Investigate the efficacy of size exclusion chromatography for the isolation of extracellular vesicles from C. elegans.

Isolation of Extracellular Vesicles (EVs) has been done extensively in the past using ultracentrifugation, a recent shift has been observed towards precipitation, and exosome isolation kits. These methods often co-elute contaminants of similar size and density which makes their detection and downstream applications quite challenging. As well as the EV yield is also compromised in some methodologies due to aggregate formation. In recent reports, size-exclusion chromatography (SEC) is replacing density gradient-based ultracentrifugation as the gold standard of exosome isolation. It outperforms in yield, purity and does not account for any physical damage to the EVs. We have standardized the methodology for an efficient pure yield of homogenous exosomes of size even smaller than 75 nm in Caenorhabditis elegans homogenate. The paper entails the application and optimization of EV isolation by SEC based on previous studies by optimizing bed size and type of sepharose column employed. We propose that this method is economically feasible in comparison with currently available approaches. A comparative study was conducted to investigate the performance of CL-6B in relation to CL-2B and further, this was combined with ultracentrifugation for higher efficacy. The methodology could be introduced in a clinical setting due to its therapeutic potential and scope. The eluted EVs were studied by flow cytometry, nanotracking and characterized for size and morphology.

Vascular Extracellular Vesicles Indicate Severe Hepatopulmonary Syndrome in Cirrhosis.

BACKGROUND: Hepatopulmonary syndrome (HPS) is a pulmonary vasculature complication in the setting of liver disease that is characterized by pathological vasodilation resulting in arterial oxygenation defects. We investigated the role of extracellular vesicles (EV) in cirrhosis patients with HPS, as well as the functional effect of EV administration in a common bile duct ligation (CBDL) HPS mouse model. METHODS: A total of 113 cirrhosis patients were studied: 42 (Gr. A) with HPS and 71 (Gr. B) without HPS, as well as 22 healthy controls. Plasma levels of EV associated with endothelial cells, epithelial cells, and hepatocytes were measured. The cytokine cargoes were estimated using ELISA. The effect of EV administered intranasally in the CBDL mouse model was investigated for its functional effect in vascular remodeling and inflammation. RESULTS: We found endothelial cells (EC) associated EV (EC-EV) were elevated in cirrhosis patients with and without HPS (p < 0.001) than controls. EC-EV levels were higher in HPS patients (p = 0.004) than in those without HPS. The epithelial cell EVs were significantly high in cirrhosis patients than controls (p < 0.001) but no changes found in patients with HPS than without. There was a progressive increase in EC-EV levels from mild to severe intrapulmonary shunting in HPS patients (p = 0.02 mild vs. severe), and we were able to predict severe HPS with an AUROC of 0.85; p < 0.001. An inverse correlation of EC-EVs was found with hemoglobin (r = -0.24; p = 0.031) and PaO2 (r = 0.690; p = 0.01) and a direct correlation with MELD (r = 0.32; p = 0.014). Further, both TNF-α (p = 0.001) and IL-1ß (p = 0.021) as cargo levels were significantly elevated inside the EVs of HPS patients than without HPS. Interestingly, upon administration of intranasal EVs, there was a significant decrease in Evans blue accumulation and lung wet-dry ratio (p = 0.042; 0.038). A significant reduction was also noticed in inflammation and cholestasis. CONCLUSION: High levels of plasma EC-EV levels were found in patients with HPS with elevated pro-inflammatory cytokine cargoes. EC-EVs were indicative of severe HPS condition. In the CBDL HPS model, we were able to prove the beneficial effects of improving vascular tone, inflammation, and liver pathogenesis.

Hepatopulmonary syndrome is associated with low sphingosine-1-phosphate levels and can be ameliorated by the functional agonist fingolimod.

BACKGROUND & AIMS: Hepatopulmonary syndrome (HPS) is characterised by a defect in arterial oxygenation induced by pulmonary vascular dilatation in patients with liver disease. Fingolimod, a sphingosine-1-phosphate (S1P) receptor modulator, suppresses vasodilation by reducing nitric oxide (NO) production. We investigated the role of S1P in patients with HPS and the role of fingolimod as a therapeutic option in an experimental model of HPS. METHODS: Patients with cirrhosis with HPS (n = 44) and without HPS (n = 89) and 25 healthy controls were studied. Plasma levels of S1P, NO, and markers of systemic inflammation were studied. In a murine model of common bile duct ligation (CBDL), variations in pulmonary vasculature, arterial oxygenation, liver fibrosis, and inflammation were estimated before and after administration of S1P and fingolimod. RESULTS: Log of plasma S1P levels was significantly lower in patients with HPS than in those without HPS (3.1 ± 1.4 vs. 4.6 ± 0.2; p <0.001) and more so in severe intrapulmonary shunting than in mild and moderate intrapulmonary shunting (p <0.001). Plasma tumour necrosis factor-α (76.5 [30.3-91.6] vs. 52.9 [25.2-82.8]; p = 0.02) and NO (152.9 ± 41.2 vs. 79.2 ± 29.2; p = 0.001) levels were higher in patients with HPS than in those without HPS. An increase in Th17 (p <0.001) and T regulatory cells (p <0.001) was observed; the latter inversely correlated with plasma S1P levels. In the CBDL HPS model, fingolimod restored pulmonary vascular injury by increasing the arterial blood gas exchange and reducing systemic and pulmonary inflammation, resulting in improved survival (p = 0.02). Compared with vehicle treatment, fingolimod reduced portal pressure (p <0.05) and hepatic fibrosis and improved hepatocyte proliferation. It also induced apoptotic death in hepatic stellate cells and reduced collagen formation. CONCLUSIONS: Plasma S1P levels are low in patients with HPS and even more so in severe cases. Fingolimod, by improving pulmonary vascular tone and oxygenation, improves survival in a murine CBDL HPS model. IMPACT AND IMPLICATIONS: A low level of plasma sphingosine-1-phosphate (S1P) is associated with severe pulmonary vascular shunting, and hence, it can serve as a marker of disease severity in patients with hepatopulmonary syndrome (HPS). Fingolimod, a functional agonist of S1P, reduces hepatic inflammation, improves vascular tone, and thus retards the progression of fibrosis in a preclinical animal model of HPS. Fingolimod is being proposed as a potential novel therapy for management of patients with HPS.

CEACAM-1 Induced CSF3-receptor Downregulation in Bone Marrow Associated With Refractory Neutropenia in Advanced Cirrhosis.

BACKGROUND AND AIMS: Cirrhosis patients exhibit cytopenia, and, at times refractory neutropenia to granulocyte colony-stimulating factor (G-CSF), which acts through the CSF3-receptor (CSF3R), and changes in CSF3R can affect the response. We conducted this study to assess the CSF3R status and its relevance in cirrhotic patients. METHODS: Cirrhotic patients (n=127) and controls (n=26) with clinically indicated bone marrow (BM) examination were studied. BM assessment was done by qRT-PCR and immunohistochemistry (IHC) for CSF3R. Circulating G-CSF, CSF3R, and carcinoembryonic antigen cell adhesion molecule-1 (CEACAM1) were measured. BM hematopoietic precursor cells and their alterations were examined by flow cytometry. The findings were validated in liver cirrhosis patients who received G-CSF for severe neutropenia. RESULTS: The mean age was 48.6±13.4 years, and 80.3% were men. Circulatory CSF3R reduction was noted with the advancement of cirrhosis, and confirmed by qRT-PCR and IHC in BM. CSF3R decline was related to decreased hematopoietic stem cells (HSCs) and downregulation of CSF3R in the remaining HSCs. Cocultures confirmed that CEACAM1 led to CSF3R downregulation in BM cells by possible lysosomal degradation. Baseline low peripheral blood-(PB)-CSF3R also predisposed development of infections on follow-up. Decreased CSF3R was also associated with nonresponse to exogenous G-CSF treatment of neutropenia. CONCLUSIONS: Advanced liver cirrhosis was associated with low CSF3R and high CEACAM1 levels in the BM and circulation, making patients prone to infection and inadequate response to exogenous G-CSF.

Circulating extracellular vesicles induce monocyte dysfunction and are associated with sepsis and high mortality in cirrhosis.

BACKGROUND: Sepsis is common in cirrhosis and is often a result of immune dysregulation. Specific stimuli and pathways of inter-cellular communications between immune cells in cirrhosis and sepsis are incompletely understood. Immune cell-derived extracellular vesicles (EV) were studied to understand mechanisms of sepsis in cirrhosis. METHODS: Immune cell-derived EV were measured in cirrhosis patients [Child-Turcotte-Pugh (Child) score A, n = 15; B n = 16; C n = 43 and Child-C with sepsis (n = 38)], and healthy controls (HC, n = 11). In vitro and in vivo functional relevance of EV in cirrhosis and associated sepsis was investigated. RESULTS: Monocyte, neutrophil and hematopoietic stem cells associated EV progressively increased with higher Child score (P < .001)and correlated with liver disease severity indices (r2  > 0.3, P < .001), which further increased in Child C sepsis than without sepsis(P < .001); monocyte EV showing the highest association with disease stage [P = .013; Odds ratio-4.14(1.34-12.42)]. A threshold level of monocyte EV of 53/µl predicted mortality in patients of Child C with sepsis [Odds ratio-6.2 (2.4-15.9), AUROC = 0.76, P < .01]. In vitro EV from cirrhotic with sepsis compared without sepsis, induced mobilization arrest in healthy monocytes within 4 hours (P = .004), reduced basal oxygen consumption rate (P < .001) and induced pro-inflammatory genes (P < .05). The septic-EV on adoptive transfer to C57/BL6J mice, induced sepsis-like condition within 24 h with leukocytopenia (P = .005), intrahepatic inflammation with increased CD11b + cells (P = .03) and bone marrow hyperplasia (P < .01). CONCLUSION: Extracellular vesicles induce functional impairment in circulating monocytes and contribute to the development and perpetuation of sepsis. High levels of monocyte EV correlate with mortality and can help early stratification of sicker patients.

Impact of temperature and sunshine duration on daily new cases and death due to COVID-19.

CONTEXT: Control of COVID-19 has now become a critical issue for public health. Many ecological factors are proven to influence the transmission and survival of the virus. However, the association between different climatic factors and spread and mortality due to COVID-19 is unknown. AIM: To determine the association of different climatic factors with the spread and mortality due to COVID-19 during January 2020 to May 2020. METHODS AND MATERIAL: The climatic indicators included in the study were duration of sunshine, average minimum temperature, and average maximum temperature, with cumulative confirmed cases, deceased, and recovered cases. The data was performed for 138 different countries of the world, from January 2020 to May 2020. STATISTICAL ANALYSIS USED: Spearman's correlation analysis was used to assess the correlation between temperature and the spread and mortality of COVID-19 cases. Both univariate and multivariate analysis was performed for cumulative and month-wise analysis, using SPSS software. RESULTS: Average maximum temperature and sunshine duration were significantly associated with COVID-19 confirmed cases, deceased, and recovered. For every 1° increase in average temperature, the confirmed, deceased, and recovered cases decreased by 2047 (P = 0.03), 157 (P = 0.016), and 743 (P = 0.005) individuals. The association remained significant even after adjusting for environmental as well as non-environmental variables. Average sunshine duration was inversely correlated with an increase in daily new cases (r = - 2261) and deaths (r = - 0.2985). CONCLUSION: Higher average temperature and longer sunshine duration are strongly associated with COVID-19 cases and deaths in 138 countries.