Resting time after phorbol 12-myristate 13-acetate in THP-1 derived macrophages provides a non-biased model for the study of NLRP3 inflammasome

Background: The activation of NLRP3 inflammasome in macrophages has been proven to play a crucial role in the development of cardiovascular diseases. THP-1 monocytes can be differentiated to macrophages by incubation with phorbol-12-myristate 13-acetate (PMA), providing a suitable model for in vitro studies. However, PMA has been shown to have effects on the levels of IL-1 beta, the main mediator of NLRP3 inflammasome, while the effects on the other mediators of the inflammasome have not been reported before. Methods: THP-1 monocytes were incubated without (THP-1), with 5ng/ml PMA for 48h (PMA48h) or with 5ng/ml PMA for 48h plus 24h in fresh medium (PMArest). Morphological changes and the expression of macrophage surface markers (CD14, CD11b, CD36 and CD204) were evaluated by flow cytometry. Changes in intracellular levels of inflammasome components (NLRP3, ASC, pro-caspase-1, pro-IL1 beta) were analyzed by western blot and release of mature IL-1 beta in cell supernatant was analyzed by ELISA. ASC speck formation was determined by immunofluorescence. Results: After 48h incubation with PMA or subsequent rest in fresh medium, cells became adherent, and the differential expression of CD36, CD11b, CD14 and CD204 compared to THP-1 cells confirmed that PMArest resemble macrophages from a molecular point of view. Changes in the levels were detected in PMA48h group for all the NLRP3-related proteins, with increase of NLRP3 and pro-IL-1 beta and secretion of mature IL-1 beta. In PMArest, no pro-IL-1 beta and lower amounts of mature IL-1 beta were detected. No ASC speck was found in PMA treated groups, but the addition of a second stimulus to PMArest resulted in ASC speck formation, together with IL-1 beta production, confirming the responsiveness of the model. Conclusion: Differentiation of THP-1 with 5ng/ml PMA followed by 24h resting period provides a model that morphologically and molecularly resembles macrophages. However, even at low concentrations, PMA induces production of IL-1 beta. The 24h rest period provides for down-regulation of pro-IL-1 beta in PMArest group, without affecting its ability to respond to a second stimulus through activation of inflammasome

Novel High Resolution Mass Spectrometry Applications for the Study of Lung Diseases

Respiratory Distress Syndrome (RDS) is a respiratory disorder that can affect preterm newborns. It is due to lung immaturity and a deficiency of lung surfactant, a thin lipoprotein layer which allows lung expansion and prevents alveolar collapse during expiration. In these babies, nasal Continuous Positive Airway Pressure (nCPAP) provides non-invasive respiratory support by alveolar recruitment and improves functional residual capacity during spontaneous breathing. In case nCPAP fails, the next step is treatment with exogenous surfactant, which improves gas exchange and survival, reducing the need for mechanical ventilation and then the incidence of chronic lung disease. Although surfactant replacement therapy remains the gold standard for the treatment of RDS, failure rate ranges from 9% to 50%. The possibility of tracing the fate of the administered drug and estimate in vivo the amount of drug reaching the lung could help in improving the therapy and allow the premise to test different delivery systems. RDS is a disorder related to prematurity. Among the factors responsible for premature birth, intrauterine infection (chorioamnionitis) is one of the main causes and it has been reported to have effects on the development of fetal organs. In particular, it has been reported that infants exposed to chorioamnionitis had a reduced risk to develop RDS but an increased risk for chronic lung diseases. Since the function of lung surfactant strictly depends on its composition, a better knowledge on surfactant metabolite and lipid changes would elucidate the effects of the exposition of the foetus to maternal intrauterine infection on RDS. The PhD project focused on 2 objectives: 1. To validate natural abundance stable isotopes approach as a reliable method to quantify the contribution of exogenous surfactant to the alveolar surfactant pool in a rabbit model of RDS treated with a porcine exogenous surfactant; 2. To assess the effect of maternal intrauterine infection on the surfactant composition of newborns affected by RDS by liquid chromatography-mass spectrometry (LC-MS/MS) based metabolomics

Novel High Resolution Mass Spectrometry Applications for the Study of Lung Diseases

Respiratory Distress Syndrome (RDS) is a respiratory disorder that can affect preterm newborns. It is due to lung immaturity and a deficiency of lung surfactant, a thin lipoprotein layer which allows lung expansion and prevents alveolar collapse during expiration. In these babies, nasal Continuous Positive Airway Pressure (nCPAP) provides non-invasive respiratory support by alveolar recruitment and improves functional residual capacity during spontaneous breathing. In case nCPAP fails, the next step is treatment with exogenous surfactant, which improves gas exchange and survival, reducing the need for mechanical ventilation and then the incidence of chronic lung disease. Although surfactant replacement therapy remains the gold standard for the treatment of RDS, failure rate ranges from 9% to 50%. The possibility of tracing the fate of the administered drug and estimate in vivo the amount of drug reaching the lung could help in improving the therapy and allow the premise to test different delivery systems. RDS is a disorder related to prematurity. Among the factors responsible for premature birth, intrauterine infection (chorioamnionitis) is one of the main causes and it has been reported to have effects on the development of fetal organs. In particular, it has been reported that infants exposed to chorioamnionitis had a reduced risk to develop RDS but an increased risk for chronic lung diseases. Since the function of lung surfactant strictly depends on its composition, a better knowledge on surfactant metabolite and lipid changes would elucidate the effects of the exposition of the foetus to maternal intrauterine infection on RDS. The PhD project focused on 2 objectives: 1. To validate natural abundance stable isotopes approach as a reliable method to quantify the contribution of exogenous surfactant to the alveolar surfactant pool in a rabbit model of RDS treated with a porcine exogenous surfactant; 2. To assess the effect of maternal intrauterine infection on the surfactant composition of newborns affected by RDS by liquid chromatography-mass spectrometry (LC-MS/MS) based metabolomics

Effetti della cattività su alcuni parametri ematologici del grifone (Gyps fulvus)

Haematological analysis is an essential field of veterinary medicine that provides inexpensive and reliable support to determinate animal health. The knowledge of how different factors affect the normal mean values of blood parameters is key to understand and improve animal health. In order to investigate how captivity can affect the haematological profile of birds of prey, the erythrocyte count, haemoglobin concentration, haematocrit, mean corpuscular volume, total leukocytes count of 123 griffon vultures (Gyps fulvus) were analysed. The birds were divided into 4 groups according to their life conditions: a control group of free-living griffons, 2 semi-captive groups held in an aviary for 15 and 30 days respectively, representing short-term captivity, and a captive group that had lived in cage for about 2 years. Our results showed that long-term captivity could influence haematocrit value and haemoglobin concentration. Furthermore, discriminant analysis highlighted significant separation between the captive birds on one hand and the control group and the semi-captive birds on the other. Instead, short-term captivity did not seem to affect prominently haemocytometric profile

Evaluation of venous blood gas analysis, blood chemistry and haemocytometric parameters in milk fed veal calves at different periods of livestock cycle

An evaluation of blood chemistry profile in relation to specific stages of livestock cycle can help better understand variations in physiological conditions in order to adjust management systems to animal needs. In addition to basal hematological investigation, the acid-base balance and blood gases are essential tools in evaluating metabolism in calves. The relationship between blood gas parameters, diet and growth should be further investigated. The aim of this study was to evaluate changes in acid-base status, blood gases, serum chemistry and hematological parameters in veal calves at different periods of livestock cycle. One hundred twenty-eight healthy cross breeding calves were enrolled in a farm in North-East Italy. Blood samplings were carried out from the jugular vein on day 1 (t1), 60 (t2) and 150 (t3) after arrival. Blood gas analysis was performed and hematological parameters were evaluated. One-way ANOVA and Tukey-Kramer post-hoc test were performed to assess differences between blood par- ameter values at the different periods. The main differences in blood gas parameter levels during the livestock cycle concerned pH, Base Excess and HCO3 with higher values recorded in t3. Urea, creatinine, gamma-glutamyl transpeptidase and bilirubin mean values were significantly higher in t1 than in t2 and t3. Aspartate aminotransferase increased from t1 to t2 and t3. Alkaline Phosphatase was higher in t2. Fe levels severely dropped in t2 and in t3, and the decrease led to a restrained but significant reduction in haemoglobin values. A correspondent decrease in the other haemocytometric parameters was found

Toll signal transduction pathway in bivalves: Complete cds of intermediate elements and related gene transcription levels in hemocytes of immune stimulated Mytilus galloprovincialis

Based on protein domain structure and organization deduced from mRNA contigs, 15 transcripts of the Toll signaling pathway have been identified in the bivalve, Mytilus galloprovincialis. Identical searches performed on publicly available Mytilus edulis ESTs revealed 11 transcripts, whereas searches performed in genomic and new transcriptome sequences of the Pacific oyster, Crassostrea gigas, identified 21 Toll-related transcripts. The remarkable molecular diversity of TRAF and IKK coding sequences of C. gigas, suggests that the sequence data inferred from Mytilus cDNAs may not be exhaustive. Most of the Toll pathway genes were constitutively and ubiquitously expressed in M. galloprovincialis, although at different levels, and clearly induced after in vivo injection with bacteria. Such over-transcription was more rapid and intense with Gram-negative than with Gram-positive bacteria. Injection of a fungus modulated the transcription of few Toll pathway genes, with the induction levels of TLR/MyD88 complex being always less intense. Purified LPS and \u3b2-glucans had marginal effect whereas peptidoglycans were ineffective. At the moment, we found no evidence of an IMD transcript in bivalves. In conclusion, mussels possess a complete Toll pathway which can be triggered either by Gram-positive or Gram-negative bacteri