Evaluation of the effectiveness of a new cryopreservation system based on a two-compartment vial for the cryopreservation of cell therapy products.

BACKGROUND AIMS: Successful cell cryopreservation and banking remain a major challenge for the manufacture of cell therapy products, particularly in relation to providing a hermetic, sterile cryovial that ensures optimal viability and stability post-thaw while minimizing exposure to toxic cryoprotective agents, typically dimethyl sulfoxide (Me2SO). METHODS: In the present study, the authors evaluated the effectiveness and functionality of Limbo technology (Cellulis S.L., Santoña, Spain). This system provides a hermetic vial with two compartments (one for adding cells with the cryoprotective agent solution and the other for the diluent solution) and an automated defrosting device. Limbo technology (Cellulis S.L.) allows reduction of the final amount of Me2SO, sidestepping washing and dilution steps and favoring standardization. The study was performed in several Good Manufacturing Practice laboratories manufacturing diverse cell therapy products (human mesenchymal stromal cells, hematopoietic progenitor cells, leukapheresis products, fibroblasts and induced pluripotent stem cells). Laboratories compared Limbo technology (Cellulis S.L.) with their standard cryopreservation procedure, analyzing cell recovery, viability, phenotype and functionality. RESULTS: Limbo technology (Cellulis S.L.) maintained the viability and functionality of most of the cell products and preserved sterility while reducing the final concentration of Me2SO. CONCLUSIONS: Results showed that use of Limbo technology (Cellulis S.L.) offers an overall safe alternative for cell banking and direct infusion of cryopreserved cell products into patients.

Probiotic Bifidobacterium breve prevents DOCA-salt hypertension.

Many probiotics that affect gut microbial ecology have been shown to produce beneficial effects on renin-angiotensin-dependent rodent models and human hypertension. We hypothesized that Bifidobacterium breve CECT7263 (BFM) would attenuate hypertension in deoxycorticosterone acetate (DOCA)-salt rats, a renin-independent model of hypertension. Rats were randomly divided into five groups: control, DOCA-salt, treated DOCA-salt-BFM, treated DOCA-salt-butyrate, and treated DOCA-salt-acetate, for 5 weeks. BFM prevented the increase in systolic blood pressure, cardiac weight, and renal damage induced by DOCA-salt. BFM increased acetate-producing bacterial population and gut acetate levels, improved colonic integrity, normalized endotoxemia, plasma trimethylamine (TMA) levels, and restored the Th17 and Treg content in mesenteric lymph nodes and aorta. Furthermore, BFM improved nitric oxide-dependent vasorelaxation induced by acetylcholine in aortic rings and reduced NADPH oxidase activity in DOCA-salt animals. These protective effects were mimicked by acetate, but not by butyrate supplementation. These data demonstrate that BFM induces changes in gut microbiota linked with attenuation of endothelial dysfunction and increase in blood pressure in this low-renin form of hypertension. These beneficial effects seem to be mediated by increased acetate and reduced TMA production by gut microbiota, thus, improving gut integrity and restoring Th17/Tregs polarization and endotoxemia.

Brain MR patterns in inherited disorders of monoamine neurotransmitters: An analysis of 70 patients.

Inherited monoamine neurotransmitter disorders (iMNDs) are rare disorders with clinical manifestations ranging from mild infantile hypotonia, movement disorders to early infantile severe encephalopathy. Neuroimaging has been reported as non-specific. We systematically analyzed brain MRIs in order to characterize and better understand neuroimaging changes and to re-evaluate the diagnostic role of brain MRI in iMNDs. 81 MRIs of 70 patients (0.1-52.9 years, 39 patients with tetrahydrobiopterin deficiencies, 31 with primary disorders of monoamine metabolism) were retrospectively analyzed and clinical records reviewed. 33/70 patients had MRI changes, most commonly atrophy (n = 24). Eight patients, six with dihydropteridine reductase deficiency (DHPR), had a common pattern of bilateral parieto-occipital and to a lesser extent frontal and/or cerebellar changes in arterial watershed zones. Two patients imaged after acute severe encephalopathy had signs of profound hypoxic-ischemic injury and a combination of deep gray matter and watershed injury (aromatic l-amino acid decarboxylase (AADCD), tyrosine hydroxylase deficiency (THD)). Four patients had myelination delay (AADCD; THD); two had changes characteristic of post-infantile onset neuronal disease (AADCD, monoamine oxidase A deficiency), and nine T2-hyperintensity of central tegmental tracts. iMNDs are associated with MRI patterns consistent with chronic effects of a neuronal disorder and signs of repetitive injury to cerebral and cerebellar watershed areas, in particular in DHPRD. These will be helpful in the (neuroradiological) differential diagnosis of children with unknown disorders and monitoring of iMNDs. We hypothesize that deficiency of catecholamines and/or tetrahydrobiopterin increase the incidence of and the CNS susceptibility to vascular dysfunction.

Lactobacillus fermentum CECT5716: a novel alternative for the prevention of vascular disorders in a mouse model of systemic lupus erythematosus.

The aim of the present study was to examine whether the immune-modulatory bacteria Lactobacillus fermentum CECT5716 (LC40) ameliorates disease activity and cardiovascular complications in a female mouse model of lupus. Eighteen-week-old NZBWF1 [systemic lupus erythematosus (SLE)] and NZW/LacJ (control) mice were treated with vehicle or LC40 (5 × 108 colony-forming units/d) for 15 wk. LC40 treatment reduced lupus disease activity, blood pressure, cardiac and renal hypertrophy, and splenomegaly in SLE mice. LC40 reduced the elevated T, B, regulatory T cells (Treg), and T helper (Th)-1 cells in mesenteric lymph nodes of lupus mice. LC40 lowered the higher plasma concentration of proinflammatory cytokines observed in lupus mice. Aortas from SLE mice showed reduced endothelium-dependent vasodilator responses to acetylcholine. Endothelial dysfunction found in SLE is related to an increase of both NADPH oxidase-driven superoxide production and eNOS phosphorylation at the inhibitory Thr495. These activities returned to normal values after a treatment with LC40. Probiotic administration to SLE mice reduced plasma LPS levels, which might be related to an improvement of the gut barrier integrity. LC40 treatment increases the Bifidobacterium count in gut microbiota of SLE mice. In conclusion, our findings identify the gut microbiota manipulation with LC40 as an alternative approach to the prevention of SLE and its associated vascular damage.-Toral, M., Robles-Vera, I., Romero, M., de la Visitación, N., Sánchez, M., O'Valle, F., Rodriguez-Nogales, A., Gálvez, J., Duarte, J., Jiménez, R. Lactobacillus fermentum CECT5716: a novel alternative for the prevention of vascular disorders in a mouse model of systemic lupus erythematosus.

Celia's encephalopathy and c.974dupG in BSCL2 gene: a hidden change in a known variant.

Celia's encephalopathy (progressive encephalopathy with/without lipodystrophy (PELD)) is a childhood neurodegenerative disorder with a fatal prognosis before the age of 10, due to the variant c.985C>T in the BSCL2 gene that causes a cryptic splicing site leading to skipping of exon 7. For years, different authors have reported cases of congenital generalized lipodystrophy due to the variant c.974dupG in BSCL2 associated with neurological manifestations of variable severity, although some of them clearly superimposable to PELD. To identify the molecular mechanisms responsible for these neurological alterations in two patients with c.974dupG. Clinical characterization, biochemistry, and neuroimaging studies of two girls carrying this variant. In silico analysis, PCR amplification, and BSCL2 cDNA sequencing. BSCL2-201 transcript expression, which lacks exon 7, by qPCR in fibroblasts from the index case, from a healthy child as a control and from two patients with PELD, and in leukocytes from the index case and her parents. One with a severe encephalopathy including a picture of intellectual deficiency, severe language impairment, myoclonic epilepsy, and lipodystrophy as described in PELD, dying at 9 years and 9 months of age. The other 2-year-old patient showed incipient signs of neurological involvement. In silico and cDNA sequencing studies showed that variant c.974dupG gives rise to skipping of exon 7. The expression of BSCL2-201 in fibroblasts was significantly higher in the index case than in the healthy child, although less than in the case with homozygous PELD due to c.985C>T variant. The expression of this transcript was approximately half in the healthy carrier parents of this patient. The c.974dupG variant leads to the skipping of exon 7 of the BSCL2 gene and is responsible for a variant of Celia's encephalopathy, with variable phenotypic expression.

Antihypertensive effects of peroxisome proliferator-activated receptor-ß/δ activation.

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily of ligand-activated transcription factors, which is composed of three members encoded by distinct genes: PPARα, PPARß/δ, and PPARγ. The biological actions of PPARα and PPARγ and their potential as a cardiovascular therapeutic target have been extensively reviewed, whereas the biological actions of PPARß/δ and its effectiveness as a therapeutic target in the treatment of hypertension remain less investigated. Preclinical studies suggest that pharmacological PPARß/δ activation induces antihypertensive effects in direct [spontaneously hypertensive rat (SHR), ANG II, and DOCA-salt] and indirect (dyslipemic and gestational) models of hypertension, associated with end-organ damage protection. This review summarizes mechanistic insights into the antihypertensive effects of PPARß/δ activators, including molecular and functional mechanisms. Pharmacological PPARß/δ activation induces genomic actions including the increase of regulators of G protein-coupled signaling (RGS), acute nongenomic vasodilator effects, as well as the ability to improve the endothelial dysfunction, reduce vascular inflammation, vasoconstrictor responses, and sympathetic outflow from central nervous system. Evidence from clinical trials is also examined. These preclinical and clinical outcomes of PPARß/δ ligands may provide a basis for the development of therapies in combating hypertension.

Antihypertensive Effects of Probiotics.

PURPOSE OF REVIEW: The present review focuses in the hypertension-associated changes in the microbiota and the current insights regarding the impact of probiotics on blood pressure in animal models and in human hypertensive patients. RECENT FINDINGS: Gut dysbiosis in hypertension is characterized by (i) the gut microbioma that is less diverse and less rich with an increased Firmicutes/Bacteroidetes ratio and (ii) a decrease in acetate- and butyrate-producing bacteria and an increase in lactate-producing bacterial populations. The meta-analysis of the human studies supports that supplementation with probiotics reduces blood pressure. The mechanism of this antihypertensive effect of probiotics and its protective effect on endothelial function has not been fully elucidated. Further investigations are needed to clarify if the effects of probiotic bacteria result from the changes in the gut microbiota and its metabolic by-products; the restoration of the gut barrier function; and the effects on endotoxemia, inflammation, and renal sympathetic nerve activity.

Vascular and Central Activation of Peroxisome Proliferator-Activated Receptor-ß Attenuates Angiotensin II-Induced Hypertension: Role of RGS-5.

Activation of peroxisome proliferator-activated receptor-ß/δ (PPARß) lowers blood pressure in genetic and mineralocorticoid-induced hypertension. Regulator of G-protein-coupled receptor signaling 5 (RGS5) protein, which interferes in angiotensin II (AngII) signaling, is a target gene to PPARß The aim of the present study was to examine whether PPARß activation in resistance arteries and brain tissues prevents the elevated blood pressure in AngII-induced hypertension and evaluate the role of RGS5 in this effect. C57BL/6J male mice were divided into five groups (control mice, PPARß agonist [4-[[[2-[3-Fluoro-4-(trifluoromethyl)phenyl]-4-methyl-5-thiazolyl]methyl]thio]-2-methylphenoxy]acetic acid (GW0742)-treated mice AngII-infused mice, GW0742-treated AngII-infused mice, and AngII-infused mice treated with GW0742 plus PPARß antagonist 3-[[[2-Methoxy-4-(phenylamino)phenyl]amino]sulfonyl]-2-thiophenecarboxylic acid methyl ester (GSK0660)) and were followed for 3 weeks. GW0742 prevented the increase in both arterial blood pressure and plasma noradrenaline levels and the higher reduction of blood pressure after ganglionic blockade, whereas it reduced the mesenteric arterial remodeling and the hyper-responsiveness to vasoconstrictors (AngII and endothelin-1) in AngII-infused mice. These effects were accompanied by an inhibition of NADPH oxidase expression and activity in the brain. Gene expression profiling revealed a marked loss of brainstem and vascular RGS5 in AngII-infused mice, which was restored by GW0742. GW0742-induced effects were abolished by GSK0660. Small interfering RNA targeting RGS5 caused augmented contractile response to AngII in resistance mesenteric arteries and blunted the inhibitory effect of GW0742 on this response. In conclusion, GW0742 exerted antihypertensive effects, restoring sympathetic tone and vascular structure and function in AngII-infused mice by PPARß activation in brain and vessels inhibiting AngII signaling as a result of RGS5 upregulation.

Larger aggregates of mutant seipin in Celia's Encephalopathy, a new protein misfolding neurodegenerative disease.

Celia's Encephalopathy (MIM #615924) is a recently discovered fatal neurodegenerative syndrome associated with a new BSCL2 mutation (c.985C>T) that results in an aberrant isoform of seipin (Celia seipin). This mutation is lethal in both homozygosity and compounded heterozygosity with a lipodystrophic BSCL2 mutation, resulting in a progressive encephalopathy with fatal outcomes at ages 6-8. Strikingly, heterozygous carriers are asymptomatic, conflicting with the gain of toxic function attributed to this mutation. Here we report new key insights about the molecular pathogenic mechanism of this new syndrome. Intranuclear inclusions containing mutant seipin were found in brain tissue from a homozygous patient suggesting a pathogenic mechanism similar to other neurodegenerative diseases featuring brain accumulation of aggregated, misfolded proteins. Sucrose gradient distribution showed that mutant seipin forms much larger aggregates as compared with wild type (wt) seipin, indicating an impaired oligomerization. On the other hand, the interaction between wt and Celia seipin confirmed by coimmunoprecipitation (CoIP) assays, together with the identification of mixed oligomers in sucrose gradient fractionation experiments can explain the lack of symptoms in heterozygous carriers. We propose that the increased aggregation and subsequent impaired oligomerization of Celia seipin leads to cell death. In heterozygous carriers, wt seipin might prevent the damage caused by mutant seipin through its sequestration into harmless mixed oligomers.

Carnitine palmitoyltransferase-1 up-regulation by PPAR-ß/δ prevents lipid-induced endothelial dysfunction.

Fatty acids cause endothelial dysfunction involving increased ROS (reactive oxygen species) and reduced NO (nitric oxide) bioavailability. We show that in MAECs (mouse aortic endothelial cells), the PPARß/δ (peroxisome- proliferator-activated receptor ß/δ) agonist GW0742 prevented the decreased A23187-stimulated NO production, phosphorylation of eNOS (endothelial nitric oxide synthase) at Ser1177 and increased intracellular ROS levels caused by exposure to palmitate in vitro. The impaired endothelium-dependent relaxation to acetylcholine in mouse aorta induced by palmitate was restored by GW0742. In vivo, GW0742 treatment prevented the reduced aortic relaxation, phosphorylation of eNOS at Ser1177, and increased ROS production and NADPH oxidase in mice fed on a high-fat diet. The PPARß/δ antagonist GSK0660 abolished all of these protective effects induced by GW0742. This agonist enhanced the expression of CPT (carnitine palmitoyltransferase)-1. The effects of GW0742 on acetylcholine- induced relaxation in aorta and on NO and ROS production in MAECs exposed to palmitate were abolished by the CPT-1 inhibitor etomoxir or by siRNA targeting CPT-1. GW0742 also inhibited the increase in DAG (diacylglycerol), PKCα/ßII (protein kinase Cα/ßII) activation, and phosphorylation of eNOS at Thr495 induced by palmitate in MAECs, which were abolished by etomoxir. In conclusion, PPARß/δ activation restored the lipid-induced endothelial dysfunction by up-regulation of CPT-1, thus reducing DAG accumulation and the subsequent PKC-mediated ROS production and eNOS inhibition.

Prospects of breeding high-quality rice using post-genomic tools.

KEY MESSAGE: The holistic understanding derived from integrating grain quality and sensory research outcomes in breeding high-quality rice in the light of post-genomics resources has been synthesized. Acceptance of new rice genotypes by producers and consumers hinges not only on their potential for higher yield but recent emphasis has also been on premium-value genotypes that have the ability to satisfy consumer preferences for grain quality. This review article provides insights into how to link grain quality attributes and sensory perception to support breeding superior rice varieties. Recent advances in quality profiling and omics technologies have provided efficient approaches to identify the key genes and biochemical markers involved in rice quality traits. Emphasis has been given to the upcoming area of holistic understanding of grain quality and attributes derived from sensory evaluation to leverage integrative gene discovery strategies that enable breeding programs to efficiently tap the huge genetic diversity in rice for novel genes that enhance rice food quality.

Array CGH detection of a novel cryptic deletion at 3q13 in a complex chromosome rearrangement.

Complex chromosome rearrangements (CCRs) are extremely rare in humans. About 20% of the apparently balanced CCRs have an abnormal phenotype and the degree of severity correlates with a higher number of breakpoints. Several studies using FISH and microarray technologies have shown that deletions in the breakpoints are common although duplications, insertions and inversions have also been detected. We report a patient with two simultaneous reciprocal translocations, t(3;4) and t(2;14;18), involving five chromosomes and six breakpoints. He showed dysmorphic features, preaxial polydactyly in the left hand, brachydactyly, postnatal growth retardation and developmental delay. The rearrangement was characterized by FISH analysis which detected an interstitial segment from chromosome 14 inserted in the derivative chromosome 2, and by whole genome array which revealed an interstitial deletion of approximately 4.5 Mb at the breakpoint site on chromosome 3. To our knowledge this microdeletion has not been previously reported and includes ~12 genes. The haploinsufficiency of one or several of these genes is likely to have contributed to the clinical phenotype of the patient.

The probiotic Lactobacillus coryniformis CECT5711 reduces the vascular pro-oxidant and pro-inflammatory status in obese mice.

Obesity is associated with intestine dysbiosis and is characterized by a low-grade inflammatory status, which affects vascular function. In the present study, we evaluated the effects of a probiotic with immunomodulatory properties, Lactobacillus coryniformis CECT5711, in obese mice fed on an HFD (high-fat diet). The probiotic treatment was given for 12 weeks, and it did not affect the weight evolution, although it reduced basal glycaemia and insulin resistance. L. coryniformis administration to HFD-induced obese mice induced marked changes in microbiota composition and reduced the metabolic endotoxaemia as it decreased the LPS (lipopolysaccharide) plasma level, which was associated with a significant improvement in gut barrier disruption. Furthermore, it lowered TNFα (tumour necrosis factor α) expression in liver, improving the inflammatory status, and thus the glucose metabolism. Additionally, the probiotic reversed the endothelial dysfunction observed in obese mice when endothelium- and NO (nitric oxide)-dependent vasodilatation induced by acetylcholine in aortic rings was studied. It also restored the increased vessel superoxide levels observed in obese mice, by reducing NADPH oxidase activity and increasing antioxidant enzymes. Moreover, chronic probiotic administration for 2 weeks also improved endothelial dysfunction and vascular oxidative stress induced by in vivo administration of LPS in control mice fed on a standard chow diet. The results of the present study demonstrate an endothelial-protective effect of L. coryniformis CECT5711 in obese mice by increasing NO bioavailability, suggesting the therapeutic potential of this gut microbiota manipulation to prevent vasculopathy in obesity.

The flavonoid quercetin induces acute vasodilator effects in healthy volunteers: correlation with beta-glucuronidase activity.

UNLABELLED: Quercetin exerts vasodilator, antiplatelet and antiproliferative effects and reduces blood pressure, oxidative status and end-organ damage in hypertensive humans and animal models. We hypothesized that oral quercetin might induce vasodilator effects in humans and that they might be related to the deconjugation of quercetin-3-O-glucuronide (Q3GA). DESIGN: double blind, randomized, placebo-controlled trial. Fifteen healthy volunteers (26±5 years, 6 female) were given a capsule containing placebo, 200 or 400mg of quercetin in random order in three consecutive weeks. At 2h a dose-dependent increase in Q3GA was observed in plasma (∼0.4 and 1µM for 200 and 400mg, respectively) with minor levels of quercetin and isorhamnetin. No changes were observed in blood pressure. At 5h quercetin induced and increase in brachial arterial diameter that correlated with the product of the levels of Q3GA by the plasma glucuronidase activity. There was an increase in urinary levels of glutathione but there was no increase in nitrites plus nitrates. Quercetin and isorhamnetin also relaxed human umbilical arteries in vitro while Q3GA was without effect. In conclusions, quercetin exerts acute vasodilator effects in vivo in normotensive, normocholesterolemic human subjects. These results are consistent with the effects being due to the deconjugation of the metabolite Q3GA.

A new seipin-associated neurodegenerative syndrome.

BACKGROUND: Seipin/BSCL2 mutations can cause type 2 congenital generalised lipodystrophy (BSCL) or dominant motor neurone diseases. Type 2 BSCL is frequently associated with some degree of intellectual impairment, but not to fatal neurodegeneration. In order to unveil the aetiology and pathogenetic mechanisms of a new neurodegenerative syndrome associated with a novel BSCL2 mutation, six children, four of them showing the BSCL features, were studied. METHODS: Mutational and splicing analyses of BSCL2 were performed. The brain of two of these children was examined postmortem. Relative expression of BSCL2 transcripts was analysed by real-time reverse transcription-polymerase chain reaction (RT-PCR) in different tissues of the index case and controls. Overexpressed mutated seipin in HeLa cells was analysed by immunofluorescence and western blotting. RESULTS: Two patients carried a novel homozygous c.985C>T mutation, which appeared in the other four patients in compound heterozygosity. Splicing analysis showed that the c.985C>T mutation causes an aberrant splicing site leading to skipping of exon 7. Expression of exon 7-skipping transcripts was very high with respect to that of the non-skipped transcripts in all the analysed tissues of the index case. Neuropathological studies showed severe neurone loss, astrogliosis and intranuclear ubiquitin(+) aggregates in neurones from multiple cortical regions and in the caudate nucleus. CONCLUSIONS: Our results suggest that exon 7 skipping in the BSCL2 gene due to the c.985C>T mutation is responsible for a novel early onset, fatal neurodegenerative syndrome involving cerebral cortex and basal ganglia.

Differing contributions of the gut microbiota to the blood pressure lowering effects induced by first-line antihypertensive drugs.

BACKGROUND AND PURPOSE: This study analyses whether first-line antihypertensive drugs ameliorate the dysbiosis state in hypertension, and to test if this modification contributes to their blood pressure (BP) lowering properties in a genetic model of neurogenic hypertension. EXPERIMENTAL APPROACH: Twenty-week-old male Wistar Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) were untreated or treated with captopril, amlodipine or hydrochlorothiazide. A faecal microbiota transplantation (FMT) experiment was also performed by gavage of faecal content from donor SHR-treated groups to SHR recipients for 3 weeks. KEY RESULTS: Faeces from SHR showed gut dysbiosis, characterized by lower acetate- and higher lactate-producing bacteria and lower strict anaerobic bacteria. All three drugs increased the anaerobic bacteria proportion, captopril and amlodipine restored the proportion of acetate-producing bacterial populations to WKY levels, whereas hydrochlorothiazide decreased butyrate-producing bacteria. Captopril and amlodipine decreased gut pathology and permeability and attenuated sympathetic drive in the gut. Both drugs decreased neuroinflammation and oxidative stress in the hypothalamic paraventricular nuclei. Hydrochlorothiazide was unable to reduce neuroinflammation, gut sympathetic tone and gut integrity. FMT from SHR-amlodipine to SHR decreased BP, ameliorated aortic endothelium-dependent relaxation to acetylcholine, lowered NADPH oxidase activity, aortic Th17 infiltration and reduced neuroinflammation, whereas FMT from SHR-hydrochlorothiazide did not have these effects. CONCLUSIONS AND IMPLICATIONS: First-line antihypertensive drugs induced different modifications of gut integrity and gut dysbiosis in SHR, which result in no contribution of microbiota in the BP lowering effects of hydrochlorothiazide, whereas the vasculo-protective effect induced by amlodipine involves gut microbiota reshaping and gut-immune system communication.

Evaluation of the MDR Direct Flow Chip Kit for the Detection of Multiple Antimicrobial Resistance Determinants.

The objective of this study was to evaluate the accuracy of the MDR Direct Flow Chip Kit for the detection of antimicrobial resistance (AMR) determinants from bacterial colonies. Ninety-two clinical isolates with known AMR determinants genotypically characterized were used. The MDR Direct Flow Chip Kit is a microarray-based assay that included 55 AMR determinants for beta-lactams (23), quinolones (13), aminoglycosides (5), macrolides (5), sulfonamides (3), colistin (2), vancomycin (2), chloramphenicol (1), and linezolid (1). The MDR Direct Flow Chip Kit correctly detects 52 of 53 AMR determinants tested. The cfr gene (linezolid resistance) was not detected. The global sensibility, specificity, positive predictive value, and the negative predictive value calculated were 98%, 100%, 100%, and 97%. The Cohen's Kappa coefficient calculated was 0.97 [95% Confidence Interval (0.90-1.03)]. In conclusion, the MDR Direct Flow Chip is an accurate assay for the detection of multiple AMR determinants in one simple reaction.

Mineralocorticoid receptor blockade improved gut microbiota dysbiosis by reducing gut sympathetic tone in spontaneously hypertensive rats.

Microbiota has a crucial role in the host blood pressure (BP) regulation. The present study analyzes whether the mineralocorticoid receptor antagonist spironolactone ameliorates the dysbiosic state in a genetic model of neurogenic hypertension. Twenty-week-old male Wistar Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) were randomly allocated into three groups: untreated WKY, untreated SHR, and SHR treated with spironolactone for 5 weeks. Spironolactone restored the Firmicutes/Bacteroidetes proportion, and acetate-producing bacteria populations to WKY levels. Spironolactone reduced the percentage of intestinal aerobic bacteria. The amelioration of gut dysbiosis was linked to a reduction in the gut pathology, an enhanced colonic integrity, a reduced gut permeability and an attenuated sympathetic drive in the gut. Spironolactone was unable to reduce neuroinflammation and oxidative stress in the paraventricular nuclei in the hypothalamus. Spironolactone reduced the higher Th17 cells proportion in mesenteric lymph nodes and Th17 infiltration in aorta, improved aortic endothelial function and reduced systolic BP. This study demonstrates for the first time that spironolactone reduces gut dysbiosis in SHR. This effect could be related to its capability to improve gut integrity and pathology due to reduced sympathetic drive in the gut.

Bioengineered tissue and cell therapy products are efficiently cryopreserved with pathogen-inactivated human platelet lysate-based solutions.

BACKGROUND: There remains much interest in improving cryopreservation techniques for advanced therapy medicinal products (ATMPs). Recently, human platelet lysate (hPL) has emerged as a promising candidate to replace fetal bovine serum (FBS) as a xeno-free culture supplement for the expansion of human cell therapy products. Whether hPL can also substitute for FBS in cryopreservation procedures remains poorly studied. Here, we evaluated several cryoprotective formulations based on a proprietary hPL for the cryopreservation of bioengineered tissues and cell therapy products. METHODS: We tested different xenogeneic-free, pathogen-inactivated hPL (ihPL)- and non-inactivated-based formulations for cryopreserving bioengineered tissue (cellularized nanostructured fibrin agarose hydrogels (NFAHs)) and common cell therapy products including bone marrow-derived mesenchymal stromal cells (BM-MSCs), human dermal fibroblasts (FBs) and neural stem cells (NSCs). To assess the tissue and cellular properties post-thaw of NFAHs, we analyzed their cell viability, identity and structural and biomechanical properties. Also, we evaluated cell viability, recovery and identity post-thaw in cryopreserved cells. Further properties like immunomodulation, apoptosis and cell proliferation were assessed in certain cell types. Additionally, we examined the stability of the formulated solutions. The formulations are under a bidding process with MD Bioproducts (Zurich, Switzerland) and are proprietary. RESULTS: Amongst the tissue-specific solutions, Ti5 (low-DMSO and ihPL-based) preserved the viability and the phenotype of embedded cells in NFAHs and preserved the matrix integrity and biomechanical properties similar to those of the standard cryopreservation solution (70% DMEM + 20% FBS + 10% DMSO). All solutions were stable at - 20 °C for at least 3 months. Regarding cell-specific solutions, CeA maintained the viability of all cell types > 80%, preserved the immunomodulatory properties of BM-MSCs and promoted good recovery post-thaw. Besides, both tested solutions were stable at - 20 °C for 18 months. Finally, we established that there is a 3-h window in which thawed NFAHs and FBs maintain optimum viability immersed in the formulated solutions and at least 2 h for BM-MSCs. CONCLUSIONS: Our results show that pathogen-inactivated solutions Ti5 allocated for bioengineered tissues and CeA allocated for cells are efficient and safe candidates to cryopreserve ATMPs and offer a xenogeneic-free and low-DMSO alternative to commercially available cryoprotective solutions.