Materials & Methods: We investigated CpG island methylation of genes known to exhibit hypermethylation in leukemia using quantitative pyrosequencing analysis., Results: Although methylation levels were reduced in remission samples, they remained significantly higher than those seen in healthy controls. This retained methylation was not related to low levels of residual leukemia cells still present at remission. Methylation levels were also stable (or increased) during continuous remission and significantly correlated with long-term survival in adult ALL patients., Conclusion: This study determined that abnormalities in DNA methylation are retained during ALL remission and may represent a novel prognostic marker for adult ALL patients.