What can nurses learn from patient's needs and wishes when developing an evidence-based quality improvement learning culture? A qualitative study.

BACKGROUND: Patient participation is fundamental in nursing care and has yielded benefits for patient outcomes. However, despite their compassionate care approach, nurses do not always incorporate patients' needs and wish into evidence-based practice, quality improvement or learning activities. Therefore, a shift to continuous quality improvement based on evidence-based practice is necessary to enhance the quality of care. The patient's opinion is an essential part of this process. To establish a more sustainable learning culture for evidence-based quality improvement, it is crucial that nurses learn alongside their patients. However, to promote this, nurses require a deeper understanding of patients' care preferences. OBJECTIVE: To explore patients' needs and wishes towards being involved in care processes that nurses can use in developing an evidence-based quality improvement learning culture. METHODS: A qualitative study was conducted in two hospital departments and one community care team. In total, 18 patients were purposefully selected for individual semi-structured interviews with an average of 15 min. A framework analysis based on the fundamental of care framework was utilised to analyse the data deductively. In addition, inductive codes were added to patients' experiences beyond the framework. For reporting this study, the SRQR guideline was used. RESULTS: Participants needed a compassionate nurse who established and sustained a trusting relationship. They wanted nurses to be present and actively involved during the care delivery. Shared decision-making improved when nurses offered fair, clear and tailored information. Mistrust or a disrupted nurse-patient relationship was found to be time-consuming and challenging to restore. CONCLUSIONS: Results confirmed the importance of a durable nurse-patient relationship and showed the consequences of nurses' communication on shared decision-making. Insights into patients' care preferences are essential to stimulate the development of an evidence-based quality improvement learning culture within nursing teams and for successful implementation processes.

G-CSF as a suitable alternative to GM-CSF to boost dinutuximab-mediated neutrophil cytotoxicity in neuroblastoma treatment.

BACKGROUND: Current immunotherapy for patients with high-risk neuroblastoma involves the therapeutic antibody dinutuximab that targets GD2, a ganglioside expressed on the majority of neuroblastoma tumors. Opsonized tumor cells are killed through antibody-dependent cellular cytotoxicity (ADCC), a process mediated by various immune cells, including neutrophils. The capacity of neutrophils to kill dinutuximab-opsonized tumor cells can be further enhanced by granulocyte-macrophage colony-stimulating factor (GM-CSF), which has been shown in the past to improve responses to anti-GD2 immunotherapy. However, access to GM-CSF (sargramostim) is limited outside of Northern America, creating a high clinical need for an alternative method to stimulate dinutuximab responsiveness in the treatment of neuroblastoma. In this in vitro study, we have investigated whether clinically well-established granulocyte colony-stimulating factor (G-CSF) can be a potentially suitable alternative for GM-CSF in the dinutuximab immunotherapy regimen of patients with neuroblastoma. METHODS: We compared the capacity of neutrophils stimulated either in vitro or in vivo with GM-CSF or G-CSF to kill dinutuximab-opsonized GD2-positive neuroblastoma cell lines and primary patient tumor material. Blocking experiments with antibodies inhibiting either respective Fc gamma receptors (FcγR) or neutrophil integrin CD11b/CD18 demonstrated the involvement of these receptors in the process of ADCC. Flow cytometry and live cell microscopy were used to quantify and visualize neutrophil-neuroblastoma interactions. RESULTS: We found that G-CSF was as potent as GM-CSF in enhancing the killing capacity of neutrophils towards neuroblastoma cells. This was observed with in vitro stimulated neutrophils, and with in vivo stimulated neutrophils from both patients with neuroblastoma and healthy donors. Enhanced killing due to GM-CSF or G-CSF stimulation was consistent regardless of dinutuximab concentration, tumor-to-neutrophil ratio and concentration of the stimulating cytokine. Both GM-CSF and G-CSF stimulated neutrophils required FcγRIIa and CD11b/CD18 integrin to perform ADCC, and this was accompanied by trogocytosis of tumor material by neutrophils and tumor cell death in both stimulation conditions. CONCLUSIONS: Our preclinical data support the use of G-CSF as an alternative stimulating cytokine to GM-CSF in the treatment of high-risk neuroblastoma with dinutuximab, warranting further testing of G-CSF in a clinical setting.

The Potential of Mesenchymal Stromal Cells in Neuroblastoma Therapy for Delivery of Anti-Cancer Agents and Hematopoietic Recovery.

Neuroblastoma is one of the most common pediatric cancers and a major cause of cancer-related death in infancy. Conventional therapies including high-dose chemotherapy, stem cell transplantation, and immunotherapy approach a limit in the treatment of high-risk neuroblastoma and prevention of relapse. In the last two decades, research unraveled a potential use of mesenchymal stromal cells in tumor therapy, as tumor-selective delivery vehicles for therapeutic compounds and oncolytic viruses and by means of supporting hematopoietic stem cell transplantation. Based on pre-clinical and clinical advances in neuroblastoma and other malignancies, we assess both the strong potential and the associated risks of using mesenchymal stromal cells in the therapy for neuroblastoma. Furthermore, we examine feasibility and safety aspects and discuss future directions for harnessing the advantageous properties of mesenchymal stromal cells for the advancement of therapy success.

Mesenchymal Stromal Cells in Neuroblastoma: Exploring Crosstalk and Therapeutic Implications.

Neuroblastoma (NB) is the second most common solid cancer in childhood, accounting for 15% of cancer-related deaths in children. In high-risk NB patients, the majority suffers from metastasis. Despite intensive multimodal treatment, long-term survival remains <40%. The bone marrow (BM) is among the most common sites of distant metastasis in patients with high-risk NB. In this environment, small populations of tumor cells can persist after treatment (minimal residual disease) and induce relapse. Therapy resistance of these residual tumor cells in BM remains a major obstacle for the cure of NB. A detailed understanding of the microenvironment and its role in tumor progression is of utmost importance for improving the treatment efficiency of NB. In BM, mesenchymal stromal cells (MSCs) constitute an important part of the microenvironment, where they support hematopoiesis and modulate immune responses. Their role in tumor progression is not completely understood, especially for NB. Although MSCs have been found to promote epithelial-mesenchymal transition, tumor growth, and metastasis and to induce chemoresistance, some reports point toward a tumor-suppressive effect of MSCs. In this review, we aim to compile current knowledge about the role of MSCs in NB development and progression. We evaluate arguments that depict tumor-supportive versus -suppressive properties of MSCs in the context of NB and give an overview of factors involved in MSC-NB crosstalk. A focus lies on the BM as a metastatic niche, since that is the predominant site for NB metastasis and relapse. Finally, we will present opportunities and challenges for therapeutic targeting of MSCs in the BM microenvironment.

The Metastatic Bone Marrow Niche in Neuroblastoma: Altered Phenotype and Function of Mesenchymal Stromal Cells.

Background: The bone marrow (BM) is the main site of metastases and relapse in patients with neuroblastoma (NB). BM-residing mesenchymal stromal cells (MSCs) were shown to promote tumor cell survival and chemoresistance. Here we characterize the MSC compartment of the metastatic NB BM niche. Methods: Fresh BM of 62 NB patients (all stages), and control fetal and adult BM were studied by flow cytometry using well-established MSC-markers (CD34-, CD45-, CD90+, CD105+), and CD146 and CD271 subtype-markers. FACS-sorted BM MSCs and tumor cells were validated by qPCR. Moreover, isolated MSCs were tested for multilineage differentiation and Colony-forming-unit-fibroblasts (CFU-Fs) capacity. Results: Metastatic BM contains a higher number of MSCs (p < 0.05) with increased differentiation capacity towards the osteoblast lineage. Diagnostic BM contains a MSC-subtype (CD146+CD271-), only detected in BM of patients with metastatic-NB, determined by flow cytometry. FACS-sorting clearly discriminated MSC(-subtypes) and NB fractions, validated by mRNA and DNA qPCR. Overall, the CD146+CD271- subtype decreased during therapy and was detected again in the majority of patients at relapse. Conclusions: We demonstrate that the neuroblastoma BM-MSC compartment is different in quantity and functionality and contains a metastatic-niche-specific MSC-subtype. Ultimately, the MSCs contribution to tumor progression could provide targets with potential for eradicating resistant metastatic disease.

Peripheral Stem Cell Apheresis is Feasible Post 131Iodine-Metaiodobenzylguanidine-Therapy in High-Risk Neuroblastoma, but Results in Delayed Platelet Reconstitution.

PURPOSE: Targeted radiotherapy with 131iodine-meta-iodobenzylguanidine (131I-MIBG) is effective for neuroblastoma (NBL), although optimal scheduling during high-risk (HR) treatment is being investigated. We aimed to evaluate the feasibility of stem cell apheresis and study hematologic reconstitution after autologous stem cell transplantation (ASCT) in patients with HR-NBL treated with upfront 131I-MIBG-therapy. EXPERIMENTAL DESIGN: In two prospective multicenter cohort studies, newly diagnosed patients with HR-NBL were treated with two courses of 131I-MIBG-therapy, followed by an HR-induction protocol. Hematopoietic stem and progenitor cell (e.g., CD34+ cell) harvest yield, required number of apheresis sessions, and time to neutrophil (>0.5 × 109/L) and platelet (>20 × 109/L) reconstitution after ASCT were analyzed and compared with "chemotherapy-only"-treated patients. Moreover, harvested CD34+ cells were functionally (viability and clonogenic capacity) and phenotypically (CD33, CD41, and CD62L) tested before cryopreservation (n = 44) and/or after thawing (n = 19). RESULTS: Thirty-eight patients (47%) were treated with 131I-MIBG-therapy, 43 (53%) only with chemotherapy. Median cumulative 131I-MIBG dose/kg was 0.81 GBq (22.1 mCi). Median CD34+ cell harvest yield and apheresis days were comparable in both groups. Post ASCT, neutrophil recovery was similar (11 days vs. 10 days), whereas platelet recovery was delayed in 131I-MIBG- compared with chemotherapy-only-treated patients (29 days vs. 15 days, P = 0.037). Testing of harvested CD34+ cells revealed a reduced post-thaw viability in the 131I-MIBG-group. Moreover, the viable CD34+ population contained fewer cells expressing CD62L (L-selectin), a marker associated with rapid platelet recovery. CONCLUSIONS: Harvesting of CD34+ cells is feasible after 131I-MIBG. Platelet recovery after ASCT was delayed in 131I-MIBG-treated patients, possibly due to reinfusion of less viable and CD62L-expressing CD34+ cells, but without clinical complications. We provide evidence that peripheral stem cell apheresis is feasible after upfront 131I-MIBG-therapy in newly diagnosed patients with NBL. However, as the harvest of 131I-MIBG-treated patients contained lower viable CD34+ cell counts after thawing and platelet recovery after reinfusion was delayed, administration of 131I-MIBG after apheresis is preferred.

Mesenchymal Neuroblastoma Cells Are Undetected by Current mRNA Marker Panels: The Development of a Specific Neuroblastoma Mesenchymal Minimal Residual Disease Panel.

Patients with neuroblastoma in molecular remission remain at considerable risk for disease recurrence. Studies have found that neuroblastoma tissue contains adrenergic (ADRN) and mesenchymal (MES) cells; the latter express low levels of commonly used markers for minimal residual disease (MRD). We identified MES-specific MRD markers and studied the dynamics of these markers during treatment. PATIENTS AND METHODS: Microarray data were used to identify genes differentially expressed between ADRN and MES cell lines. Candidate genes were then studied using real-time quantitative polymerase chain reaction in cell lines and control bone marrow and peripheral blood samples. After selecting a panel of markers, serial bone marrow, peripheral blood, and peripheral blood stem cell samples were obtained from patients with high-risk neuroblastoma and tested for marker expression; survival analyses were also performed. RESULTS: PRRX1, POSTN, and FMO3 mRNAs were used as a panel for specifically detecting MES mRNA in patient samples. MES mRNA was detected only rarely in peripheral blood; moreover, the presence of MES mRNA in peripheral blood stem cell samples was associated with low event-free survival and overall survival. Of note, during treatment, serial bone marrow samples obtained from 29 patients revealed a difference in dynamics between MES mRNA markers and ADRN mRNA markers. Furthermore, MES mRNA was detected in a higher percentage of patients with recurrent disease than in those who remained disease free (53% v 32%, respectively; P = .03). CONCLUSION: We propose that the markers POSTN and PRRX1, in combination with FMO3, be used for real-time quantitative polymerase chain reaction-based detection of MES neuroblastoma mRNA in patient samples because these markers have a unique pattern during treatment and are more prevalent in patients with poor outcome. Together with existing markers of MRD, these new markers should be investigated further in large prospective studies.

Leukocytes Crossing the Endothelium: A Matter of Communication.

Leukocytes cross the endothelial vessel wall in a process called transendothelial migration (TEM). The purpose of leukocyte TEM is to clear the causing agents of inflammation in underlying tissues, for example, bacteria and viruses. During TEM, endothelial cells initiate signals that attract and guide leukocytes to sites of tissue damage. Leukocytes react by attaching to these sites and signal their readiness to move back to endothelial cells. Endothelial cells in turn respond by facilitating the passage of leukocytes while retaining overall integrity. In this review, we present recent findings in the field and we have endeavored to synthesize a coherent picture of the intricate interplay between endothelial cells and leukocytes during TEM.

Small Rho GTPase-mediated actin dynamics at endothelial adherens junctions.

VE-cadherin-based cell-cell junctions form the major restrictive barrier of the endothelium to plasma proteins and blood cells. The function of VE-cadherin and the actin cytoskeleton are intimately linked. Vascular permeability factors and adherent leukocytes signal through small Rho GTPases to tightly regulate actin cytoskeletal rearrangements in order to open and re-assemble endothelial cell-cell junctions in a rapid and controlled manner. The Rho GTPases are activated by guanine nucleotide exchange factors (GEFs), conferring specificity and context-dependent control of cell-cell junctions. Although the molecular mechanisms that couple cadherins to actin filaments are beginning to be elucidated, specific stimulus-dependent regulation of the actin cytoskeleton at VE-cadherin-based junctions remains unexplained. Accumulating evidence has suggested that depending on the vascular permeability factor and on the subcellular localization of GEFs, cell-cell junction dynamics and organization are differentially regulated by one specific Rho GTPase. In this Commentary, we focus on new insights how the junctional actin cytoskeleton is specifically and locally regulated by Rho GTPases and GEFs in the endothelium.

Plasminogen Activator Inhibitor-1 Controls Vascular Integrity by Regulating VE-Cadherin Trafficking.

BACKGROUND: Plasminogen activator inhibitor-1 (PAI-1), a serine protease inhibitor, is expressed and secreted by endothelial cells. Patients with PAI-1 deficiency show a mild to moderate bleeding diathesis, which has been exclusively ascribed to the function of PAI-1 in down-regulating fibrinolysis. We tested the hypothesis that PAI-1 function plays a direct role in controlling vascular integrity and permeability by keeping endothelial cell-cell junctions intact. METHODOLOGY/PRINCIPAL FINDINGS: We utilized PAI-039, a specific small molecule inhibitor of PAI-1, to investigate the role of PAI-1 in protecting endothelial integrity. In vivo inhibition of PAI-1 resulted in vascular leakage from intersegmental vessels and in the hindbrain of zebrafish embryos. In addition PAI-1 inhibition in human umbilical vein endothelial cell (HUVEC) monolayers leads to a marked decrease of transendothelial resistance and disrupted endothelial junctions. The total level of the endothelial junction regulator VE-cadherin was reduced, whereas surface VE-cadherin expression was unaltered. Moreover, PAI-1 inhibition reduced the shedding of VE-cadherin. Finally, we detected an accumulation of VE-cadherin at the Golgi apparatus. CONCLUSIONS/SIGNIFICANCE: Our findings indicate that PAI-1 function is important for the maintenance of endothelial monolayer and vascular integrity by controlling VE-cadherin trafficking to and from the plasma membrane. Our data further suggest that therapies using PAI-1 antagonists like PAI-039 ought to be used with caution to avoid disruption of the vessel wall.

A local VE-cadherin and Trio-based signaling complex stabilizes endothelial junctions through Rac1.

Endothelial cell-cell junctions maintain a restrictive barrier that is tightly regulated to allow dynamic responses to permeability-inducing angiogenic factors, as well as to inflammatory agents and adherent leukocytes. The ability of these stimuli to transiently remodel adherens junctions depends on Rho-GTPase-controlled cytoskeletal rearrangements. How the activity of Rho-GTPases is spatio-temporally controlled at endothelial adherens junctions by guanine-nucleotide exchange factors (GEFs) is incompletely understood. Here, we identify a crucial role for the Rho-GEF Trio in stabilizing junctions based around vascular endothelial (VE)-cadherin (also known as CDH5). Trio interacts with VE-cadherin and locally activates Rac1 at adherens junctions during the formation of nascent contacts, as assessed using a novel FRET-based Rac1 biosensor and biochemical assays. The Rac-GEF domain of Trio is responsible for the remodeling of junctional actin from radial into cortical actin bundles, a crucial step for junction stabilization. This promotes the formation of linear adherens junctions and increases endothelial monolayer resistance. Collectively, our data show the importance of spatio-temporal regulation of the actin cytoskeleton through Trio and Rac1 at VE-cadherin-based cell-cell junctions in the maintenance of the endothelial barrier.

The Rho-GEF Trio regulates a novel pro-inflammatory pathway through the transcription factor Ets2.

Inflammation is characterized by endothelium that highly expresses numerous adhesion molecules to trigger leukocyte extravasation. Central to this event is increased gene transcription. Small Rho-GTPases not only control the actin cytoskeleton, but are also implicated in gene regulation. However, in inflammation, it is not clear how this is regulated. Here, we show that the guanine-nucleotide exchange factor Trio expression is increased upon inflammatory stimuli in endothelium. Additionally, increased Trio expression was found in the vessel wall of rheumatoid arthritis patients. Trio silencing impaired VCAM-1 expression. Finally, we excluded that Trio-controlled VCAM-1 expression used the classical NFκB or MAP-kinase pathways, but rather acts on the transcriptional level by increasing phosphorylation and nuclear translocalization of Ets2. These data implicate Trio in regulating inflammation and provide novel targets for therapeutic purposes to treat inflammatory diseases such as rheumatoid arthritis.

The tyrosine phosphatase SHP2 regulates recovery of endothelial adherens junctions through control of ß-catenin phosphorylation.

Impaired endothelial barrier function results in a persistent increase in endothelial permeability and vascular leakage. Repair of a dysfunctional endothelial barrier requires controlled restoration of adherens junctions, comprising vascular endothelial (VE)-cadherin and associated ß-, γ-, α-, and p120-catenins. Little is known about the mechanisms by which recovery of VE-cadherin-mediated cell-cell junctions is regulated. Using the inflammatory mediator thrombin, we demonstrate an important role for the Src homology 2-domain containing tyrosine phosphatase (SHP2) in mediating recovery of the VE-cadherin-controlled endothelial barrier. Using SHP2 substrate-trapping mutants and an in vitro phosphatase activity assay, we validate ß-catenin as a bona fide SHP2 substrate. SHP2 silencing and SHP2 inhibition both result in delayed recovery of endothelial barrier function after thrombin stimulation. Moreover, on thrombin challenge, we find prolonged elevation in tyrosine phosphorylation levels of VE-cadherin-associated ß-catenin in SHP2-depleted cells. No disassembly of the VE-cadherin complex is observed throughout the thrombin response. Using fluorescence recovery after photobleaching, we show that loss of SHP2 reduces the mobility of VE-cadherin at recovered cell-cell junctions. In conclusion, our data show that the SHP2 phosphatase plays an important role in the recovery of disrupted endothelial cell-cell junctions by dephosphorylating VE-cadherin-associated ß-catenin and promoting the mobility of VE-cadherin at the plasma membrane.

Repression of MAL tumour suppressor activity by promoter methylation during cervical carcinogenesis.

We recently identified MAL (T-lymphocyte maturation associated protein) as the most down-regulated gene in cervical oncogenesis. Here, we examined the mechanism underlying MAL silencing, its functional role in cervical carcinogenesis, and the relevance of detecting MAL alterations for risk assessment of hrHPV-positive women. MAL mRNA expression and promoter methylation were analysed in primary keratinocytes, hrHPV-immortalized keratinocytes, cervical cancer cell lines, biopsies, and scrapings by quantitative (methylation-specific) PCR. SiHa cells were transfected with MAL cDNA and assayed for proliferation, migration, and anchorage-independent growth. MAL mRNA was (nearly) undetectable in all HPV-immortalized and cervical cancer cells, but could be up-regulated upon methylation inhibition. MAL promoter methylation at two promoter regions (M1 and M2) was detected in all HPV-immortalized cells and cancer cells. Ectopic expression of MAL in SiHa cells suppressed proliferation, migration, and anchorage-independent growth. None (0/22) of normal cervical biopsies, 9% (6/66) of CIN1 lesions, 53% (34/64) of CIN3 lesions, 90% (85/94) of cervical squamous cell carcinomas (SCCs), and 93% (26/28) of cervical adenocarcinomas (AdCAs) demonstrated MAL promoter methylation at both promoter regions. Moreover, detection of MAL promoter methylation in cervical scrapings was predictive for underlying high-grade lesions. Both in biopsies and in scrapings, MAL promoter methylation was significantly correlated with reduced mRNA expression. MAL gene silencing by promoter methylation is a frequent and biologically essential event in HPV-induced cervical carcinogenesis. Hence, MAL promoter methylation and/or mRNA expression analysis on cervical scrapings may provide a valuable diagnostic tool to improve the detection of CIN3, SCC, and AdCA.