Potentiality of Food-Isolated Lentilactobacillus kefiri Strains as Probiotics: State-of-Art and Perspectives.

Lentilactobacillus kefiri is one of the main lactic acid bacteria species in kefir and it was also isolated from other fermented foods. Numerous strains have been isolated and characterized regarding its potential as probiotics for the development of novel functional foods. To our knowledge this is the first review focused on highlighting safety aspects and health beneficial effects reported for L. kefiri strains. Several L. kefiri strains lack of transmissible antibiotic resistance genes, are tolerant to the harsh conditions of the gastrointestinal environment, and could resist different preservation procedures. Moreover, many of the isolated strains have shown antimicrobial activity against pathogens and their toxins, exhibited immunomodulatory activity as well as induced some beneficial effects at metabolic level. Regarding all the scientific evidence, certain L. kefiri strains emerge as excellent candidates to be applied to the development of both food supplements and new fermented foods with health-promoting properties. However, the availability of genomic information is still very limited, so much more work must be done in order to explore the potentiality of L. kefiri as a probiotic and a source of bioactive metabolites.

[Dream content in different sleep disorders: sleep apnoea and hypopnoea syndrome, primary insomnia, idiopathic REM sleep behaviour disorder and narcolepsy type 1]. / Contenido onírico en diferentes trastornos del sueño: síndrome de apnea e hipopnea del sueño, insomnio primario, trastorno de la conducta del sueño REM idiopático y narcolepsia de tipo 1.

AIM: The aim of this study is to determine whether there are any differences in the dream content in different sleep disorders and to describe their characteristics. PATIENTS AND METHODS: We studied four sleep disorders: sleep apnoea and hypopnoea syndrome (SAHS), primary insomnia (PI), idiopathic REM sleep behaviour disorder (IRBD) and narcolepsy type I. Each patient was asked to keep a dream diary for two weeks. The content of the diaries was transcribed and analysed for length, mental content, complexity and threat. The results were compared to establish differences. RESULTS: Eighty-nine patients were studied: 23 with SAHS without continuous positive airway pressure (CPAP) who had the highest number of dreams involving threats (32.5%); 19 with SAHS treated with CPAP who had the highest number of dreams involving objects (64.8%), descriptive elements (38%) and higher complexity (9.5%); 22 with primary insomnia who had the highest number of dreams with threatening events in the social sphere (57.7%); 12 with IRBD who had the highest number of dreams with failures (14%) and lower complexity (71.7%); and 13 with narcolepsy type I who had the highest number of dreams related to activities (84.3%) and threats to life (41.4%) These differences were statistically significant (p <0.05). CONCLUSIONS: Different sleep disorders are associated with different dream contents, which would be translating different underlying neurological processes. These findings should be replicated in studies that analyse more patients and add a control group without sleep disorders.

TITLE: Contenido onírico en diferentes trastornos del sueño: síndrome de apnea e hipopnea del sueño, insomnio primario, trastorno de la conducta del sueño REM idiopático y narcolepsia de tipo 1.Objetivo. Determinar si existen diferencias en el contenido onírico en diferentes trastornos del sueño y describir sus características. Pacientes y métodos. Estudiamos cuatro trastornos del sueño: síndrome de apnea e hipopnea del sueño (SAHS), insomnio primario (IP), trastorno de conducta del sueño REM idiopático (TCSRI) y narcolepsia de tipo 1. Se solicitó a cada paciente que llenara un diario de sus sueños durante dos semanas. El contenido de los diarios fue transcrito y analizado en longitud, contenido mental, complejidad y amenaza. Los resultados se compararon para establecer diferencias. Resultados. Se estudió a 89 pacientes: 23 con SAHS sin presión positiva continua de la vía aérea (CPAP) que tuvieron la mayor cantidad de sueños con participación en amenazas (32,5%); 19 con SAHS tratados con CPAP que tuvieron la mayor cantidad de sueños con objetos (64,8%), elementos descriptivos (38%) y de más alta complejidad (9,5%); 22 con insomnio primario con la mayor cantidad de sueños con eventos amenazantes al ámbito social (57,7%); 12 con TCSRI que tuvieron en sus sueños la más alta cantidad de fracasos (14%) y menor complejidad (71,7%), y 13 con narcolepsia de tipo 1 que tuvieron la mayor cantidad de sueños relacionados con actividades (84,3%) y amenazas hacia la vida (41,4 %). Estas diferencias fueron estadísticamente significativas (p menor de 0,05). Conclusiones. Los distintos trastornos del sueño sí se asocian a contenidos oníricos diferentes que traducirían distintos procesos neurológicos subyacentes. Estos hallazgos deberían replicarse en estudios que analicen más pacientes y añadan un grupo control sin trastornos del sueño.

Cortical gray matter progression in idiopathic REM sleep behavior disorder and its relation to cognitive decline.

BACKGROUND: Idiopathic Rapid eye movement sleep behavior disorder (IRBD) is recognized as the prodromal stage of the alpha-Synucleinopathies. Although some studies have addressed the characterization of brain structure in IRBD, little is known about its progression. OBJECTIVE: The present work aims at further characterizing gray matter progression throughout IRBD relative to normal aging and investigating how these changes are associated with cognitive decline. METHODS: Fourteen patients with polysomnography-confirmed IRBD and 18 age-matched healthy controls (HC) underwent neuropsychological, olfactory, motor, and T1-weighted MRI evaluation at baseline and follow-up. We compared the evolution of cortical thickness (CTh), subcortical volumes, smell, motor and cognitive performance in IRBD and HC after a mean of 1.6 years. FreeSurfer was used for CTh and volumetry preprocessing and analyses. The symmetrized percent of change (SPC) of the CTh was correlated with the SPC of motor and neuropsychological performance. RESULTS: IRBD and HC differed significantly in the cortical thinning progression in regions encompassing bilateral superior parietal and precuneus, the right cuneus, the left occipital pole and lateral orbitofrontal gyri (FWE corrected, p < 0.05). The Visual form discrimination test showed worse progression in the IRBD relative to HC, that was associated with gray matter loss in the right superior parietal and the left precuneus. Increasing motor signs in IRBD were related to cortical thinning mainly involving frontal regions, and late-onset IRBD was associated with cortical thinning involving posterior areas (FWE corrected, p < 0.05). Despite finding olfactory identification deficits in IRBD, results did not show decline over the disease course. CONCLUSION: Progression in IRBD patients is characterized by parieto-occipital and orbitofrontal thinning and visuospatial loss. The cognitive decline in IRBD is associated with degeneration in parietal regions.

Disruption of posterior brain functional connectivity and its relation to cognitive impairment in idiopathic REM sleep behavior disorder.

BACKGROUND: Resting-state functional MRI has been proposed as a new biomarker of prodromal neurodegenerative disorders, but it has been poorly investigated in the idiopathic form of rapid-eye-movement sleep behavior disorder (IRBD), a clinical harbinger of subsequent synucleinopathy. Particularly, a complex-network approach has not been tested to study brain functional connectivity in IRBD patients. OBJECTIVES: The aim of the current work is to characterize resting-state functional connectivity in IRBD patients using a complex-network approach and to determine its possible relation to cognitive impairment. METHOD: Twenty patients with IRBD and 27 matched healthy controls (HC) underwent resting-state functional MRI with a 3T scanner and a comprehensive neuropsychological battery. The functional connectome was studied using threshold-free network-based statistics. Global and local network parameters were calculated based on graph theory and compared between groups. Head motion, age and sex were introduced as covariates in all analyses. RESULTS: IRBD patients showed reduced cortico-cortical functional connectivity strength in comparison with HC in edges located in posterior regions (p <0.05, FWE corrected). This regional pattern was also shown in an independent analysis comprising posterior areas where a decreased connectivity in 51 edges was found, whereas no significant results were detected when an anterior network was considered (p <0.05, FWE corrected). In the posterior network, the left superior parietal lobule had reduced centrality in IRBD. Functional connectivity strength between left inferior temporal lobe and right superior parietal lobule positively correlated with mental processing speed in IRBD (r = .633; p = .003). No significant correlations were found in the HC group. CONCLUSION: Our findings support the presence of disrupted posterior functional brain connectivity of IRBD patients similar to that found in synucleinopathies. Moreover, connectivity reductions in IRBD were associated with lower performance in mental processing speed domain.

Cholinergic denervation in patients with idiopathic rapid eye movement sleep behaviour disorder.

BACKGROUND AND PURPOSE: Cholinergic dysfunction appears to play a role in the cognitive impairment observed in Parkinson's disease and dementia with Lewy bodies. The occurrence of cholinergic dysfunction in the early stages of these conditions, however, has not been investigated. The objective of this study was to investigate cholinergic function in patients with idiopathic rapid eye movement sleep behaviour disorder (iRBD), a disorder recognized to be an early stage of both Parkinson's disease and dementia with Lewy bodies. METHODS: A total of 21 patients with polysomnography-confirmed iRBD with no evidence of parkinsonism and cognitive impairment and 10 controls underwent positron emission tomography (PET) to assess brain acetylcholinesterase levels (11 C-donepezil PET) and nigrostriatal dopaminergic function (18 F-DOPA PET). Clinical examination included the Movement Disorder Society-Unified Parkinson's Disease Rating Scale part III, Mini Mental State Examination and Montreal Cognitive Assessment. RESULTS: The 11 C-donepezil PET was successfully performed in 17 patients with iRBD and nine controls. Compared with controls, patients with iRBD showed a mean 7.65% reduction in neocortical 11 C-donepezil levels (P = 0.005). Bilateral superior temporal cortex, occipital cortex, cingulate cortex and dorsolateral prefrontal cortex showed the most significant reductions at voxel level. CONCLUSION: Reduced neocortical 11 C-donepezil binding in our patients indicates cholinergic denervation and suggests that the projections from the nucleus basalis of Meynert, which supplies cholinergic innervation to the neocortex, are dysfunctional in iRBD. Longitudinal studies will clarify if these changes are predictive of future cognitive impairment in these patients.

Comparing the accuracy and neuroanatomical correlates of the UPSIT-40 and the Sniffin' Sticks test in REM sleep behavior disorder.

BACKGROUND: Olfactory impairment increases the risk of developing neurodegenerative diseases in patients with idiopathic REM sleep behavior disorder (IRBD). Knowing the test properties of distinct olfactory measures could contribute to their selection for clinical or research purposes. OBJECTIVE: To compare the accuracy in distinguishing IRBD patients from controls with the University of Pennsylvania Smell Identification Test (UPSIT-40) and Sniffin' Sticks Extended test, and to assess the gray-matter volume correlates of these tests. METHOD: Twenty-one patients with IRBD and 27 healthy controls were assessed using both olfactory tests. Independent logistic regressions were computed with diagnosis as a dependent variable and olfactory measures as predictive variables. Receiver operating characteristic curves were computed for each olfactory subtest. Diagnostic accuracy for IRBD was calculated according to the resulting optimal cut-off score. Structural MRI data, acquired with a 3T scanner, were analyzed with voxel-based morphometry. RESULTS: Patients differed from controls in all olfactory measures. The Sniffin-Identification correctly classified 89.1% of cases; the UPSIT-40, 85.4%; the Sniffin-Discrimination, 82.6%; the Sniffin-Total, 81.8%; and the Sniffin-Threshold, 77.3%. Respective AUROC, optimal cut-off, sensitivity, and specificity for each test were: 0.902, ≤26, 85.7%, and 85.2% for the UPSIT-40; 0.884, ≤29, 89.5%, and 76.0% for the Sniffin-Total; 0.922, ≤11, 90.5%, and 88.0% for the Sniffin-Identification; 0.739, ≤4, 73.7%, and 76.0% for the Sniffin-Threshold; and 0.838, ≤11, 85.7%, and 76.0% for the Sniffin-Discrimination. UPSIT-40 scores correlated with gray-matter volumes in orbitofrontal regions in anosmic patients. CONCLUSIONS: UPSIT-40 and Sniffin' Identification showed similar discrimination accuracy, but only the UPSIT-40 showed structural correlates (p ≤ .05 FDR-corrected).

A glycoproteomic approach reveals that the S-layer glycoprotein of Lactobacillus kefiri CIDCA 83111 is O- and N-glycosylated.

In Gram-positive bacteria, such as lactic acid bacteria, general glycosylation systems have not been documented so far. The aim of this work was to characterize in detail the glycosylation of the S-layer protein of Lactobacillus kefiri CIDCA 83111. A reductive ß-elimination treatment followed by anion exchange high performance liquid chromatography analysis was useful to characterize the O-glycosidic structures. MALDI-TOF mass spectrometry analysis confirmed the presence of oligosaccharides bearing from 5 to 8 glucose units carrying galacturonic acid. Further nanoHPLC-ESI analysis of the glycopeptides showed two O-glycosylated peptides: the peptide sequence SSASSASSA already identified as a signature glycosylation motif in L. buchneri, substituted on average with eight glucose residues and decorated with galacturonic acid and another O-glycosylated site on peptide 471-476, with a Glc5-8GalA2 structure. As ten characteristic sequons (Asn-X-Ser/Thr) are present in the S-layer amino acid sequence, we performed a PNGase F digestion to release N-linked oligosaccharides. Anion exchange chromatography analysis showed mainly short N-linked chains. NanoHPLC-ESI in the positive and negative ion modes were useful to determine two different peptides substituted with short N-glycan structures. To our knowledge, this is the first description of the structure of N-glycans in S-layer glycoproteins from Lactobacillus species. SIGNIFICANCE: A detailed characterization of protein glycosylation is essential to establish the basis for understanding and investigating its biological role. It is known that S-layer proteins from kefir-isolated L. kefiri strains are involved in the interaction of bacterial cells with yeasts present in kefir grains and are also capable to antagonize the adverse effects of different enteric pathogens. Therefore, characterization of type and site of glycosidic chains in this protein may help to understand these important properties. Furthermore, this is the first description of N-glycosidic chains in S-layer glycoprotein from Lactobacillus spp.

Morphology and properties of neutralized chitosan-cellulose nanocrystals biocomposite films.

Chitosan/cellulose nanocrystals (CH-CN) films were obtained by casting of dispersions, and treated with NaOH for neutralization proposes. The composition of films was varied from 1 to 10wt.% of CN. Changes in the morphology of the systems were correlated with the different properties studied. FTIR revealed the presence of a weak interaction between the polymer matrix and nanofiller, confirmed by a slightly increase in thermal stability. SEM images suggested that incorporating CN amounts higher than 3wt.% generates phase-segregated systems, and SAXS showed that CH avoid the typical organization of CN at concentrations below 5wt.%. Improved performance against water was obtained in composite materials comparing to the pure polymer matrix, as well as CH and CH-CN films did not show antibacterial activity demonstrating that remnant acetic acid, when no neutralization step is done, plays an important role in this property.

Kefir-isolated bacteria and yeasts inhibit Shigella flexneri invasion and modulate pro-inflammatory response on intestinal epithelial cells.

The aim of this work was to evaluate the ability of a kefir-isolated microbial mixture containing three bacterial and two yeast strains (MM) to protect intestinal epithelial cells against Shigella flexneri invasion, as well as to analyse the effect on pro-inflammatory response elicited by this pathogen. A significant decrease in S. flexneri strain 72 invasion was observed on both HT-29 and Caco-2 cells pre-incubated with MM. Pre-incubation with the individual strains Saccharomyces cerevisiae CIDCA 8112 or Lactococcus lactis subsp. lactis CIDCA 8221 also reduced the internalisation of S. flexneri into HT-29 cells although in a lesser extent than MM. Interestingly, Lactobacillus plantarum CIDCA 83114 exerted a protective effect on the invasion of Caco-2 and HT-29 cells by S. flexneri. Regarding the pro-inflammatory response on HT-29 cells, S. flexneri infection induced a significant activation of the expression of interleukin 8 (IL-8), chemokine (C-C motif) ligand 20 (CCL20) and tumour necrosis factor alpha (TNF-α) encoding genes (P<0.05), whereas incubation of cells with MM did not induce the expression of any of the mediators assessed. Interestingly, pre-incubation of HT-29 monolayer with MM produced an inhibition of S. flexneri-induced IL-8, CCL20 and TNF-α mRNA expression. In order to gain insight on the effect of MM (or the individual strains) on this pro-inflammatory response, a series of experiments using a HT-29-NF-κB-hrGFP reporter system were performed. Pre-incubation of HT-29-NF-κB-hrGFP cells with MM significantly dampened Shigella-induced activation. Our results showed that the contribution of yeast strain Kluyveromyces marxianus CIDCA 8154 seems to be crucial in the observed effect. In conclusion, results presented in this study demonstrate that pre-treatment with a microbial mixture containing bacteria and yeasts isolated from kefir, resulted in inhibition of S. flexneri internalisation into human intestinal epithelial cells, along with the inhibition of the signalling via NF-κB that in turn led to the attenuation of the inflammatory response.

Role of S-layer proteins in bacteria.

S-layers are paracrystalline bidimensional arrays of proteins or glycoproteins that overlay the cell surface of several genus and species of bacteria and archaea. As the outermost layer of several genus and species of microorganisms, S-layer proteins (SLP) are in direct contact with bacterial environment and thus may be involved in many of their surface properties, including adherence to various substrates, mucins and eukaryotic cells, aggregation and coaggregation with yeasts and other bacteria. In addition, SLP have been reported to be responsible for the bacterial protection against detrimental environmental conditions and to play an important role in surface recognition or as carriers of virulence factors. In this mini-review, we bring together the latest evidences about functional and mechanical properties of bacterial SLP from two different perspectives: (A) their role on bacterial adherence to different substrates and surfaces, and (B) their role as mechanical barriers in bacterial harmful environments.

Impact of kefir derived Lactobacillus kefiri on the mucosal immune response and gut microbiota.

The evaluation of the impact of probiotics on host health could help to understand how they can be used in the prevention of diseases. On the basis of our previous studies and in vitro assays on PBMC and Caco-2 ccl20:luc reporter system presented in this work, the strain Lactobacillus kefiri CIDCA 8348 was selected and administrated to healthy Swiss mice daily for 21 days. The probiotic treatment increased IgA in feces and reduced expression of proinflammatory mediators in Peyer Patches and mesenteric lymph nodes, where it also increased IL-10. In ileum IL-10, CXCL-1 and mucin 6 genes were upregulated; meanwhile in colon mucin 4 was induced whereas IFN-γ, GM-CSF, and IL-1ß genes were downregulated. Moreover, ileum and colon explants showed the anti-inflammatory effect of L. kefiri since the LPS-induced increment of IL-6 and GM-CSF levels in control mice was significantly attenuated in L. kefiri treated mice. Regarding fecal microbiota, DGGE profiles allowed differentiation of experimental groups in two separated clusters. Quantitative PCR analysis of different bacterial groups revealed only significant changes in Lactobacillus population. In conclusion, L. kefiri is a good candidate to be used in gut inflammatory disorders.

Immunization with crude antigens plus aluminium hydroxide protects cattle from Fasciola hepatica infection.

The ability of total homogenate (TH) of Fasciola hepatica conjugated with aluminium hydroxide (alum) or Freund's complete adjuvant (FCA) to protect cattle against experimental fasciolosis was evaluated. Compared with the infected group, the immunized animals with alum-TH and FCA-TH presented a significant reduction in fluke burden (85.9% and 96.8%, respectively), a higher percentage of short-sized worms, a marked reduction in the released eggs in faeces (89% and 57%, respectively), as well as an increased production of specific antibodies before infection. The alum-TH immunized group also showed a significant increase in the antigen-specific proliferation of peripheral blood mononuclear cells (PBMC) as early as 4 weeks before infection. Although both immunized groups (alum-TH and FCA-TH) were able to develop an efficient protective immune response to metacercarial challenge, an earlier PBMC response, lower hepatic damage and less effect on weight gain were found in alum-immunized animals. Therefore, alum is a good candidate for future immunization against bovine fasciolosis.

[Standard values of frontal cognitive functioning tests for those over 60 years of age]. / Valores normativos de tests de funcion cognitiva frontal para la poblacion mayor de 60 anos.

INTRODUCTION: There are some neuropsychological test with a good sensibility for cognitive disorders affecting frontal lobe damage. AIM. To provide normative data for test that assess frontal cognitive function in population over 60 years old. SUBJECTS AND METHODS: 110 neurologically healthy volunteers over 60 years old, recruited from different primary care centers from Barcelona, participated in the study and were assessed by the Trail Making Test-A (TTA), the verbal fluency test FAS and the similarities test. RESULTS: The mean age of the sample was 71.5 years-old (DE: 6.7). The mean years of education was 8.6 (DE: 4.4). 60% of the subjects were women. The mean score obtained in TTA was 64.2 (DE: 26.0), 24.5 (DE: 11.8) in FAS and 12.5 (DE: 5.2) in similarities test. Years of education result a high significant factor for predicting the performance in all the frontal test. Gender only influenced the performance in TTA. Age did not influence the performance in any of these test. CONCLUSIONS: These findings support the idea that years of education is the most influencing factor in the performance in frontal cognitive test in normal aged population.

[Perception Digital Test (PDT) for the assessment of incipient visual disorder in initial Alzheimer's disease]. / Test Digital de Percepción (TDP) para la valoración de trastornos perceptivos incipientes en la enfermedad de Alzheimer inicial.

INTRODUCTION: Visual perception disorders are frequent in degenerative diseases with posterior cerebral lobe involvement, such as Alzheimer's disease (AD). The main aim of this study is to create a Perception Digital Test (PDT) based on the treatment of digital photographs, and to present normative values in the elderly Spanish population. The second objective is to validate the test in a group of patients with mild AD, stage 4 in the Global Deterioration Scale (GDS) (AD-4). SUBJECTS: The group were constituted by 86 healthy controls (CTR) and 27 AD patients. METHOD: VPT, which was formed by 15 items with four options of answer, was administered to all subjects. RESULTS: Mean time of administration of PDT in CTR was 85 seconds (standard desviation: 33). Mean result obtained for each groups was significantly different (CTR: 13.5 [1.4]; AD-4: 10.6 (2.9); p = 0.05). Age did not significantly influence PDT performance in any of the groups. However, years of education influenced significantly the CTR group score. CONCLUSION: PDT is an easy, quick and sensitive test to assess visual perception disorders in initial AD.

Antithrombotic effect of a new nitric oxide donor (LA419) on experimental thrombogenesis.

BACKGROUND: The ability of nitrous compounds to donate nitric oxide (NO), an agent with vasodilating and inhibitory effects on platelet function, has been considered a useful pharmacologic strategy for cardiovascular complications. The purpose of this study was to investigate the effects of a new NO donor, LA419, on platelet interaction in an ex vivo model with human blood circulating through collagen-rich surfaces. MATERIALS AND METHODS: Platelet adhesive and cohesive function were analyzed by morphometric procedures after perfusion techniques. Treated blood was exposed to thrombogenic surfaces and platelet interactions were morphometrically evaluated. RESULTS: All the concentrations studied of LA419 (10 microM, 20 microM and 100 microM) reduced overall platelet interaction with a collagen surface (27.19 +/- 4.72; 25.52 +/- 3.52; and 23.44 +/- 3.01, P < 0.05, respectively, vs. 32.31 +/- 1.61% in the control). The antithrombotic effect was confirmed by results in cross-sectional studies performed in arterial vessels exposed to circulating blood. Values of thrombus and covered surface at 20 microM LA419 were, respectively, 13.67 +/- 4.97% and 19.01 +/- 5.89%; respect to controls 34.80 +/- 5.29% and 37.93 +/- 5.34% (P < 0.05). Moreover, LA419 reduced significantly thrombus area (88.45 +/- 21.97 microm(2); P < 0.05) with respect to controls (168.45 +/- 21.97 microm(2)) and thrombus height, from an average of 10.27 +/- 1.05 microm in nontreated blood to 7.16 +/- 0.6 microm in treated samples (P < 0.05). CONCLUSION: From the present data we can conclude that LA419 possesses a strong antiplatelet action, as demonstrated by its ability to significantly inhibit the interaction of platelet with highly thrombogenic collagen surfaces.

Evaluation of effects of rofecoxib on platelet function in an in vitro model of thrombosis with circulating human blood.

BACKGROUND: Cyclooxygenase (COX)-2-selective non-steroidal anti-inflammatory drugs have been used for anti-inflammatory therapy. However, it has also been described that they may increase risk of cardiovascular events. OBJECTIVES: To study the effects of COX2 inhibitor rofecoxib on platelet function using in vitro tests. Results were compared with those obtained in a parallel experiment with acetyl salicylic acid (ASA). METHODS: Studies of platelet aggregation, using different agonists, were performed by a turbidimetric method. Adhesive and cohesive function of platelets were analyzed by perfusion techniques, treated blood was exposed to thrombogenic surfaces and platelet interaction was morphometrically evaluated. RESULTS: Twenty-five micro M of rofecoxib induced a prolonged lag time and a reduction in the percentage of aggregation when arachidonic acid, ADP or collagen were used as agonists. In perfusion studies with parallel chamber rofecoxib 50 microM and ASA 500 microM reduced overall platelet interaction with the collagen surface (17.4 +/- 3.7, P < 0.05; vs. 32.1 +/- 2.6%P < 0.05 and 17.9 +/- 2.4, vs. 31.9 +/- 3.24, P < 0.05, respectively). In studies performed on annular chambers, 25 micro M of rofecoxib reduced platelet interaction; values of the thrombus and covered surface were 17.4 +/- 4.5%; P < 0.05 and 21.1 +/- 4.1%; P < 0.05, respectively, vs. 30.4 +/- 7.5% and 33.5 +/- 6.5 in the control. ASA did also impair thrombus formation but differences did not reach the levels of statistical significance. Moreover, rofecoxib but not ASA reduced significantly thrombus height and thrombus area (7.4 +/- 0.5 microM; P < 0.005 and 96.0 +/- 21.2 microM(2); P < 0.05 vs. control 11.2 +/- 0.9 microM and 220.0 +/- 47.7 microM(2), respectively). CONCLUSION: We conclude that under our experimental conditions, rofecoxib diminished platelet aggregation induced by different agonists and inhibited platelet-mediated thrombogenesis in an in vitro model of thrombosis.

[Uremic media affects hemostatic properties of human endothelial cells in culture and increases the production of von Willebrand factor]. / El medio urémico altera las propiedades hemostáticas de células endoteliales humanas en cultivo y aumenta la producción de factor von Willebrand.

We have investigated the ability of serum from uremic patients to modify the thrombogenic properties of the endothelium. The effect of the uremic media on the morphology of ECs, and their resistance to flow was analyzed. The reactivity of the extracellular matrix (ECM) generated by ECs towards normal platelets was evaluated in a parallel-plate perfusion chamber. Exposure of ECs to uremic media resulted in abnormal morphology and signs of accelerated growth. Detachment of ECs exposed to circulating blood was increased when cells had been grown with media supplemented with uremic serum (22% vs 13%). Platelet deposition and formation of aggregates were significantly elevated on ECMs generated in the presence of uremic media (40.23 +/- 6.43% vs 25.42 +/- 2.69%, p < 0.05, n = 5). Immunocytochemical methods detected an enhanced expression of von Willebrand factor antigen on uremic ECMs (uremic 17.1 +/- 4.2% vs control 13.57 +/- 3.98%, p < 0.05) and its mRNA expression in endothelial cells (uremic 213.24 +/- 6.13 vs control 200.77 +/- 7.52, p < 0.05). These results suggest that uremic medium alters endothelial function and impairs the antithrombotic functions of cultured endothelial cells. This effect may contribute to the increased cardiovascular and thrombotic risk reported in ESRD patients.

Uremic medium disturbs the hemostatic balance of cultured human endothelial cells.

We have investigated the ability of serum from uremic patients to modify the thrombogenic properties of the endothelium. The effects of uremic medium on the morphology of endothelial cells (ECs), and their resistance to flow was analyzed. The influence of uremic media on the reactivity of the extracellular matrix (ECM) generated by ECs towards normal platelets was evaluated in a parallel-plate perfusion chamber. Exposure of ECs to uremic medium resulted in abnormal cell morphology and signs of an accelerated growth. Detachment of ECs exposed to circulating blood was increased when cells had been grown with media supplemented with uremic serum (21% vs. 14% non exposed). Platelet deposition was significantly elevated on ECMs generated in the presence of uremic media (uremicECMs) (p<0.01 vs. control studies). Effects of uremic serum were not observed at short incubation periods (5 h) but were evident after 24 or 72 h of incubation. Northern blot analysis revealed increased expression of tissue factor (TF) mRNA in ECs exposed to uremic conditions. Immunocytochemical methods detected an augmented expression of TF antigen on uremic ECMs. Incubation of ECMs with an antibody to human tissue factor prevented the increase in platelet deposition observed in uremic ECMs, suggesting that the presence of TF in ECM could be responsible for the enhanced platelet deposition. Results from our study indicate that uremic medium impairs the antithrombotic functions of cultured endothelial cells.