Low-density lipoproteins (LDL) are circulating inhibitors of the pro-coagulant activity of tissue factor

Las lipoproteínas de baja densidad (LDL) se cuentan entre los inhibidores circulantes de la actividad procoagulante del factor tisular. El objetivo del trabajo fue explorar el comportamiento electroforético de las proteínas involucradas y la cinética de inhibición que permitieran desarrollar un ensayo cuantitativo in vitro capaz de poner en evidencia dicha capacidad. El ensayo cinético consistió en incubar tromboplastina cálcica comercial a 37 ºC con volúmenes crecientes de LDL aislada por precipitación selectiva redisuelta; se realizó a distintos tiempos el tiempo de protrombina en una etapa; se calcularon las actividades respectivas y el porcentaje de inhibición de la coagulabilidad respecto a un blanco. Los resultados muestran inhibición creciente hasta los 60 min en el intervalo entre 0,013 y 0,026 nmol de apoB, parámetros que definen las condiciones seleccionadas del ensayo que aplicado sobre 30 muestras de personas normolipémicas de ambos sexos (edades entre 30 y 70 años) presentó una media +/- SEM de 2.512 +/- 101,4 expresadas en porcentaje de inhibición de la coagulabilidad / nmol de apoB, hallando diferencia significativa en la condición diabética. El comportamiento electroforético, evaluado a tiempo cero mostró dos bandas pre-beta y beta, atribuibles a la tromboplastina y a la LDL respectivamente; en tanto que luego de incubar 60 min se observó una única banda intensificada de migración pre-beta que se podría atribuir a la presencia del complejo LDL - factor tisular. Son necesarios estudios clínicos que permitan evaluar los alcances de aplicación de los ensayos propuestos.Low-density lipoproteins (LDL) are circulating inhibitors of the pro-coagulant activity of tissue factor. The objective of the present work was to explore the electrophoretic mobility of the involved proteins and the kinetic of pro - coagulant activity inhibition, that allow us to propose a quantitative in vitro assay of potential clinical use. For the kinetic assay, commercial calcic thromboplastine was incubated with increasing volumes of redisolved - selectively precipitated LDL at 37 ºC. Then the prothrombine time in one step was made; the inhibitory activity and percentage of inhibition against a blank were calculated. The results show an increasing inhibition of procoagulant activity until 60 min between 0.013 – 0.026 nmol apoB. The conditions for the assay of 30 samples of normolipemic individuals (ages between 30 – 70 years old) were selected on the basis of these results. The assay of % coagulation inhibition / nmol apoB showed signficative differences for control group versus diabetic one. The protein electrophoresis showed two bands in pre-beta and beta positions corresponding to thromboplastine and LDL respectively at time zero. After 60 min of incubation one intense band corresponding to LDL – tissue factor complex can be observed. This work proposes a simple assay for the evaluation of LDL role in haemostatic control in pathological conditions, although further clinical assays are still required.Facultad de Ciencias Exacta

Biological variations in a long-term ex situ cultivation: a Mediterranean demosponge as model system

The demosponge Tethya citrina Sarà & Melone was subjected to long-term aquarium farming, considering that several species belonging to this genus are a rich source of useful bioactive metabolites. The rearing method in the present research was mainly based on applying parameters taken from the current literature. The sponge's biological activity was assessed in terms of survival and growth over time; status of filtering apparatus; protein banding profiles. Farmed sponges showed a high survival rate and marked reduction in size. In addition, they were frequently affected by massive production of asexual buds. Histological and ultrastructural observations showed a gradual disorganization of the sponge choanocyte chambers, with a drastic reduction in choanocytes and a progressive increase in spherulous cells. Comparative electrophoretic analysis of the protein profiles of wild and reared specimens showed differences in protein composition and abundance between the tested groups. Such data are consistent with the increase in spherulous cells whose content was markedly electron-dense, a feature stressing the proteinaceous nature of these inclusions. Experimental tank-rearing of Tethya provided promising responses in terms of high survival rate of the species and easy reproduction, though the rearing protocol used led to profound morpho-functional changes in the sponge. This confirms the inadequate state of knowledge on farming techniques for this taxon, while also highlighting potential applications in biotechnology of ex situ breeding techniques, in order to modify the sponges' biological responses

A simple method to assess the oxidative susceptibility of low density lipoproteins

Background: Oxidative modification of low density lipoproteins (LDL) is recognized as one of the major processes involved in atherogenesis. The in vitro standardized measurement of LDL oxidative susceptibility could thus be of clinical significance. The aim of the present study was to establish a method which would allow the evaluation of oxidative susceptibility of LDL in the general clinical laboratory. Results: LDL was isolated from human plasma by selective precipitation with amphipathic polymers. The ability of LDL to form peroxides was assessed by measuring thiobarbituric acid reactive substances (TBARS) after incubation with Cu2+ and H2O2. Reaction kinetics showed a three-phase pattern (latency, propagation and decomposition phases) which allowed us to select 150 min as the time point to stop the incubation by cooling and EDTA addition. The mixture Cu 2+/H2O2 yielded more lipoperoxides than each one on its own at the same time end-point. Induced peroxidation was measured in normal subjects and in type 2 diabetic patients. In the control group, results were 21.7 ± 1.5 nmol MDA/mg LDL protein, while in the diabetic group results were significantly increased (39.0 ± 3.0 nmol MDA/mg LDL protein; p < 0.001). Conclusion: a simple and useful method is presented for the routine determination of LDL susceptibility to peroxidation in a clinical laboratory.Facultad de Ciencias Exacta

Is the Salmonella contamination of swine carcasses at slaughter related to the Salmonella load in caecum?

The aim of this study was to examine the relationship between the load of Salmonella spp. in caeca and the carcass contamination in an Italian slaughterhouse. The sampling scheme was designed to be representative of the pigs slaughtered in a day and to estimate a 12% prevalence of pigs highly contaminated by Salmonella spp. (HCP, cecal load ≥3log). Environmental swabs were taken before slaughter. Cecal contents and carcass swabs were collected from the same pig. Salmonella MPN were estimated according to ISO6579- 2:2012/A1 and ISO7218:2007/E. The overall Salmonella prevalence were 34.64% and 7.19% for ceca and carcasses respectively, with S. Derby and S. 4,[5],12:i:- being the prevalent serotypes. The HCP prevalence was 11.44%. 7/59 environmental swabs tested positive; when the same serotype was isolated from the environment and from carcasses, the samples were excluded from further analysis. Statistical analysis was performed to investigate the relationship between Salmonella spp. loads in the cecum and contamination of the carcass of the same pig and the prevalence of HCP and the contamination of carcasses on the same day. For this purpose, the days were classified as “high prevalence days” depending on the proportion of caeca resulted positive (≥36%) and as “high load” days depending on the prevalence of HCP (≥10%). A correlation between the contamination of carcasses and the cecal Salmonella loads of the same animal was found (Spearman’s correlation coefficient: 0.2254; p-value=0.0001). No correlation was found between the contamination of carcasses and the categorization of the day of sampling as “high prevalence day”. Conversely, a correlation was found between the contamination of carcasses and the “high load” category of the sampling day (Wilcoxon test, p=0.0011). Notably, not the prevalence of pigs carrying Salmonella spp. but the prevalence of highly contaminated pigs was shown to be related to the contamination of carcasses

In vitro glycation of low-density lipoproteins and atherogenicity in cell culture

La hiperglucemia sostenida incrementa la glicación de proteínas. En particular, las modificaciones en las lipoproteínas de baja densidad (LDL) aumentan su potencial aterogénico. En este trabajo se comparan las modificaciones producidas por la glicación in vitro de LDL aisladas por dos métodos: precipitación selectiva (PS) y ultracentrifugación (UC). Para ello, se determinó el incremento de fructosamina, el consumo de los grupos ε-amino de lisina, guanidinio de arginina y la disminución de residuos de triptofano. Para todos los analitos, los resultados cinéticos indicaron diferencias significativas con relación al basal (p<0,05), coincidentes para ambos métodos en el tiempo de aparición y en el porcentaje de variación. La aterogenicidad de las LDL glicadas separadas por PS fue estudiada en cultivos de macrófagos RAW 264.7 evaluando la formación de células espumosas y cuantificando la incorporación de LDL por tinción de los depósitos lipídicos con Oil Red. Los resultados indican que la captación de LDL modificadas aumentó con el tiempo de incubación, siendo mayor la aterogenicidad de las LDL glicadas respecto de las nativas (p<0,001, 1 h a 37 °C). El procedimiento de PS seleccionado, accesible al laboratorio bioquímico clínico, permite evaluar las modificaciones por glicación que sufren las LDL en pacientes diabéticos.Long-term hyperglycemia increases protein glycation. In particular, modifications in the low-density lipoproteins (LDL) increase their atherogenic potential. In this study, the modifications caused by in vitro glycation of LDL separated by two methods: selective precipitation (SP) and ultracentrifugation (UC) were compared. Increase fructosamine level, decrease of ε-amino group of lysine, guanidinio of arginine and triptophan fluorescence were determined. Results showed significant differences vs. basal (p<0.05) for all the tested parameters, with coincidence for the two separation methods both in time and grade of modifications. The atherogenicity of glycated LDL separated by SP was studied in macrophages RAW 264.7 in culture, through the formation of foam cells and the quantification of the dye taken up by the cellular storage lipids. Results show that the uptake of modified LDL by macrophages increased with the time of incubation, being the atherogenicity of glycated LDL greater than native LDL (p<0.001, 1 h at 37 °C). The selected SP procedure, within the facilities of routine biochemical laboratory, enables the evaluation of the modifications caused by glycation in the LDL of diabetic patients.Facultad de Ciencias Exacta

Modification of low-density lipoproteins in diabetic patients

En los pacientes diabéticos, las lipoproteínas presentan frecuentemente cambios cualitativos y cuantitativos en su composición y varios pasos del metabolismo están alterados. Estas anormalidades contribuyen al incremento del riesgo de enfermedad cardiovascular y se la conoce como “dislipemia aterogénica”. En este trabajo se estudiaron en una población de pacientes diabéticos las modificaciones fisicoquímicas por glicación de sus lipoproteínas de baja densidad (LDL), los resultados se compararon con una población control y con ensayos de glicación in vitro. Las LDL se aislaron por precipitación selectiva y las modificaciones se evaluaron por el incremento de fructosamina, el consumo de los residuos e-amino de lisina, guanidinio de arginina y la disminución de la fluorescencia del grupo indol del triptofano. Los procedimientos seleccionados resultan accesibles al laboratorio clínico. Los valores medios para todos los analitos medidos fueron significativamente diferentes de los de la población control (p<0,0001); de la comparación del ensayo in vitro pudo deducirse que las alteraciones de los residuos de arginina serían un marcador temprano de las modificaciones por glicación, en tanto que las producidas en los residuos de lisina y triptofano representarían indicadores de las alteraciones de mediano plazo. Vale considerar la utilidad de evaluar simultáneamente en una misma molécula, indicadores de control glucémico tanto de corto como de mediano plazo, teniendo en cuenta su relevancia en la fisiopatología de la ateroesclerosis.In diabetic patients, lipoproteins usually show qualitative and quantitative changes in their composition and as a consequence, several metabolic pathways are altered. These alterations contribute to an increase in the risk of cardiovascular disease, also known as “atherogenic dyslipidemia”. The present workstudied the physicochemical modifications by glycation of low density lipoproteins (LDL) in a population of diabetic patients. The results were compared with a control population and with the results obtained from an in vitro glycation assay. LDL were isolated byselective precipitation and the modifications were assessed by the increase in fructosamine level, the decrease of e-amino group of lysine, guanidinio of arginine and indol fluorescence of tryptophan residues. The select methods are available for clinical laboratories. The mean valúes for all the measured analytes were signi- ficantly different from those obtained for the control population (p&lt; 0.0001). Taking into account the results of the in vitro kinetics assays, it can be assumed that the modifications of the arginine residues would be an early glycation marker while the changes in the lysine and triptophan residues may be considered middle term alterations. It is worth remarking the usefulness of evaluating, early and middle term indicators ofglycaemic control simultaneously in the same molecule. These findings are relevant for the pathophysiology of athero- genesis.Facultad de Ciencias Exacta

Reproductive cycle, number of parities and faecal Salmonella spp. excretion in sows: a longitudinal study

The aim of the study was to evaluate the fecal excretion of Salmonella spp in sows with different number of parties, in distinct periods of the reproductive cycle. Two integrated farms in central Italy have been included in the study. The target populations were primaparous and multiparous (1-5 and more than 5 parities) sows, tested in different periods of the reprouductive cycle: 14 days before parturition (pre-partum), 2-5 days (post-partum), 20 days after parturition (post-partum 2) and gestation (24-31 days post-partum)

Impact of chronic obstructive pulmonary disease on short-term outcome in patients with ST-elevation myocardial infarction during COVID-19 pandemic: insights from the international multicenter ISACS-STEMI registry

Background: Chronic obstructive pulmonary disease (COPD) is projected to become the third cause of mortality worldwide. COPD shares several pathophysiological mechanisms with cardiovascular disease, especially atherosclerosis. However, no definite answers are available on the prognostic role of COPD in the setting of ST elevation myocardial infarction (STEMI), especially during COVID-19 pandemic, among patients undergoing primary angioplasty, that is therefore the aim of the current study. Methods: In the ISACS-STEMI COVID-19 registry we included retrospectively patients with STEMI treated with primary percutaneous coronary intervention (PCI) between March and June of 2019 and 2020 from 109 high-volume primary PCI centers in 4 continents. Results: A total of 15,686 patients were included in this analysis. Of them, 810 (5.2%) subjects had a COPD diagnosis. They were more often elderly and with a more pronounced cardiovascular risk profile. No preminent procedural dissimilarities were noticed except for a lower proportion of dual antiplatelet therapy at discharge among COPD patients (98.9% vs. 98.1%, P = 0.038). With regards to short-term fatal outcomes, both in-hospital and 30-days mortality occurred more frequently among COPD patients, similarly in pre-COVID-19 and COVID-19 era. However, after adjustment for main baseline differences, COPD did not result as independent predictor for in-hospital death (adjusted OR [95% CI] = 0.913[0.658–1.266], P = 0.585) nor for 30-days mortality (adjusted OR [95% CI] = 0.850 [0.620–1.164], P = 0.310). No significant differences were detected in terms of SARS-CoV-2 positivity between the two groups. Conclusion: This is one of the largest studies investigating characteristics and outcome of COPD patients with STEMI undergoing primary angioplasty, especially during COVID pandemic. COPD was associated with significantly higher rates of in-hospital and 30-days mortality. However, this association disappeared after adjustment for baseline characteristics. Furthermore, COPD did not significantly affect SARS-CoV-2 positivity. Trial registration number: NCT 04412655 (2nd June 2020)

Gender Difference in the Effects of COVID-19 Pandemic on Mechanical Reperfusion and 30-Day Mortality for STEMI: Results of the ISACS-STEMI COVID-19 Registry

Background. Several reports have demonstrated the impact of the COVID-19 pandemic on the management and outcome of patients with ST-segment elevation myocardial infarction (STEMI). The aim of the current analysis is to investigate the potential gender difference in the effects of the COVID-19 pandemic on mechanical reperfusion and 30-day mortality for STEMI patients within the ISACS-STEMI COVID-19 Registry. Methods. This retrospective multicenter registry was performed in high-volume primary percutaneous coronary intervention (PPCI) centers on four continents and included STEMI patients undergoing PPCIs in March–June 2019 and 2020. Patients were divided according to gender. The main outcomes were the incidence and timing of the PPCI, (ischemia time ≥ 12 h and door-to-balloon ≥ 30 min) and in-hospital or 30-day mortality. Results. We included 16683 STEMI patients undergoing PPCIs in 109 centers. In 2020 during the pandemic, there was a significant reduction in PPCIs compared to 2019 (IRR 0.843 (95% CI: 0.825–0.861, p < 0.0001). We did not find a significant gender difference in the effects of the COVID-19 pandemic on the numbers of STEMI patients, which were similarly reduced from 2019 to 2020 in both groups, or in the mortality rates. Compared to prepandemia, 30-day mortality was significantly higher during the pandemic period among female (12.1% vs. 8.7%; adjusted HR [95% CI] = 1.66 [1.31–2.11], p < 0.001) but not male patients (5.8% vs. 6.7%; adjusted HR [95% CI] = 1.14 [0.96–1.34], p = 0.12). Conclusions. The COVID-19 pandemic had a significant impact on the treatment of patients with STEMI, with a 16% reduction in PPCI procedures similarly observed in both genders. Furthermore, we observed significantly increased in-hospital and 30-day mortality rates during the pandemic only among females. Trial registration number: NCT 04412655