Concurrent transposon engineering and CRISPR/Cas9 genome editing of primary CLL-1 chimeric antigen receptor-natural killer cells.

BACKGROUND: Natural killer (NK) cell genome editing promises to enhance the innate and alloreactive anti-tumor potential of NK cell adoptive transfer. DNA transposons are versatile non-viral gene vectors now being adapted to primary NK cells, representing important tools for research and clinical product development. AIMS AND METHODS: We set out to generate donor-derived, primary chimeric antigen receptor (CAR)-NK cells by combining the TcBuster transposon system with Epstein-Barr virus-transformed lymphoblastoid feeder cell-mediated activation and expansion. RESULTS: This approach allowed for clinically relevant NK-cell expansion capability and CAR expression, which was further enhanced by immunomagnetic selection based on binding to the CAR target protein.The resulting CAR-NK cells targeting the myeloid associated antigen CLL-1 efficiently targeted CLL-1-positive AML cell lines and primary AML populations, including a population enriched for leukemia stem cells. Subsequently, concurrent delivery of CRISPR/Cas9 cargo was applied to knockout the NK cell cytokine checkpoint cytokine-inducible SH2-containing protein (CIS, product of the CISH gene), resulting in enhanced cytotoxicity and an altered NK cell phenotype. CONCLUSIONS: This report contributes a promising application of transposon engineering to donor-derived NK cells and emphasizes the importance of feeder mediated NK cell activation and expansion to current protocols.

Marrow changes and reduced proliferative capacity of mesenchymal stromal cells from patients with "no-option" critical limb ischemia; observations on feasibility of the autologous approach from a clinical trial.

BACKGROUND AIMS: Approximately 1 in 3 patients with critical limb ischemia (CLI) are not suitable for surgical or endovascular revascularization. Those "no-option" patients are at high risk of amputation and death. Autologous bone marrow mesenchymal stromal cells (MSCs) may provide a limb salvage option. In this study, bone marrow characteristics and expansion potentials of CLI-derived MSCs produced during a phase 1b clinical trial were compared with young healthy donor MSCs to determine the feasibility of an autologous approach. Cells were produced under Good Manufacturing Practice conditions and underwent appropriate release testing. METHODS: Five bone marrow aspirates derived from patients with CLI were compared with six young healthy donor marrows in terms of number of colony-forming units-fibroblast (CFUF) and mononuclear cells. The mean population doubling times and final cell yields were used to evaluate expansion potential. The effect of increasing the volume of marrow on the CFUF count and final cell yield was evaluated by comparing 5 CLI-derived MSCs batches produced from a targeted 30 mL of marrow aspirate to five batches produced from a targeted 100 mL of marrow. RESULTS: CLI-derived marrow aspirate showed significantly lower numbers of mononuclear cells with no difference in the number of CFUFs when compared with healthy donors' marrow aspirate. CLI-derived MSCs showed a significantly longer population doubling time and reduced final cell yield compared with young healthy donors' MSCs. The poor growth kinetics of CLI MSCs were not mitigated by increasing the bone marrow aspirate from 30 to 100 mL. CONCLUSIONS: In addition to the previously reported karyotype abnormalities in MSCs isolated from patients with CLI, but not in cells from healthy donors, the feasibility of autologous transplantation of bone marrow MSCs for patients with no-option CLI is further limited by the increased expansion time and the reduced cell yield.

Phase 2 studies of lenalidomide, subcutaneous bortezomib, and dexamethasone as induction therapy in patients with newly diagnosed multiple myeloma.

There are limited prospective data on lenalidomide, subcutaneous bortezomib, and dexamethasone (RsqVd) in transplant-eligible/transplant-ineligible patients with newly diagnosed multiple myeloma. Reliable biomarkers for efficacy and toxicity are required to better tailor therapy. Two parallel studies were conducted by Cancer Trials Ireland (CTI; NCT02219178) and the Dana-Farber Cancer Institute (DFCI; NCT02441686). Patients received four 21-day cycles of RsqVd and could then receive either another 4 cycles of RsqVd or undergo autologous stem cell transplant. Postinduction/posttransplant, patients received lenalidomide maintenance, with bortezomib included for high-risk patients. The primary endpoint was overall response rate (ORR) after 4 cycles of RsqVd. Eighty-eight patients were enrolled and 84 treated across the two studies; median age was 64.7 (CTI study) and 60.0 years (DFCI study), and 59% and 57% had stage II-III disease. Pooled ORR after 4 cycles in evaluable patients was 93.5%, including 48.1% complete or very good partial responses (CTI study: 91.9%, 59.5%; DFCI study: 95.0%, 37.5%), and in the all-treated population was 85.7% (44.0%). Patients received a median of 4 (CTI study) and 8 (DFCI study) RsqVd cycles; 60% and 31% of patients (CTI study) and 33% and 51% of patients (DFCI study) underwent transplant or received further RsqVd induction, respectively. The most common toxicity was peripheral neuropathy (pooled: 68%, 7% grade 3-4; CTI study: 57%, 7%; DFCI study: 79%, 7%). Proteomics analyses indicated elevated kallikrein-6 in good versus poor responders, decreased midkine in good responders, and elevated macrophage inflammatory protein 1-alpha in patients who stopped treatment from neurotoxicity, suggesting predictive biomarkers warranting further investigation.

NRXN1α+/- is associated with increased excitability in ASD iPSC-derived neurons.

BACKGROUND: NRXN1 deletions are identified as one of major rare risk factors for autism spectrum disorder (ASD) and other neurodevelopmental disorders. ASD has 30% co-morbidity with epilepsy, and the latter is associated with excessive neuronal firing. NRXN1 encodes hundreds of presynaptic neuro-adhesion proteins categorized as NRXN1α/ß/γ. Previous studies on cultured cells show that the short NRXN1ß primarily exerts excitation effect, whereas the long NRXN1α which is more commonly deleted in patients involves in both excitation and inhibition. However, patient-derived models are essential for understanding functional consequences of NRXN1α deletions in human neurons. We recently derived induced pluripotent stem cells (iPSCs) from five controls and three ASD patients carrying NRXN1α+/- and showed increased calcium transients in patient neurons. METHODS: In this study we investigated the electrophysiological properties of iPSC-derived cortical neurons in control and ASD patients carrying NRXN1α+/- using patch clamping. Whole genome RNA sequencing was carried out to further understand the potential underlying molecular mechanism. RESULTS: NRXN1α+/- cortical neurons were shown to display larger sodium currents, higher AP amplitude and accelerated depolarization time. RNASeq analyses revealed transcriptomic changes with significant upregulation glutamatergic synapse and ion channels/transporter activity including voltage-gated potassium channels (GRIN1, GRIN3B, SLC17A6, CACNG3, CACNA1A, SHANK1), which are likely to couple with the increased excitability in NRXN1α+/- cortical neurons. CONCLUSIONS: Together with recent evidence of increased calcium transients, our results showed that human NRXN1α+/- isoform deletions altered neuronal excitability and non-synaptic function, and NRXN1α+/- patient iPSCs may be used as an ASD model for therapeutic development with calcium transients and excitability as readouts.

Serum brain-derived neurotrophic factor (BDNF) concentration predicts polyneuropathy and overall survival in multiple myeloma patients.

Brain-derived neurotrophic factor (BDNF) is a protein with a potent influence on several aspects of neuronal and blood vessel functions. However, its prognostic potential and functional role in multiple myeloma (MM) remain largely unknown. In this study, we investigated the influence of BDNF on the risk of chemotherapy-induced peripheral neuropathy (CIPN) and clinical outcome. Study group consisted of 91 newly-diagnosed MM patients treated with bortezomib and/or thalidomide-based chemotherapy. Detection of BDNF in serum was performed using ELISA. Polyneuropathy was assessed according to the CTCAE Criteria v5. We observed that BDNF concentration correlated with the severity of polyneuropathy (P = 0·0463). Higher BDNF values were noted in patients who responded to treatment (P = 0·0326), and BDNF proved to be a useful marker to predict lack of response after eight cycles of treatment (sensitivity - 100%, specificity - 61·5%, P = 0·0142). Moreover this marker showed significant diagnostic usefulness in diagnosis of CIPN (sensitivity - 76%, specificity - 71·43%; area under the curve (AUC)= 0·77, 95%, confidence interval (CI): 0·64-0·88; P < 0·0001). Low BDNF was an independent, unfavourable prognostic factor associated with reduced overall survival (OS) (hazard ratio (HR) = 2·79, P = 0·0470). In conclusion, BDNF level may play a prognostic role and constitute a useful biomarker in predicting CIPN in MM patients.

Autologous bone marrow mesenchymal stromal cell therapy for "no-option" critical limb ischemia is limited by karyotype abnormalities.

BACKGROUND: Critical limb ischemia (CLI) is the most severe manifestation of peripheral vascular disease. Revascularization is the preferred therapy, but it is not achievable in 25%-40% of patients due to diffuse anatomic distribution of the disease or medical comorbidities. No-option CLI represents an unmet medical need. Mesenchymal stromal cells (MSCs) may provide salvage therapy through their angiogenic and tissue-trophic properties. This article reports a phase 1b clinical study examining the safety and feasibility of intramuscular transplantation of autologous bone-marrow MSCs for patients with no-option CLI. METHODS: Twelve patients were enrolled in the clinical trial, and nine proceeded to bone marrow aspiration and culture expansion of MSCs. RESULTS: A high rate of karyotype abnormality (>30%) was detected in the produced cell batches, resulting in failure of release for clinical administration. Four patients were treated with the investigational medicinal product (IMP), three with a low dose of 20 × 106 MSCs and one with a mid-dose of 40 × 106 MSCs. There were no serious adverse events related to trial interventions, including bone marrow aspiration, IMP injection or therapy. CONCLUSIONS: The results of this trial conclude that an autologous cell therapy approach with MSCs for critical limb ischemia is limited by the high rate of karyotype abnormalities.

A Novel High-Throughput Screening Platform Identifies Itaconate Derivatives from Marine Penicillium antarcticum as Inhibitors of Mesenchymal Stem Cell Differentiation.

Worldwide diffused diseases such as osteoarthritis, atherosclerosis or chronic kidney disease are associated with a tissue calcification process which may involve unexpected local stem cell differentiation. Current pharmacological treatments for such musculoskeletal conditions are weakly effective, sometimes extremely expensive and often absent. The potential to develop new therapies is represented by the discovery of small molecules modulating resident progenitor cell differentiation to prevent aberrant tissue calcification. The marine environment is a rich reserve of compounds with pharmaceutical potential and many novel molecules are isolated from macro and microorganisms annually. The potential of small molecules synthetized by marine filamentous fungi to influence the osteogenic and chondrogenic differentiation of human mesenchymal stem/stromal cells (hMSCs) was investigated using a novel, high-throughput automated screening platform. Metabolites synthetized by the marine-derived fungus Penicillium antarcticum were evaluated on the platform. Itaconic acid derivatives were identified as inhibitors of calcium elaboration into the matrix of osteogenically differentiated hMSCs and also inhibited hMSC chondrogenic differentiation, highlighting their capacity to impair ectopic calcification. Bioactive small molecule discovery is critical to address ectopic tissue calcification and the use of biologically relevant assays to identify naturally occurring metabolites from marine sources represents a strategy that can contribute to this effort.

The role of inflammation in leukaemia.

Acute leukaemias are a group of malignancies characterised by the invasion of the bone marrow by immature haematopoietic precursors and differentiation arrest at various maturation steps. Multiplicity of intrinsic and extrinsic factors influences the transformation and progression of leukaemia. The intrinsic factors encompass genetic alterations of cellular pathways leading to the activation of, among others, inflammatory pathways (such as nuclear factor kappa B). The extrinsic components include, among others, the inflammatory pathways activated by the bone marrow microenvironment and include chemokines, cytokines and adhesion molecules. In this chapter, we review the role of inflammatory processes in the transformation, survival and proliferation of leukaemias, particularly the role of nuclear factor kappa B and its downstream signalling in leukaemias and the novel therapeutic strategies that exploit potentially unique properties of inflammatory signalling that offer interesting options for future therapeutic interventions.

A multidisciplinary group-based survivorship intervention for those living with multiple myeloma: a feasibility study.

BACKGROUND: While multiple myeloma continues to be an incurable cancer, advances in its understanding and management have led to significantly improved survival rates. Survivorship interventions for those living with multiple myeloma remain scarce, despite mounting evidence for multiple unmet support needs among multiple myeloma survivors. The current study aimed to evaluate the feasibility and preliminary effectiveness of a novel multidisciplinary group-based multiple myeloma survivorship intervention. METHODS: A mixed-method, repeated measures feasibility study was conducted within a routine cancer support service. Seven participants, aged over 18, who had a multiple myeloma diagnosis and were clinically assessed as suitable for the intervention by their haemato-oncologist, attended online for six weekly group sessions of physical exercise and self-management input, completing qualitative, physical and self-report measures at baseline, post-intervention and follow-up. RESULTS: The intervention was deemed overall feasible, with relatively high uptake, participants describing it as largely acceptable and appropriate and providing recommendations for feasibility-enhancing intervention refinements. Findings regarding the preliminary effectiveness of the intervention were mixed. While qualitative analyses stressed the benefits of the intervention (e.g. peer support, connectedness, improved well-being) and large effect sizes were observed for most physical outcomes, no improvements in self-reported outcomes (i.e. quality of life, fatigue) were reported. CONCLUSIONS: This study represents the first investigation of a promising novel survivorship intervention for those living with multiple myeloma, highlighting the importance of peer support in particular, on which future clinical trials, aiming to establish the intervention's effectiveness for routine care, will be able to build.

Extreme Symptom Burden for Patients With COVID-19 at the End of Life; Extrapolation of Knowledge Gained to Achieve Sustained Comfort and Dignity for all Patients in Their Last Days of Life1.

BACKGROUND: We describe two complex cases in the setting of COVID-19 at the End of Life, to enhance learning for all patients. CASE PRESENTATION: Maintenance of sustained comfort in two cases required multiple drugs, specifically selected for symptoms that necessitated three separate pumps delivering continuous 24-hour subcutaneous infusion. CASE MANAGEMENT: Management of sustained comfort included opioid, midazolam, anti-secretory, diclofenac for intractable temperature, phenobarbital for extreme agitation, in one, where seizure activity was present, while insomnia, was a prominent feature of another. Management of Akatasia was also required. CASE OUTCOME: Attention to each individual patient's rapidly evolving symptoms, during the dying phase, with a thorough differential diagnosis, wa s vitally important in the context of a 'Good Death'. This was achieved in both cases, reflected by evidence at the bedside of comfort and a minimum need for 'as required' drugs in the last days of life. CONCLUSIONS: COVID-19 being a new illness, we need to prospectively study the symptom burden/clustering at End of Life and learn from management of this new disease for other illnesses also. Further research is required to develop protocols on; when does Midazolam dose reach tolerance and when should an alternative drug such as phenobarbital for sustained Gamma-Aminobutyric Acid effects be initiated; examine the optimal approach to sustained temperature control; be cognisant of extrapyramidal side effects of drugs used at End of Life and consider looking at a lack of need for 'as required' drugs in the last days of life as an outcome measure of sustained comfort.

Generation of three induced pluripotent stem cell lines from a patient with KCNQ2 developmental and epileptic encephalopathy as a result of the pathogenic variant c.881C > T; p.Ala294Val (NUIGi059-A, NUIGi059-B, NUIGi059-C) and 3 healthy controls (NUIGi060-A, NUIGi060-B, NUIGi060-C).

Developmental and epileptic encephalopathies (DEEs) are a group of severe, early-onset epilepsies which are often caused by genetic mutations in ion channels. Mutations in KCNQ2, which encodes the voltage-gated potassium channel Kv7.2, is known to cause DEE. Here, we generated three iPSC lines from dermal fibroblasts of a 5 year-old male patient with the KCNQ2 c.881C > T (p.Ala294Val) pathogenic heterozygous variant and three iPSC lines from a healthy sibling control. These iPSC lines have been validated by SNP karyotyping, STR analysis, expression of pluripotent genes, the capacity to differentiate into three germ layers and confirmation of the mutation in the patient.

Generation of three induced pluripotent stem cell lines from a patient with KCNQ2 developmental and epileptic encephalopathy as a result of the pathogenic variant c.638C > T; p.Arg213Gln (NUIGi063-A, NUIGi063-B, NUIGi063-C) and 3 healthy controls (NUIGi064-A, NUIGi064-B, NUIGi064-C).

KCNQ2 encodes the potassium-gated voltage channel Kv7.2, responsible for the M-current, which contributes to neuronal resting membrane potential. Pathogenic variants in KCNQ2 cause early onset epilepsies, developmental and epileptic encephalopathies. In this study, we generated three iPSC lines from dermal fibroblasts of a 5 year-old female patient with the KCNQ2 c.638C > T (p.Arg213Gln) pathogenic heterozygous variant and three iPSC lines from a healthy sibling control. These iPSC lines were validated by confirming the targeted mutation, SNP karyotyping, STR analysis, pluripotent gene expression, differentiation capacity into three germ layers, and were free of transgene integration and Mycoplasma.

Tool Wear Issues in Hot Forging of Steel.

Steel forging tools are subjected to a number of tribological wear mechanisms depending on the geometry and surface of the tool and the flow of the material. Thus, there is no single general tribological wear mechanism, and only the predominant wear mechanisms in this case can be indicated. The problem has been known for years, but due to its complexity research on it is still relevant. In this study, the various wear mechanisms of hot work tools are analyzed on the basis of original research. Moreover, the influence of the micro- and macrostructure of the material and of its mechanical, physical, and technological characteristics on susceptibility to a given type of wear is considered. Adhesive wear, wear caused by plastic deformation, mechanical fatigue, thermal fatigue, the influence of hardness, heat treatment, and impact strength on tool wear and the mechanisms causing this wear are discussed in addition to tribological wear mechanisms such as abrasive wear. The influence of thermomechanical history and the characteristics of the tool material, including structural anisotropy, on the wear of these tools is indicated. The analysis of wear mechanisms performed will enable more precise definition of the principles of tool material selection and tool material condition for the hot forging of steel.

On the Influence of Heat Input on Ni-WC GMAW Hardfaced Coating Properties.

Hardfacing is one of the techniques used for part lifecycle elongation. Despite being used for over 100 years, there still is much to discover, as modern metallurgy provides more and more sophisticated alloys, which then have to be studied to find the best technological parameters in order to fully utilize complex material properties. One of the most efficient and versatile hardfacing approaches is Gas Metal Arc Welding technology (GMAW) and its cored-wire equivalent, known as FCAW (Flux-Cored/Cored Arc Welding). In this paper, the authors study the influence of heat input on the geometrical properties and hardness of stringer weld beads fabricated from cored wire consisting of macrocrystalline tungsten carbides in a nickel matrix. The aim is to establish a set of parameters which allow to manufacture wear-resistant overlays with high deposition rates, preserving all possible benefits of this heterogenic material. This study shows, that for a given diameter of the Ni-WC wire, there exists an upper limit of heat input beyond which the tungsten carbide crystals may exhibit undesired segregation at the root.

The miR-17-92 cluster: Yin and Yang in human cancers.

MicroRNAs (miRNAs) are non-coding RNAs which modulate gene expression via multiple post-transcriptional mechanisms. They are involved in a variety of biological processes, including cell proliferation, metastasis, metabolism, tumorigenesis, and apoptosis. Dysregulation of miRNA expression has been implicated in human cancers, and they may also serve as biomarkers of disease progression and prognosis. The miR-17-92 cluster is one of the most widely studied miRNA clusters, which was initially reported as an oncogene, but was later reported to exhibit tumour suppressive effects in some human cancers. This review summarizes the recent progress and context-dependant role of this cluster in various cancers. We summarize the known mechanisms which regulate miR-17-92 expression and molecular pathways that are in turn controlled by it. We discuss examples where it acts as an oncogene or a tumour suppressor along with key targets affecting hallmarks of cancer. We discuss how cellular contexts regulate the biological effects of miR-17-92. The plausible mechanisms of its paradoxical roles are explained, and mechanisms are described that may contribute to cell fate regulation by miR-17-92. Further, we discuss recently developed strategies to target miR-17-92 cluster in human cancers. MiR-17-92 may serve as a potential biomarker for prognosis and response to therapy as well as a target for cancer prevention and therapeutics.

Hot Deformation Behavior of 4130 High-Strength Steel.

Hot deformation behavior of 4130 steel and optimization of its processing parameters are presented in this paper. Compression tests were performed at temperatures ranging from 800 to 1200 °C and at the strain rates in the range from 0.01 to 100 s-1. A comprehensive analysis of the material behavior at different temperature and strain-rate ranges was performed taking into account various criteria of stability and instability of the material flow under various thermomechanical conditions. The flow-stress curves obtained during compression tests, as well as the processing maps elaborated on the basis of various flow-stability criteria, are discussed. Processing parameters developed according to the Prasad's and Murty's criteria are recommended for designing the technology of forging of the investigated steel. Such parameters ensure the homogeneity and stability of the material flow in a forged part, what was confirmed by successful forging of 4130 steel in industrial conditions. The processing map developed according to Gegel's approach, as compared to the processing maps obtained in accordance with the Prasad's and Murty's criteria, should be treated as general support for determining the thermomechanical processing parameters.