Regenerative and Anti-Inflammatory Potential of Regularly Fed, Starved Cells and Extracellular Vesicles In Vivo.

BACKGROUND: Mesenchymal stem/stromal cells (MSC) have been employed successfully in immunotherapy and regenerative medicine, but their therapeutic potential is reduced considerably by the ischemic environment that exists after transplantation. The assumption that preconditioning MSC to promote quiescence may result in increased survival and regenerative potential upon transplantation is gaining popularity. METHODS: The purpose of this work was to evaluate the anti-inflammatory and regenerative effects of human bone marrow MSC (hBM-MSC) and their extracellular vesicles (EVs) grown and isolated in a serum-free medium, as compared to starved hBM-MSC (preconditioned) in streptozotocin-induced diabetic fractured male C57BL/6J mice. RESULTS: Blood samples taken four hours and five days after injection revealed that cells, whether starved or not, generated similar plasma levels of inflammatory-related cytokines but lower levels than animals treated with EVs. Nonetheless, starved cells prompted the highest production of IL-17, IL-6, IL-13, eotaxin and keratinocyte-derived chemokines and induced an earlier soft callus formation and mineralization of the fracture site compared to EVs and regularly fed cells five days after administration. CONCLUSIONS: Preconditioning may be crucial for refining and defining new criteria for future MSC therapies. Additionally, the elucidation of mechanisms underpinning an MSC's survival/adaptive processes may result in increased cell survival and enhanced therapeutic efficacy following transplantation.

Baseline Characteristics, Evaluation, and Management of Women With Complaints of Recurrent Urinary Tract Infections.

OBJECTIVE: The aims of this study were to determine the proportion of women presenting for recurrent urinary tract infections (UTIs) who met the diagnostic criteria (culture-proven UTI ≥3 in 1 year or ≥2 in 6 months) and to assess advanced testing utilization, preventive therapy use, and risk factors. METHODS: This is a retrospective chart review of women seen as new urogynecology consults for recurrent UTI (rUTI) between April 1, 2017, and April 1, 2018, followed through April 1, 2019. Exclusion criteria included catheter use, cancer treatment within 2 years, and prior organ transplant, urinary diversion, conduit, or bladder augmentation. RESULTS: Of 600 women, 71% had follow-up with a median of 179 days. Urinary tract infection symptoms included frequency (50%), dysuria (46%), urgency (43%), and malodorous urine (7%). One third met the rUTI diagnostic criteria. Two hundred thirty-four (39%) underwent advanced testing, and 9% (21/234) of women who underwent advanced testing had a change in clinical care. Preventive therapy use increased after consultation (P < 0.001), with vaginal estrogen (47%) being most common. Compared with women not meeting the rUTI criteria, women meeting the rUTI criteria were more likely to be older (adjusted odds ratio [aOR], 1.03/year; 95% confidence interval [CI], 1.02-1.04), have a prior history of gynecologic cancer (aOR, 4.07; 95% CI, 1.02-16.25), or report UTI symptoms of dysuria (aOR, 2.27; 95% CI, 1.57-3.27), or malodorous urine (aOR, 2.96; 95% CI, 1.47-5.94) and, while equally likely to be receiving preventive treatment prior to consultation, were more likely after consultation (OR, 3.06; 95% CI, 2.05-4.55). DISCUSSION: Thirty-seven percent of women seen for rUTI met the diagnostic criteria. Advanced imaging rarely changed care. Education about diagnostic criteria and preventive therapy is warranted.

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.

Optimizing parameters of an open-source airway segmentation algorithm using different CT images.

BACKGROUND: Computed tomography (CT) helps physicians locate and diagnose pathological conditions. In some conditions, having an airway segmentation method which facilitates reconstruction of the airway from chest CT images can help hugely in the assessment of lung diseases. Many efforts have been made to develop airway segmentation algorithms, but methods are usually not optimized to be reliable across different CT scan parameters. METHODS: In this paper, we present a simple and reliable semi-automatic algorithm which can segment tracheal and bronchial anatomy using the open-source 3D Slicer platform. The method is based on a region growing approach where trachea, right and left bronchi are cropped and segmented independently using three different thresholds. The algorithm and its parameters have been optimized to be efficient across different CT scan acquisition parameters. The performance of the proposed method has been evaluated on EXACT'09 cases and local clinical cases as well as on a breathing pig lung phantom using multiple scans and changing parameters. In particular, to investigate multiple scan parameters reconstruction kernel, radiation dose and slice thickness have been considered. Volume, branch count, branch length and leakage presence have been evaluated. A new method for leakage evaluation has been developed and correlation between segmentation metrics and CT acquisition parameters has been considered. RESULTS: All the considered cases have been segmented successfully with good results in terms of leakage presence. Results on clinical data are comparable to other teams' methods, as obtained by evaluation against the EXACT09 challenge, whereas results obtained from the phantom prove the reliability of the method across multiple CT platforms and acquisition parameters. As expected, slice thickness is the parameter affecting the results the most, whereas reconstruction kernel and radiation dose seem not to particularly affect airway segmentation. CONCLUSION: The system represents the first open-source airway segmentation platform. The quantitative evaluation approach presented represents the first repeatable system evaluation tool for like-for-like comparison between different airway segmentation platforms. Results suggest that the algorithm can be considered stable across multiple CT platforms and acquisition parameters and can be considered as a starting point for the development of a complete airway segmentation algorithm.

High levels of ephrinB2 over-expression increases the osteogenic differentiation of human mesenchymal stem cells and promotes enhanced cell mediated mineralisation in a polyethyleneimine-ephrinB2 gene-activated matrix.

Gene therapy can be combined with tissue engineering constructs to produce gene-activated matrices (GAMs) with enhanced capacity for repair. Polyethyleneimine (PEI), a non-viral vector, has previously been optimised for high efficiency gene transfer in rat mesenchymal stem cells (rMSCs). The use of PEI to transfect human MSCs (hMSCs) with ephrinB2 is assessed here. Recently a role for the ephrinB2 ligand and EphB4 receptor duo has been proposed in bone remodelling. Herein, over-expression of the ephrinB2 ligand resulted in increased osteogenic differentiation in hMSCs. As ephrinB2 is a cell surface anchored ligand which only interacts with cells expressing the cognate EphB4 receptor through direct contact, we have shown that direct cell-cell contact between two neighbouring cells is responsible for enhanced osteogenesis. In an effort to begin to elucidate the molecular mechanisms at play downstream of ephrinB2 over-expression, RT-PCR was performed on the GAMs which revealed no significant changes in runx2 or BMP2 expression but an upregulation of osterix (Osx) and Dlx5 expression prompting the belief that the mode of osteogenesis is independent of the BMP2 pathway. This select interaction, coupled with the transient gene expression profile of PEI, makes the PEI-ephrinB2 GAM an ideal candidate matrix for a bone targeted GAM.

Development of a thermoresponsive chitosan gel combined with human mesenchymal stem cells and desferrioxamine as a multimodal pro-angiogenic therapeutic for the treatment of critical limb ischaemia.

Critical limb ischaemia (CLI) is a debilitating ischaemic disease caused by vascular occlusion. Pro-angiogenic therapeutics have the potential to produce collateral vasculature, delaying or negating the need for amputation or invasive revascularisation. Thermoresponsive hydrogels can provide an in situ depot for the sustained release of drugs and provide protection and cohesion for encapsulated cells. Human mesenchymal stem cells (hMSCs) have demonstrated strong angiogenic potential in vitro and angiogenic efficacy in vivo. Desferrioxamine (DFO), a pharmacological activator of the pro-angiogenic hypoxia inducible factor-1α pathway, has shown pro-angiogenic efficacy in vivo. This study combined hMSCs and DFO with a thermoresponsive chitosan/ß-glycerophosphate (ß-GP) gel, to function as an injectable, multimodal, pro-angiogenic therapeutic for the treatment of CLI. This gel underwent a thermogelation beginning at 33°C, and provided a sustained, biologically active release of DFO over the space of seven days, whilst permitting the survival, proliferation and migration of encapsulated hMSCs. hMSCs encapsulated in gel containing a 100µM concentration of DFO displayed an upregulation in VEGF expression. The combination of hMSCs and DFO within the gel resulted in a synergistic enhancement in bioactivity, as measured by increased VEGF expression in gel-exposed human umbilical vein endothelial cells. This formulation displays significant potential as an injectable pro-angiogenic therapeutic for the treatment of CLI.

On the application of active learning and Gaussian processes in postcryopreservation cell membrane integrity experiments.

Biological cell cryopreservation permits storage of specimens for future use. Stem cell cryostorage in particular is fast becoming a broadly spread practice due to their potential for use in regenerative medicine. For the optimal cryopreservation process, ultralow temperatures are needed. However, elevated temperatures are often unavoidable in a typical sample handling cycle which in turn negatively affects the postcryopreservation integrity of cells. In this paper, we present an application of active learning using an underlying Gaussian Process (GP) model in an experimental study on postcryopreservation membrane integrity response to a range of elevated temperature conditions. We tailored this technique for the current investigation and developed an algorithm which enabled identification of the sampling locations for the experiments in order to obtain the highest information return about the process from a limited size sample set. We applied this algorithm in the experimental study investigating the effects of severe temperature elevation (ranging from -40 to 20 °C) over a short term event (48 hours) on the postcryopreservation membrane integrity of Mesenchymal Stem Cells (MSCs) derived from human bone marrow. The algorithm showed excellent performance by selecting the locations which maximized the reduction of variance of the process response estimate. An approximating GP model developed from this experimental data shows that the elevated temperatures during cryopreservation have an imminent detrimental effect on cell integrity.

Investigation of the role of computed tomography as an adjunct to autopsy in the evaluation of stillbirth.

INTRODUCTION: The number of parents agreeing to autopsy following stillbirth is declining, which has undermined clinicians' ability to assess causes of intrauterine death and can impact counselling regarding recurrence. Post-mortem radiological imaging is a potential alternative method of investigating perinatal loss. The aim of this study was to assess the role of multi-detector computed tomography (MDCT) in the investigation of stillbirth. STUDY DESIGN: Following ethical approval and written consent, parents were offered MDCT of the stillborn infant. MDCT was performed with 3D reconstruction, and images were analysed for image quality, anthropomorphic measurements and pathologic findings. Body part and organ-specific measurements were performed; including head, chest and abdominal circumferences, and muscle and liver mass was also measured. Findings were correlated with obstetric history, post-mortem skeletal survey (plain radiography), and formal autopsy. RESULTS: Fourteen third-trimester stillborn infants were scanned. Image quality was moderate to excellent for most body structures. CT was better than plain radiography for imaging skeletal structures and large solid organs and demonstrated a range of pathologies including renal vein thrombosis, mesenteric calcification and skeletal hyperostosis that were not seen on plain radiographs. MDCT did not overlook autopsy findings and provided some additional information. CONCLUSION: This study confirms the feasibility of MDCT in the investigation of third trimester stillbirth. MDCT image quality is acceptable and the examination can demonstrate a range of anatomic and pathologic findings. Initially, its value may be as an important adjunct to conventional autopsy.

Management of the coinfected patient: human immunodeficiency virus/hepatitis B and human immunodeficiency virus/hepatitis C.

Deaths from liver disease have increased in persons infected with human immunodeficiency virus (HIV) because of coinfection with chronic hepatitis B and C; consequently, all HIV-infected patients should be screened for hepatitis B and C, and all those susceptible should be vaccinated for hepatitis B. Hepatitis A vaccination is indicated for susceptible coinfected patients. It is also important to stress other means of preventing the transmission of hepatitis, such as safe sex and avoidance of blood exposures. Three oral agents, lamivudine, adefovir, and tenofovir, are active against hepatitis B infection. The need for highly active antiretroviral therapy and hepatitis B therapy should be addressed in a coordinated fashion, since two of these agents are active against both HIV and hepatitis B virus. Oral combination therapy for hepatitis B infection looks promising but needs further study. Combination therapy for chronic hepatitis C with pegylated interferon plus ribavirin is the most effective available therapy and the current standard of care. Prior to therapy, patients should be evaluated for contraindications to therapy. During treatment, they should be closely monitored for adverse events.

Single-dose (30 mg/kg) azithromycin compared with 10-day amoxicillin/clavulanate for the treatment of uncomplicated acute otitis media: a double-blind, placebo-controlled, randomized clinical trial.

BACKGROUND: The long half-life of azithromycin allows for single-dose oral therapy for acute otitis media (AOM). OBJECTIVE: This study was designed to compare the efficacy and tolerability of single-dose azithromycin with 10-day, twice-daily amoxicillin/clavulanate for the treatment of new-onset, uncomplicated AOM in children. METHODS: Children aged 6 months to 12 years with new-onset AOM were randomly assigned to receive either a single 30-mg/kg dose of azithromycin or standard-dose amoxicillin/clavulanate (45 mg/kg administered BID for 10 days) in a double-blind, double-placebo, multicenter clinical trial. The diagnosis of AOM was based on specific clinical signs and symptoms, and was confirmed by pneumatic otoscopy and acoustic reflectometry (level ≥3). Clinical response was assessed on days 12-16 and 28-32. RESULTS: Mean (SD) age of children receiving azithromycin (n = 173) or amoxicillin/clavulanate (n = 173) was 2.7 (2.3) years and 3.4 (2.8) years, respectively, with 43% and 36% ≤2 years of age. Most (53.2%) of the children were boys, and most (51.2%) were white. Clinical success rates (intent-to-treat) for azithromycin and amoxicillin/clavulanate, respectively, were 87% and 88% (95% CI, -9.2 to 6.5) on day 12-16 and 75% and 75% (95% CI, -10.2 to 10.5) on day 28-32. The incidences of treatment-related adverse events for azithromycin and amoxicillin/clavulanate were 16.8% and 22.5%, respectively. Corresponding rates of diarrhea were 6.4% and 12.7%, respectively. Vomiting, which was generally mild, occurred in 7 children in each group. One azithromycin patient and 5 amoxicillin/clavulanate patients discontinued treatment because of adverse events. The compliance rate for azithromycin was significantly higher than that for amoxicillin/clavulanate (99% vs 83%; P<0.001). CONCLUSIONS: In this trial comparing the efficacy of single-dose azithromycin (30 mg/kg) with twice-daily amoxicillin/clavulanate (45 mg/kg) for the treatment of new-onset, uncomplicated AOM, no differences were detected between the 2 regimens. Single-dose azithromycin was generally well tolerated and provides an alternative to conventional oral regimens for AOM.