"Diving in the deep-end and swimming": a mixed methods study using normalization process theory to evaluate a learning collaborative approach for the implementation of palliative care practices in hemodialysis centers.

BACKGROUND: Normalization Process Theory (NPT) is an implementation theory that can be used to explain how and why implementation strategies work or not in particular circumstances. We used it to understand the mechanisms that lead to the adoption and routinization of palliative care within hemodialysis centers. METHODS: We employed a longitudinal, mixed methods approach to comprehensively evaluate the implementation of palliative care practices among ten hemodialysis centers participating in an Institute for Healthcare Improvement Breakthrough- Series learning collaborative. Qualitative methods included longitudinal observations of collaborative activities, and interviews with implementers at the end of the study. We used an inductive and deductive approach to thematic analysis informed by NPT constructs (coherence, cognitive participation, collective action, reflexive monitoring) and implementation outcomes. The NoMAD survey, which measures NPT constructs, was completed by implementers at each hemodialysis center during early and late implementation. RESULTS: The four mechanisms posited in NPT had a dynamic and layered relationship during the implementation process. Collaborative participants participated because they believed in the value and legitimacy of palliative care for patients receiving hemodialysis and thus had high levels of cognitive participation at the start. Didactic Learning Sessions were important for building practice coherence, and sense-making was solidified through testing new skills in practice and first-hand observation during coaching visits by an expert. Collective action was hampered by limited time among team members and practical issues such as arranging meetings with patients. Reflexive monitoring of the positive benefit to patient and family experiences was key in shifting mindsets from disease-centric towards a patient-centered model of care. NoMAD survey scores showed modest improvement over time, with collective action having the lowest scores. CONCLUSIONS: NPT was a useful framework for understanding the implementation of palliative care practices within hemodialysis centers. We found a nonlinear relationship among the mechanisms which is reflected in our model of implementation of palliative care practices through a learning collaborative. These findings suggest that the implementation of complex practices such as palliative care may be more successful through iterative learning and practice opportunities as the mechanisms for change are layered and mutually reinforcing. TRIAL REGISTRATION: ClinicalTrials.gov, NCT04125537 . Registered 14 October 2019 - Retrospectively registered.

Patient and surgeon experiences with video visits in plastic surgery-toward a data-informed scheduling triage tool.

BACKGROUND: Coronavirus disease 2019 provided the impetus for unprecedented adoption of telemedicine. This study aimed to understand video visit adoption by plastic surgery providers; and patient and surgeon perceptions about its efficacy, value, accessibility, and long-term viability. A secondary aim was to develop the proposed 'Triage Tool for Video Visits in Plastic Surgery' to help determine visit video eligibility. METHODS: This mixed-methods evaluation assessed provider-level scheduling data from the Division of Plastic and Reconstructive Surgery at Stanford Health Care to quantify telemedicine adoption and semi-structured phone interviews with patients (n = 20) and surgeons (n = 10) to explore stakeholder perspectives on video visits. RESULTS: During the 13-week period after the local stay-at-home orders due to coronavirus disease 2019, 21.4% of preoperative visits and 45.5% of postoperative visits were performed via video. Video visits were considered acceptable by patients and surgeons in plastic surgery in terms of quality of care but were limited by the inability to perform a physical examination. Interviewed clinicians reported that long-term viability needs to be centered around technology (eg, connection, video quality, etc) and physical examinations. Our findings informed a proposed triage tool to determine the appropriateness of video visits for individual patients that incorporates visit type, anesthesia, case, surgeon's role, and patient characteristics. CONCLUSION: Video technology has the potential to facilitate and improve preoperative and postoperative patient care in plastic surgery but the following components are needed: patient education on taking high-quality photos; standardized clinical guidelines for conducting video visits; and an algorithm-assisted triage tool to support scheduling.

Factors associated with antibiotic prescribing in patients with acute respiratory tract complaints in Malta: a 1-year repeated cross-sectional surveillance study.

OBJECTIVE: To identify factors that influence general practitioners' (GPs') oral antibiotic prescribing for acute respiratory tract complaints (aRTCs) in Malta. DESIGN: Repeated, cross-sectional surveillance. SETTING: Maltese general practice; both public health centres and private GP clinics. PARTICIPANTS: 30 GPs registered on the Malta Medical Council's Specialist Register and 3 GP trainees registered data of 4831 patients of all ages suffering from any aRTC. Data were collected monthly between May 2015 and April 2016 during predetermined 1-week periods. OUTCOME MEASURES: The outcome of interest was antibiotic prescription (yes/no), defined as an oral antibiotic prescription issued for an aRTC during an in-person consultation, irrespective of the number of antibiotics given. The association between GP, practice and consultation-level factors, patient sociodemographic factors and patient health status factors, and antibiotic prescription was investigated. RESULTS: The antibiotic prescription rate was 45.0%. Independent factors positively associated with antibiotic prescribing included female GP sex (OR 2.3, 95% CI 1.22 to 4.26), GP age with GPs ≥60 being the most likely (OR 34.7, 95% CI 14.14 to 84.98), patient age with patients ≥65 being the most likely (OR 2.3, 95% CI 1.71 to 3.18), number of signs and/or symptoms with patients having ≥4 being the most likely (OR 9.6, 95% CI 5.78 to 15.99), fever (OR 2.6, 95% CI 2.08 to 3.26), productive cough (OR 1.3, 95% CI 1.03 to 1.61), otalgia (OR 1.3, 95% CI 1.01 to 1.76), tender cervical nodes (OR 2.2, 95% CI 1.57 to 3.05), regular clients (OR 1.3, 95% CI 1.05 to 1.66), antibiotic requests (OR 4.8, 95% CI 2.52 to 8.99) and smoking (OR 1.4, 95% CI 1.13 to 1.71). Conversely, patients with non-productive cough (OR 0.3, 95% CI 0.26 to 0.41), sore throat (OR 0.6, 95% CI 0.53 to 0.78), rhinorrhoea (OR 0.3, 95% CI 0.23 to 0.36) or dyspnoea (OR 0.6, 95% CI 0.41 to 0.83) were less likely to receive an antibiotic prescription. CONCLUSION: Antibiotic prescribing for aRTCs was high and influenced by a number of factors. Potentially inappropriate prescribing in primary care can be addressed through multifaceted interventions addressing modifiable factors associated with prescription. TRIAL REGISTRATION NUMBER: NCT03218930.

Locally delivered CD40 agonist antibody accumulates in secondary lymphoid organs and eradicates experimental disseminated bladder cancer.

Immunotherapy with intratumoral injection of adenoviral vectors expressing CD40L has yielded positive results in experimental and clinical bladder cancer. We therefore hypothesized that anti-CD40 antibody would be effective in this setting. Agonistic CD40 antibodies were developed as vaccine adjuvants but have later been used as treatment of advanced solid tumors and hematologic cancers. Systemic anti-CD40 therapy has been associated with immune-related adverse events, such as cytokine release syndrome and liver toxicity, and local delivery is an attractive approach that could reduce toxicity. Herein, we compared local and systemic anti-CD40 antibody delivery to evaluate efficacy, toxicity, and biodistribution in the experimental MB49 bladder cancer model. Antitumor effects were confirmed in the B16 model. In terms of antitumor efficacy, local anti-CD40 antibody stimulation was superior to systemic therapy at an equivalent dose and CD8 T cells were crucial for tumor growth inhibition. Both administration routes were dependent on host CD40 expression for therapeutic efficacy. In vivo biodistribution studies revealed CD40-specific antibody accumulation in the tumor-draining lymph nodes and the spleen, most likely reflecting organs with frequent target antigen-expressing immune cells. Systemic administration led to higher antibody concentrations in the liver and blood compared with local delivery, and was associated with elevated levels of serum haptoglobin. Despite the lack of a slow-release system, local anti-CD40 therapy was dependent on tumor antigen at the injection site for clearance of distant tumors. To summarize, local low-dose administration of anti-CD40 antibody mediates antitumor effects in murine models with reduced toxicity and may represent an attractive treatment alternative in the clinic.

Perlecan maintains microvessel integrity in vivo and modulates their formation in vitro.

Perlecan is a heparan sulfate proteoglycan assembled into the vascular basement membranes (BMs) during vasculogenesis. In the present study we have investigated vessel formation in mice, teratomas and embryoid bodies (EBs) in the absence of perlecan. We found that perlecan was dispensable for blood vessel formation and maturation until embryonic day (E) 12.5. At later stages of development 40% of mutant embryos showed dilated microvessels in brain and skin, which ruptured and led to severe bleedings. Surprisingly, teratomas derived from perlecan-null ES cells showed efficient contribution of perlecan-deficient endothelial cells to an apparently normal tumor vasculature. However, in perlecan-deficient EBs the area occupied by an endothelial network and the number of vessel branches were significantly diminished. Addition of FGF-2 but not VEGF(165) rescued the in vitro deficiency of the mutant ES cells. Furthermore, in the absence of perlecan in the EB matrix lower levels of FGFs are bound, stored and available for cell surface presentation. Altogether these findings suggest that perlecan supports the maintenance of brain and skin subendothelial BMs and promotes vasculo- and angiogenesis by modulating FGF-2 function.

Identification of conformational epitopes for human IgG on Chemotaxis inhibitory protein of Staphylococcus aureus.

BACKGROUND: The Chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS) blocks the Complement fragment C5a receptor (C5aR) and formylated peptide receptor (FPR) and is thereby a potent inhibitor of neutrophil chemotaxis and activation of inflammatory responses. The majority of the healthy human population has antibodies against CHIPS that have been shown to interfere with its function in vitro. The aim of this study was to define potential epitopes for human antibodies on the CHIPS surface. We also initiate the process to identify a mutated CHIPS molecule that is not efficiently recognized by preformed anti-CHIPS antibodies and retains anti-inflammatory activity. RESULTS: In this paper, we panned peptide displaying phage libraries against a pool of CHIPS specific affinity-purified polyclonal human IgG. The selected peptides could be divided into two groups of sequences. The first group was the most dominant with 36 of the 48 sequenced clones represented. Binding to human affinity-purified IgG was verified by ELISA for a selection of peptide sequences in phage format. For further analysis, one peptide was chemically synthesized and antibodies affinity-purified on this peptide were found to bind the CHIPS molecule as studied by ELISA and Surface Plasmon Resonance. Furthermore, seven potential conformational epitopes responsible for antibody recognition were identified by mapping phage selected peptide sequences on the CHIPS surface as defined in the NMR structure of the recombinant CHIPS31-121 protein. Mapped epitopes were verified by in vitro mutational analysis of the CHIPS molecule. Single mutations introduced in the proposed antibody epitopes were shown to decrease antibody binding to CHIPS. The biological function in terms of C5aR signaling was studied by flow cytometry. A few mutations were shown to affect this biological function as well as the antibody binding. CONCLUSION: Conformational epitopes recognized by human antibodies have been mapped on the CHIPS surface and amino acid residues involved in both antibody and C5aR interaction could be defined. This information has implications for the development of an effective anti-inflammatory agent based on a functional CHIPS molecule with low interaction with human IgG.

Serum response factor is required for sprouting angiogenesis and vascular integrity.

Serum response factor (SRF) is a transcription factor that controls the expression of cytoskeletal proteins and immediate early genes in different cell types. Here, we found that SRF expression is restricted to endothelial cells (ECs) of small vessels such as capillaries in the mouse embryo. EC-specific Srf deletion led to aneurysms and hemorrhages from 11.5 days of mouse development (E11.5) and lethality at E14.5. Mutant embryos presented a reduced capillary density and defects in EC migration, with fewer numbers of filopodia in tip cells and ECs showing defects in actin polymerization and intercellular junctions. We show that SRF is essential for the expression of VE-cadherin and beta-actin in ECs both in vivo and in vitro. Moreover, knockdown of SRF in ECs impaired VEGF- and FGF-induced in vitro angiogenesis. Taken together, our results demonstrate that SRF plays an important role in sprouting angiogenesis and small vessel integrity in the mouse embryo.

Perlecan is critical for heart stability.

AIMS: Perlecan is a heparansulfate proteoglycan found in basement membranes, cartilage, and several mesenchymal tissues that form during development, tumour growth, and tissue repair. Loss-of-function mutations in the perlecan gene in mice are associated with embryonic lethality caused primarily by cardiac abnormalities probably due to hemopericards. The aim of the present study was to investigate the mechanism underlying the early embryonic lethality and the pathophysiological relevance of perlecan for heart function. METHODS AND RESULTS: Perlecan-deficient murine embryonic stem cells were used to investigate the myofibrillar network and the electrophysiological properties of single cardiomyocytes. The mechanical stability of the developing perlecan-deficient mouse hearts was analysed by microinjecting fluorescent-labelled dextran. Maturation and formation of basement membranes and cell-cell contacts were investigated by electron microscopy, immunohistochemistry, and western blotting. Sarcomere formation and cellular functional properties were unaffected in perlecan-deficient cardiomyocytes. However, the intraventricular dye injection experiments revealed mechanical instability of the early embryonic mouse heart muscle wall before embryonic day 10.5 (E10.5). Accordingly, perlecan-null embryonic hearts contained lower amounts of the critical basement membrane components, collagen IV and laminins. Furthermore, basement membranes were absent in perlecan-null cardiomoycytes whereas adherens junctions formed and matured around E9.5. Infarcted hearts from perlecan heterozygous mice displayed reduced heart function when compared with wild-type hearts. CONCLUSION: We propose that perlecan plays an important role in maintaining the integrity during cardiac development and is important for heart function in the adult heart after injury.

Endothelial-to-mesenchymal transition contributes to cardiac fibrosis.

Cardiac fibrosis, associated with a decreased extent of microvasculature and with disruption of normal myocardial structures, results from excessive deposition of extracellular matrix, which is mediated by the recruitment of fibroblasts. The source of these fibroblasts is unclear and specific anti-fibrotic therapies are not currently available. Here we show that cardiac fibrosis is associated with the emergence of fibroblasts originating from endothelial cells, suggesting an endothelial-mesenchymal transition (EndMT) similar to events that occur during formation of the atrioventricular cushion in the embryonic heart. Transforming growth factor-beta1 (TGF-beta1) induced endothelial cells to undergo EndMT, whereas bone morphogenic protein 7 (BMP-7) preserved the endothelial phenotype. The systemic administration of recombinant human BMP-7 (rhBMP-7) significantly inhibited EndMT and the progression of cardiac fibrosis in mouse models of pressure overload and chronic allograft rejection. Our findings show that EndMT contributes to the progression of cardiac fibrosis and that rhBMP-7 can be used to inhibit EndMT and to intervene in the progression of chronic heart disease associated with fibrosis.

Caspase-8 serves both apoptotic and nonapoptotic roles.

Knockout of caspase-8, a cysteine protease that participates in the signaling for cell death by receptors of the TNF/nerve growth factor family, is lethal to mice in utero. To explore tissue-specific roles of this enzyme, we established its conditional knockout using the Cre/loxP recombination system. Consistent with its role in cell death induction, deletion of caspase-8 in hepatocytes protected them from Fas-induced caspase activation and death. However, application of the conditional knockout approach to investigate the cause of death of caspase-8 knockout embryos revealed that this enzyme also serves cellular functions that are nonapoptotic. Its deletion in endothelial cells resulted in degeneration of the yolk sac vasculature and embryonal death due to circulatory failure. Caspase-8 deletion in bone-marrow cells resulted in arrest of hemopoietic progenitor functioning, and in cells of the myelomonocytic lineage, its deletion led to arrest of differentiation into macrophages and to cell death. Thus, besides participating in cell death induction by receptors of the TNF/nerve growth factor family, caspase-8, apparently independently of these receptors, also mediates nonapoptotic and perhaps even antiapoptotic activities.

Endothelium-specific ablation of PDGFB leads to pericyte loss and glomerular, cardiac and placental abnormalities.

Platelet-derived growth factor-B (PDGFB) is necessary for normal cardiovascular development, but the relative importance of different cellular sources of PDGFB has not been established. Using Cre-lox techniques, we show here that genetic ablation of Pdgfb in endothelial cells leads to impaired recruitment of pericytes to blood vessels. The endothelium-restricted Pdgfb knockout mutants also developed organ defects including cardiac, placental and renal abnormalities. These defects were similar to those observed in Pdgfb null mice. However, in marked contrast to the embryonic lethality of Pdgfb null mutants, the endothelium-specific mutants survived into adulthood with persistent pathological changes, including brain microhemorrhages, focal astrogliosis, and kidney glomerulus abnormalities. This spectrum of pathological changes is reminiscent of diabetic microangiopathy, suggesting that the endothelium-restricted Pdgfb knockouts may serve as models for some of the pathogenic events of vascular complications to diabetes.

Role of collagen type II and perlecan in skeletal development.

The cartilage extracellular matrix is composed of a dense collagen network that entraps a range of other specialized proteins important for the proper formation and function of the tissue. Loss of two abundant cartilage components, type II collagen and perlecan, has drastic effects on skeletal development. Both collagen II and perlecan mutants have severe and lethal chondrodysplasia characterized by disorganized growth plate, lack of collagen network, defective endochondral bone formation, and abnormal intervertebral disk development. To test whether the reduced collagen density in the perlecan-null cartilage is due to enhanced activity of collagen-degrading proteinases, we have analyzed gelatinase expression and activity in the mutant tissue. Immunohistochemical analysis revealed a weak, but clear, expansion of MMP-9 deposition into the hypertrophic zone of the perlecan-null growth plate. However, in situ and SDS-PAGE zymography showed that the activity of gelatinases (MMP-2 and MMP-9) is not altered in perlecan-null cartilage, suggesting that they are not primarily linked to the reduced fibrillar network observed in the mutant. Likewise, intercrossing of perlecan mutants onto an MMP-9-null background could not rescue the ultrastructural abnormalities of the perlecan-deficient cartilage.

Targeted disruption of the murine zyxin gene.

Zyxin is an evolutionarily conserved protein that is concentrated at sites of cell adhesion, where it associates with members of the Enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP) family of cytoskeletal regulators and is postulated to play a role in cytoskeletal dynamics and signaling. Zyxin transcripts are detected throughout murine embryonic development, and the protein is widely expressed in adults. Here we used a reverse genetic approach to examine the consequences of loss of zyxin function in the mouse. Mice that lack zyxin function are viable and fertile and display no obvious histological abnormalities in any of the organs examined. Because zyxin contributes to the localization of Ena/VASP family members at certain subcellular locations, we carefully examined the zyxin(-/-) mice for evidence of defects that have been observed when Ena/VASP proteins are compromised in the mouse. Specifically, we evaluated blood platelet function, nervous system development, and skin architecture but did not detect any defects in these systems. Zyxin is the founding member of a family of proteins that also includes the lipoma preferred partner (LPP) and thyroid receptor-interacting protein 6 (TRIP6). These zyxin family members display patterns of expression that significantly overlap that of zyxin. Western blot analysis indicates that there is no detectable upregulation of either LPP or TRIP6 expression in tissues derived from zyxin-null mice. Because zyxin family members may have overlapping functions, a comprehensive understanding of the role of these proteins in the mouse will require the generation of compound mutations in which multiple zyxin family members are simultaneously compromised.

Endothelium-specific platelet-derived growth factor-B ablation mimics diabetic retinopathy.

Loss of pericytes from the capillary wall is a hallmark of diabetic retinopathy, however, the pathogenic significance of this phenomenon is unclear. In previous mouse gene knockout models leading to pericyte deficiency, prenatal lethality has so far precluded analysis of postnatal consequences in the retina. We now report that endothelium-restricted ablation of platelet-derived growth factor-B generates viable mice with extensive inter- and intra-individual variation in the density of pericytes throughout the CNS. We found a strong inverse correlation between pericyte density and the formation of a range of retinal microvascular abnormalities strongly reminiscent of those seen in diabetic humans. Proliferative retinopathy invariably developed when pericyte density was <50% of normal. Our data suggest that a reduction of the pericyte density is sufficient to cause retinopathy in mice, implying that pericyte loss may also be a causal pathogenic event in human diabetic retinopathy.