Heparin versus citrate anticoagulation for continuous renal replacement therapy in intensive care: the RRAM observational study

Background: In the UK, 10% of admissions to intensive care units receive continuous renal replacement therapy with regional citrate anticoagulation replacing systemic heparin anticoagulation over the last decade. Regional citrate anticoagulation is now used in > 50% of intensive care units, despite little evidence of safety or effectiveness. Aim: The aim of the Renal Replacement Anticoagulant Management study was to evaluate the clinical and health economic impacts of intensive care units moving from systemic heparin anticoagulation to regional citrate anticoagulation for continuous renal replacement therapy. Design: This was an observational comparative effectiveness study. Setting: The setting was NHS adult general intensive care units in England and Wales. Participants: Participants were adults receiving continuous renal replacement therapy in an intensive care unit participating in the Intensive Care National Audit & Research Centre Case Mix Programme national clinical audit between 1 April 2009 and 31 March 2017. Interventions: Exposure – continuous renal replacement therapy in an intensive care unit after completion of transition to regional citrate anticoagulation. Comparator – continuous renal replacement therapy in an intensive care unit before starting transition to regional citrate anticoagulation or had not transitioned. Outcome measures: Primary effectiveness – all-cause mortality at 90 days. Primary economic – incremental net monetary benefit at 1 year. Secondary outcomes – mortality at hospital discharge, 30 days and 1 year; days of renal, cardiovascular and advanced respiratory support in intensive care unit; length of stay in intensive care unit and hospital; bleeding and thromboembolic events; prevalence of end-stage renal disease at 1 year; and estimated lifetime incremental net monetary benefit. Data sources: Individual patient data from the Intensive Care National Audit & Research Centre Case Mix Programme were linked with the UK Renal Registry, Hospital Episode Statistics (for England), Patient Episodes Data for Wales and Civil Registrations (Deaths) data sets, and combined with identified periods of systemic heparin anticoagulation and regional citrate anticoagulation (survey of intensive care units). Staff time and consumables were obtained from micro-costing. Continuous renal replacement therapy system failures were estimated from the Post-Intensive Care Risk-adjusted Alerting and Monitoring data set. EuroQol-3 Dimensions, three-level version, health-related quality of life was obtained from the Intensive Care Outcomes Network study. Results: Out of the 188 (94.9%) units that responded to the survey, 182 (96.8%) use continuous renal replacement therapy. After linkage, data were available from 69,001 patients across 181 intensive care units (60,416 during periods of systemic heparin anticoagulation use and 8585 during regional citrate anticoagulation use). The change to regional citrate anticoagulation was not associated with a step change in 90-day mortality (odds ratio 0.98, 95% confidence interval 0.89 to 1.08). Secondary outcomes showed step increases in days of renal support (difference in means 0.53 days, 95% confidence interval 0.28 to 0.79 days), advanced cardiovascular support (difference in means 0.23 days, 95% confidence interval 0.09 to 0.38 days) and advanced respiratory support (difference in means, 0.53 days, 95% CI 0.03 to 1.03 days) with a trend toward fewer bleeding episodes (odds ratio 0.90, 95% confidence interval 0.76 to 1.06) with transition to regional citrate anticoagulation. The micro-costing study indicated that regional citrate anticoagulation was more expensive and was associated with an estimated incremental net monetary loss (step change) of –£2376 (95% confidence interval –£3841 to –£911). The estimated likelihood of cost-effectiveness at 1 year was less than 0.1%. Limitations: Lack of patient-level treatment data means that the results represent average effects of changing to regional citrate anticoagulation in intensive care units. Administrative data are subject to variation in data quality over time, which may contribute to observed trends. Conclusions: The introduction of regional citrate anticoagulation has not improved outcomes for patients and is likely to have substantially increased costs. This study demonstrates the feasibility of evaluating effects of changes in practice using routinely collected data. Future work: (1) Prioritise other changes in clinical practice for evaluation and (2) methodological research to understand potential implications of trends in data quality. Trial registration: This trial is registered as ClinicalTrials.gov NCT03545750

Human airway xenograft models of epithelial cell regeneration

Regeneration and restoration of the airway epithelium after mechanical, viral or bacterial injury have a determinant role in the evolution of numerous respiratory diseases such as chronic bronchitis, asthma and cystic fibrosis. The study in vivo of epithelial regeneration in animal models has shown that airway epithelial cells are able to dedifferentiate, spread, migrate over the denuded basement membrane and progressively redifferentiate to restore a functional respiratory epithelium after several weeks. Recently, human tracheal xenografts have been developed in immunodeficient severe combined immunodeficiency (SCID) and nude mice. In this review we recall that human airway cells implanted in such conditioned host grafts can regenerate a well-differentiated and functional human epithelium; we stress the interest in these humanized mice in assaying candidate progenitor and stem cells of the human airway mucosa

Following damage, the majority of bone marrow-derived airway cells express an epithelial marker

<p>Abstract</p> <p>Background</p> <p>Adult-derived bone marrow stem cells are capable of reconstituting the haematopoietic system. However there is ongoing debate in the literature as to whether bone marrow derived cells have the ability to populate other tissues and express tissue specific markers. The airway has been an organ of major interest and was one of the first where this was demonstrated. We have previously demonstrated that the mouse airway can be repopulated by side population bone marrow transplanted cells. Here we investigate the frequency and phenotypic nature of these bone marrow derived cells.</p> <p>Methods</p> <p>Female mice were engrafted with male whole bone marrow or side population (SP) cells and subjected to detergent-induced damage after 3 months. Donor cells were identified by Y chromosome fluorescence in situ hybridisation and their phenotype was assessed by immunohistochemistry on the same sections. Slides were visualised by a combination of widefield and deconvolved microscopy and whole cells were analysed on cytospin preparations.</p> <p>Results</p> <p>The frequencies of engraftment of male cells in the airway of mice that show this (9/10), range from 1.0 – 1.6% with whole marrow and 0.6 – 1.5% with SP cells. Undamaged controls have only between 0.1 and 0.2% male cells in the trachea. By widefield microscopy analysis we find 60.2% (53/88) of male donor derived cells express cytokeratins as a marker of epithelial cells. These results were reinforced using deconvolved microscopy and scored by two independent investigators. In addition cytospin analysis of cells dissociated from the damaged trachea of engrafted mice also reveals donor derived Y chromosome positive cells that are immunopositive for cytokeratin. Using cytokeratin and the universal haematopoietic marker CD45 immunohistochemistry, we find the donor derived cells fall into four phenotypic classes. We do not detect cytokeratin positive cells in whole bone marrow using cytokeratin immunostaining and we do not detect any cytokeratin mRNA in SP or bone marrow samples by RT-PCR.</p> <p>Conclusion</p> <p>The appearance of bone marrow derived cells in the tracheal epithelium is enriched by detergent-induced tissue damage and the majority of these cells express an epithelial marker. The cytokeratin positive donor derived cells in the tracheal epithelium are not present in the injected donor cells and must have acquired this novel phenotype <it>in vivo</it>.</p

Comparative study of fungal cell disruption—scope and limitations of the methods

Simple and effective protocols of cell wall disruption were elaborated for tested fungal strains: Penicillium citrinum, Aspergillus fumigatus, Rhodotorula gracilis. Several techniques of cell wall disintegration were studied, including ultrasound disintegration, homogenization in bead mill, application of chemicals of various types, and osmotic shock. The release of proteins from fungal cells and the activity of a cytosolic enzyme, glucose-6-phosphate dehydrogenase, in the crude extracts were assayed to determine and compare the efficacy of each method. The presented studies allowed adjusting the particular method to a particular strain. The mechanical methods of disintegration appeared to be the most effective for the disintegration of yeast, R. gracilis, and filamentous fungi, A. fumigatus and P. citrinum. Ultrasonication and bead milling led to obtaining fungal cell-free extracts containing high concentrations of soluble proteins and active glucose-6-phosphate dehydrogenase systems

Molecular properties of CD133+ glioblastoma stem cells derived from treatment-refractory recurrent brain tumors

Glioblastoma multiforme (GBM) remains refractory to conventional therapy. CD133+ GBM cells have been recently isolated and characterized as chemo-/radio-resistant tumor-initiating cells and are hypothesized to be responsible for post-treatment recurrence. In order to explore the molecular properties of tumorigenic CD133+ GBM cells that resist treatment, we isolated CD133+ GBM cells from tumors that are recurrent and have previously received chemo-/radio-therapy. We found that the purified CD133+ GBM cells sorted from the CD133+ GBM spheres express SOX2 and CD44 and are capable of clonal self-renewal and dividing to produce fast-growing CD133− progeny, which form the major cell population within GBM spheres. Intracranial injection of purified CD133+, not CD133− GBM daughter cells, can lead to the development of YKL-40+ infiltrating tumors that display hypervascularity and pseudopalisading necrosis-like features in mouse brain. The molecular profile of purified CD133+ GBM cells revealed characteristics of neuroectoderm-like cells, expressing both radial glial and neural crest cell developmental genes, and portraying a slow-growing, non-differentiated, polarized/migratory, astrogliogenic, and chondrogenic phenotype. These data suggest that at least a subset of treated and recurrent GBM tumors may be seeded by CD133+ GBM cells with neural and mesenchymal properties. The data also imply that CD133+ GBM cells may be clinically indolent/quiescent prior to undergoing proliferative cell division (PCD) to produce CD133− GBM effector progeny. Identifying intrinsic and extrinsic cues, which promote CD133+ GBM cell self-renewal and PCD to support ongoing tumor regeneration may highlight novel therapeutic strategies to greatly diminish the recurrence rate of GBM

Lung epithelial stem cells and their niches : Fgf10 takes center stage

Throughout life adult animals crucially depend on stem cell populations to maintain and repair their tissues to ensure life-long organ function. Stem cells are characterized by their capacity to extensively self-renew and give rise to one or more differentiated cell types. These powerful stem cell properties are key to meet the changing demand for tissue replacement during normal lung homeostasis and regeneration after lung injury. Great strides have been made over the last few years to identify and characterize lung epithelial stem cells as well as their lineage relationships. Unfortunately, knowledge on what regulates the behavior and fate specification of lung epithelial stem cells is still limited, but involves communication with their microenvironment or niche, a local tissue environment that hosts and influences the behaviors or characteristics of stem cells and that comprises other cell types and extracellular matrix. As such, an intimate and dynamic epithelial-mesenchymal cross-talk, which is also essential during lung development, is required for normal homeostasis and to mount an appropriate regenerative response after lung injury. Fibroblast growth factor 10 (Fgf10) signaling in particular seems to be a well-conserved signaling pathway governing epithelial-mesenchymal interactions during lung development as well as between different adult lung epithelial stem cells and their niches. On the other hand, disruption of these reciprocal interactions leads to a dysfunctional epithelial stem cell-niche unit, which may culminate in chronic lung diseases such as chronic obstructive pulmonary disease (COPD), chronic asthma and idiopathic pulmonary fibrosis (IPF)