Rare Variants in Genes of the Cholesterol Pathway Are Present in 60% of Patients with Acute Myocardial Infarction.

Acute myocardial infarction (AMI) is a pandemic in which conventional risk factors are inadequate to detect who is at risk early in the asymptomatic stage. Although gene variants in genes related to cholesterol, which may increase the risk of AMI, have been identified, no studies have systematically screened the genes involved in this pathway. In this study, we included 105 patients diagnosed with AMI with an elevation of the ST segment (STEMI) and treated with primary percutaneous coronary intervention (PPCI). Using next-generation sequencing, we examined the presence of rare variants in 40 genes proposed to be involved in lipid metabolism and we found that 60% of AMI patients had a rare variant in the genes involved in the cholesterol pathway. Our data show the importance of considering the wide scope of the cholesterol pathway in order to assess the genetic risk related to AMI.

Mass Spectrometry Analysis of the Exhaled Breath Condensate and Proposal of Dermcidin and S100A9 as Possible Markers for Lung Cancer Prognosis.

INTRODUCTION: New sampling techniques to analyse lung diseases, such as exhaled breath condensate (EBC), are a breakthrough in research field since they are less invasive and less traumatic for the patients compared to lung biopsies. Nevertheless, there is an increasing need to optimize not only the sampling protocols but the storage and processing of specimens to get accurate results. METHODS: Exhaled breath condensate was sampled employing the ECoScreen device. Concentrated protein was obtained after ultracentrifugation, lyophilization and reversed-phase chromatography. MALDI-time of flight (TOF)/TOF mass spectrometry (MS) was applied to determine the protein profile in EBC. Commercially available ELISA kits were used to detect the selected biomarker in the EBC after MALDI-MS proteins identification. RESULTS: The obtained EBC volume after two periods of 10 min doubled the amount obtained after 20 min. One hundred peptides were detected by MALDI-MS, and 18 proteins were identified after reversed-phase chromatography concentration. Dermcidin (P81605), S100A9 (P06702) and Cathepsin G (P08311) were selected to be analysed by ELISA. Dermcidin and S100A9 expression were statistically higher in lung cancer versus healthy volunteers. VEGF concentrations decreased, respectively, by 5.94 and 11.42-fold after 1 and 2 years of frozen EBC preservation in parallel with the declined number of proteins identified by MALDI-MS. CONCLUSION: Exhaled breath condensate analysis combined with MS technique may become a valuable method for lung cancer screening and Dermcidin and S100A9 may serve as biomarkers for lung cancer diagnosis or prognosis.

Dopamine mobilizes mesenchymal progenitor cells through D2-class receptors and their PI3K/AKT pathway.

As the nervous system exerts direct and indirect effects on stem cells mobilization and catecholamines mobilize hematopoietic stem cells, we hypothesized that dopamine might induce mesenchymal progenitor cells (MPCs) mobilization. We show that dopamine induced in vitro MPCs migration through D2-class receptors, and their alternative phosphoinositide 3-kinase/Akt pathways. Also, administration of catecholamines induced in vivo mobilization of colony-forming unit-fibroblast in mice. In contrast, in vitro and in vivo MPCs migration was suppressed by D2-class receptors antagonists and blocking antibodies, consistent with dopamine signaling pathway implication. In humans, patients treated with L-dopa or catecholaminergic agonists showed a significant increase of a MPC-like population (CD45-CD31-CD34-CD105+) in their peripheral blood. These findings reveal a new link between catecholamines and MPCs mobilization and suggest the potential use of D2-class receptors agonists for mobilization of MPCs in clinical settings.

Preliminary Investigation on Efficacy and Safety of Substance P-Coated Stent for Promoting Re-Endothelialization: A Porcine Coronary Artery Restenosis Model.

BACKGROUND: Current polymer-based drug-eluting stents (DESs) have fundamental issues about inflammation and delayed re-endothelializaton of the vessel wall. Substance-P (SP), which plays an important role in inflammation and endothelial cells, has not yet been applied to coronary stents. Therefore, this study compares poly lactic-co-glycolic acid (PLGA)-based everolimus-eluting stents (PLGA-EESs) versus 2-methacryloyloxyethyl phosphorylcholine (MPC)-based SP-eluting stents (MPC-SPs) in in-vitro and in-vivo models. METHODS: The morphology of the stent surface and peptide/drug release kinetics from stents were evaluated. The in-vitro proliferative effect of SP released from MPC-SP is evaluated using human umbilical vein endothelial cell. Finally, the safety and efficacy of the stent are evaluated after inserting it into a pig's coronary artery. RESULTS: Similar to PLGA-EES, MPC-SP had a uniform surface morphology with very thin coating layer thickness (2.074 µm). MPC-SP showed sustained drug release of SP for over 2 weeks. Endothelial cell proliferation was significantly increased in groups treated with SP (n = 3) compared with the control (n = 3) and those with everolimus (n = 3) (SP: 118.9 ± 7.61% vs. everolimus: 64.3 ± 12.37% vs. the control: 100 ± 6.64%, p < 0.05). In the animal study, the percent stenosis was higher in MPC-SP group (n = 7) compared to PLGA-EES group (n = 7) (MPC-SP: 28.6 ± 10.7% vs. PLGA-EES: 16.7 ± 6.3%, p < 0.05). MPC-SP group showed, however, lower inflammation (MPC-SP: 0.3 ± 0.26 vs. PLGA-EES: 1.2 ± 0.48, p < 0.05) and fibrin deposition (MPC-SP: 1.0 ± 0.73 vs. PLGA-EES: 1.5 ± 0.59, p < 0.05) around the stent strut. MPC-SP showed more increased expression of cluster of differentiation 31, suggesting enhanced re-endothelialization. CONCLUSION: Compared to PLGA-EES, MPC-SP demonstrated more decreased inflammation of the vascular wall and enhanced re-endothelialization and stent coverage. Hence, MPC-SP has the potential therapeutic benefits for the treatment of coronary artery disease by solving limitations of currently available DESs.

In vivo genotoxicity assessment in rats exposed to Prestige-like oil by inhalation.

One of the largest oil spill disasters in recent times was the accident of the oil tanker Prestige in front of the Galician coast in 2002. Thousands of people participated in the cleanup of the contaminated areas, being exposed to a complex mixture of toxic substances. Acute and prolonged respiratory symptoms and genotoxic effects were reported, although environmental exposure measurements were restricted to current determinations, such that attribution of effects observed to oil exposure is difficult to establish. The aim of this study was to analyze peripheral blood leukocytes (PBL) harvested from a rat model of subchronic exposure to a fuel oil with similar characteristics to that spilled by the Prestige tanker, in order to determine potential genotoxic effects under strictly controlled, in vivo exposure. Wistar Han and Brown Norway rats were exposed to the oil for 3 wk, and micronucleus test (MN) and comet assay, standard and modified with 8-oxoguanine DNA glycosylase (OGG1) enzyme, were employed to assess genotoxicity 72 h and 15 d after the last exposure. In addition, the potential effects of oil exposure on DNA repair capacity were determined by means of mutagen sensitivity assay. Results obtained from this study showed that inhalation oil exposure induced DNA damage in both Brown Norway and Wistar Han rats, especially in those animals evaluated 15 d after exposure. Although alterations in the DNA repair responses were noted, the sensitivity to oil substances varied depending on rat strain. Data support previous positive genotoxicity results reported in humans exposed to Prestige oil during cleanup tasks.

Systemic Treatment of Immune-Mediated Keratoconjunctivitis Sicca with Allogeneic Stem Cells Improves the Schirmer Tear Test Score in a Canine Spontaneous Model of Disease.

Keratoconjunctivitis sicca (KCS) is characterized by ocular discomfort, conjunctival hyperaemia, and corneal scarring, causing reduced aqueous tear production that can be measured using the standard Schirmer tear test (STT). Canine adipose tissue-derived MSCs (cATMSCs) have been proposed as treatment due to their anti-inflammatory effect, by releasing cytokines and immunomodulatory soluble factors. PURPOSE: The aim of this study was to evaluate the effect of the systemic administration of cATMSCs on tear production in dogs with immune-mediated KCS, compared to classical Cyclosporine A (CsA) treatment. METHODS: Twenty-eight client-owned dogs with spontaneous KCS were allocated in the experimental group (n = 14, treated with systemic cATMSCs or control group (n = 14, treated with CsA). SST values increased significantly at days 15 (p = 0.002), 45 (p = 0.042) and 180 (p = 0.005) with no observed side-effects in the experimental group. Eyes with an initial STT value of 11-14 mm/min maintained significant improvement at day 180, needing only artificial tears as treatment. Eyes with an initial STT value <11 mm/min needed cyclosporin treatment at day 45, so follow-up was stopped. Control animals treated with CsA did not improve their STT at day 180. RESULTS AND CONCLUSIONS: Systemic allogeneic cATMSCs application appeared to be a feasible and effective therapy with positive outcome in dogs with initial STT between 11-14 mm/min, with a significant improvement in tear production. The STT increment was maintained for at least 180 days, without needing additional medication, thus suggesting it could constitute an alternative therapy to classical immunosuppressive treatments.

Airway Hyperresponsiveness, Inflammation, and Pulmonary Emphysema in Rodent Models Designed to Mimic Exposure to Fuel Oil-Derived Volatile Organic Compounds Encountered during an Experimental Oil Spill.

BACKGROUND: Fuel oil-derived volatile organic compounds (VOCs) inhalation is associated with accidental marine spills. After the Prestige petroleum tanker sank off northern Spain in 2002 and the Deepwater Horizon oil rig catastrophe in 2009, subjects involved in environmental decontamination showed signs of ongoing or residual lung disease up to 5 y after the exposure. OBJECTIVES: We aimed at investigating mechanisms driving persistent respiratory disease by developing an animal model of inhalational exposure to fuel oil-derived VOCs. METHODS: Female Wistar and Brown Norway (BN) rats and C57BL mice were exposed to VOCs produced from fuel oil mimicking the Prestige spill. Exposed animals inhaled the VOCs 2 h daily, 5 d per week, for 3 wk. Airway responsiveness to methacholine (MCh) was assessed, and bronchoalveolar lavage (BAL) and lung tissues were analyzed after the exposure and following a 2-wk washout. RESULTS: Consistent with data from human studies, both strains of rats that inhaled fuel oil-derived VOCs developed airway hyperresponsiveness that persisted after the washout period, in the absence of detectable inflammation in any lung compartment. Histopathology and quantitative morphology revealed the development of peripherally distributed pulmonary emphysema, which persisted after the washout period, associated with increased alveolar septal cell apoptosis, microvascular endothelial damage of the lung parenchyma, and inhibited expression of vascular endothelial growth factor (VEGF). DISCUSSION: In this rat model, fuel oil VOCs inhalation elicited alveolar septal cell apoptosis, likely due to DNA damage. In turn, the development of a peculiar pulmonary emphysema pattern altered lung mechanics and caused persistent noninflammatory airway hyperresponsiveness. Such findings suggest to us that humans might also respond to VOCs through physiopathological pathways different from those chiefly involved in typical cigarette smoke-driven emphysema in chronic obstructive pulmonary disease (COPD). If so, this study could form the basis for a novel disease mechanism for lasting respiratory disease following inhalational exposure to catastrophic fuel oil spills. https://doi.org/10.1289/EHP4178.

Allogeneic Adipose-Derived Mesenchymal Stem Cells (Horse Allo 20) for the Treatment of Osteoarthritis-Associated Lameness in Horses: Characterization, Safety, and Efficacy of Intra-Articular Treatment.

Osteoarthritis commonly causes lameness in the horse and has a great impact in performance animals. Due to the limitations of current medical therapies, allogenic mesenchymal stem cells (MSCs) may become an alternative method to control inflammation, reduce tissue damage and pain, and therefore improve lameness. We present the results of a regulatory clinical trial testing adipose-derived MSCs (Horse Allo 20) in veterinary (Agencia Española del Medicamento y Productos Sanitarios, Spanish Medicines Agency, Reference number 325/ECV) involving a total number of 80 participants and with 90 days of follow-up period. The manufacturing process of Horse Allo 20 was robust with no influence of the adipose tissue donor (gender, age, or breed), sample origin (intraperitoneal or subcutaneous), or storage conditions (fresh vs. frozen product presentations) on the quality, safety, and efficacy of the drug product. An in vivo safety study showed that local and systemic tolerance was safe even after repeated intra-articular administration (three injections). An in vivo efficacy study demonstrated the efficacy of the treatment after one or two injections by a reduction in lameness (P < 0.05) for an extended period of time (90 days), decreasing the need for prolonged local and/or systemic anti-inflammatory therapies and their well-known deleterious effects and toxicities.

Allogeneic adipose-derived mesenchymal stem cell therapy in dogs with refractory atopic dermatitis: clinical efficacy and safety.

Canine atopic dermatitis (AD) is a common skin disease with a 10-15 per cent prevalence. Current treatments vary in their efficacy and safety. The immunomodulatory properties of mesenchymal stem cells (MSCs) make them a promising alternative treatment. The aim of this study was to evaluate the therapeutic efficacy and safety of allogeneic canine adipose MSCs (cAd-MSCs) in dogs with refractory AD. Twenty-six dogs, suffering from AD for at least 12 months, not responding to conventional therapy, received an intravenous dose of 1.5×106 cAd-MSCs/kg bodyweight. Clinical signs, haematological and biochemistry profiles, and AD severity were assessed in a six-month follow-up using a validated scoring system (Canine Atopic Dermatitis Extent and Severity Index, version 4 (CADESI-04)). The degree of pruritus was quantified using a validated visual analogue scale, and also owner's global assessment of treatment efficacy. Twenty-two animals completed the study. Pruritus and CADESI-04 scores decreased significantly after one week or month of treatment, respectively, and remained stable for six months. Owner's global assessment score was 2.15±1.15 for all the animals in the study. In conclusion, systemic administration of allogeneic cAd-MSCs appeared to be a simple therapy with positive outcome in the remission of clinical signs for AD refractory to conventional medications, for at least six months and with no adverse events.

Macrophagic enhancement in optical coherence tomography imaging by means of superparamagnetic iron oxide nanoparticles.

BACKGROUND: The ability of optical coherence tomography (OCT) to visualise macrophages in vivo in coronary arteries is still controversial. We hypothesise that imaging of macrophages in OCT could be enhanced by means of superparamagnetic nanoparticles. METHODS: We compared the optical backscattering and attenuation of cell pellets containing RAW 264.7 macrophages with those of macrophagic cell pellets labelled with very small superparamagnetic oxydised nanoparticles (VSOP) by means of light intensity analysis in OCT. The labelled macrophages were incubated with VSOP at a concentration of 1 mM Fe, corresponding to intracellular iron concentrations of 8.8 pg/cell. To study the effect of intracellular accumulation on the backscattering, VSOP dilutions without cells were also compared. OCT pullbacks of the PCR tubes containing the cell pellets were obtained and light intensity analysis was performed on raw OCT images in polar view, after normalisation by the backscattering of the PCR tube. The backscattering was estimated by the peak normalised intensity, whilst the attenuation was estimated by the number of pixels between the peak and the normalised intensity 1 (peak-to-one). RESULTS: VSOP-loaded macrophages have higher backscattering than the corresponding unlabelled macrophages (peak normalised intensity 6.30 vs. 3.15) with also slightly higher attenuation (peak-toone 61 vs. 66 pixels). The backscattering of the nanoparticles in suspension was negligible in the light intensity analysis. CONCLUSIONS: VSOP increase significantly the optical backscattering of macrophages in the nearinfrared region, with minimal increase in signal attenuation. This finding enables the enhancement of macrophages in conventional OCT imaging with an easily implementable methodology.

Determination of ELISA reproducibility to detect protein markers in exhaled breath condensate.

Exhaled breath condensate (EBC) is a representative sample from the lungs that may be used to detect different markers, but the reproducibility of these determinations is unknown over time. The aim of this paper is to assess the reproducibility of protein marker determination in EBC using samples collected at two different time points. EBC and blood were collected from 16 healthy subjects, smokers and non-smokers by using the ECoScreen device. EBC was collected on two separate occasions within ten days. Enzyme-linked immunosorbent assay (ELISA) was performed to measure angiogenesis and hypoxia markers. Blood and EBC samples were analyzed by ELISA to detect angiogenesis markers: vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and endostatin. A hypoxia marker, the anhydrase IX, was also determined. Biomarker concentration was higher in plasma samples compared to EBC. bFGF determination was higher in women (39.47 ± 3.914 versus 27.15 ± 3.145; p < 0.05). There were no significant differences among the averages of detection for any of the markers. The Bland-Altman method showed that the average of the differences or biases in EBC for every biomarker was close to zero, indicating a good reproducibility of the measurements. Nevertheless, the VEGF showed wide limits of agreement. EBC is suitable to detect biomarkers by ELISA and the measurements are reproducible over time. Nevertheless, some factors such as sex should be taken into account when analyzing the results.

Enrichment of neural-related genes in human mesenchymal stem cells from neuroblastoma patients.

Neuroblastoma (NB) is one of the most common pediatric solid tumors and, like most human cancers, is characterized by a broad variety of genomic alterations. Although mesenchymal stem cells (MSCs) are known to interact with cancer cells, the relationship between MSCs and metastatic NB cancer cells in bone marrow (BM) is unknown. To obtain genetic evidence about this interaction, we isolated ΒΜ-derived MSCs from children with NB and compared their global expression patterns with MSCs obtained from normal pediatric donors, using the Agilent 44K microarrays. Significance analysis of microarray results with a false discovery rate (FDR) <5% identified 496 differentially expressed genes showing either a 2-fold upregulation or downregulation between both groups of samples. Comparison of gene ontology categories of differentially expressed genes revealed the upregulation of genes categorized as 'neurological system process', 'cell adhesion', 'apoptosis', 'cell surface receptor linked signal transduction', 'intrinsic to membrane' and 'extracellular region'. Among the downregulated genes, several immunology-related terms were the most abundant. These findings provide preliminary genetic evidence of the interaction between MSCs and NB cancer cells in ΒΜ as well as identify relevant biological processes potentially altered in MSCs in response to NB.

Mesenchymal niches of bone marrow in cancer.

Over the last decade, genetic and cell biology studies have indicated that tumour growth is not only determined by malignant cancer cells themselves, but also by the tumour microenvironment. Cells present in the tumour microenvironment include fibroblasts, vascular, smooth muscle, adipocytes, immune cells and mesenchymal stem cells (MSC). The nature of the relationship between MSC and tumour cells appears dual and whether MSC are pro- or anti-tumorigenic is a subject of controversial reports. This review is focused on the role of MSC and bone marrow (BM) niches in cancer.