Priming Mesenchymal Stem Cells with Lipopolysaccharide Boosts the Immunomodulatory and Regenerative Activity of Secreted Extracellular Vesicles.

Background: Mesenchymal stem cells (MSCs)-derived extracellular vesicles (EVs) have been proposed as an alternative to live-cell administration for Acute Respiratory Distress Syndrome (ARDS). MSC-EVs can be chiefly influenced by the environment to which the MSCs are exposed. Here, lipopolysaccharide (LPS) priming of MSCs was used as a strategy to boost the natural therapeutic potential of the EVs in acute lung injury (ALI). Methods: The regenerative and immunemodulatory effect of LPS-primed MSC-EVs (LPS-EVs) and non-primed MSC-EVs (C-EVs) were evaluated in vitro on alveolar epithelial cells and macrophage-like THP-1 cells. In vivo, ALI was induced in adult male rats by the intrapulmonary instillation of HCl and LPS. Rats (n = 8 to 22/group) were randomized to receive a single bolus (1 × 108 particles) of LPS-EVs, C-EVs, or saline. Lung injury severity was assessed at 72 h in lung tissue and bronchoalveolar lavage. Results: In vitro, LPS-EVs improved wound regeneration and attenuated the inflammatory response triggered by the P. aeruginosa infection, enhancing the M2 macrophage phenotype. In in vivo studies, LPS-EVs, but not C-EVs, significantly decreased the neutrophilic infiltration and myeloperoxidase (MPO) activity in lung tissue. Alveolar macrophages from LPS-EVs-treated animals exhibited a reduced expression of CXCL-1, a key neutrophil chemoattractant. However, both C-EVs and LPS-EVs reduced alveolar epithelial and endothelial permeability, mitigating lung damage. Conclusions: EVs from LPS-primed MSCs resulted in a better resolution of ALI, achieving a greater balance in neutrophil infiltration and activation, while avoiding the complete disruption of the alveolar barrier. This opens new avenues, paving the way for the clinical implementation of cell-based therapies.

Clustering COVID-19 ARDS patients through the first days of ICU admission. An analysis of the CIBERESUCICOVID Cohort.

BACKGROUND: Acute respiratory distress syndrome (ARDS) can be classified into sub-phenotypes according to different inflammatory/clinical status. Prognostic enrichment was achieved by grouping patients into hypoinflammatory or hyperinflammatory sub-phenotypes, even though the time of analysis may change the classification according to treatment response or disease evolution. We aimed to evaluate when patients can be clustered in more than 1 group, and how they may change the clustering of patients using data of baseline or day 3, and the prognosis of patients according to their evolution by changing or not the cluster. METHODS: Multicenter, observational prospective, and retrospective study of patients admitted due to ARDS related to COVID-19 infection in Spain. Patients were grouped according to a clustering mixed-type data algorithm (k-prototypes) using continuous and categorical readily available variables at baseline and day 3. RESULTS: Of 6205 patients, 3743 (60%) were included in the study. According to silhouette analysis, patients were grouped in two clusters. At baseline, 1402 (37%) patients were included in cluster 1 and 2341(63%) in cluster 2. On day 3, 1557(42%) patients were included in cluster 1 and 2086 (57%) in cluster 2. The patients included in cluster 2 were older and more frequently hypertensive and had a higher prevalence of shock, organ dysfunction, inflammatory biomarkers, and worst respiratory indexes at both time points. The 90-day mortality was higher in cluster 2 at both clustering processes (43.8% [n = 1025] versus 27.3% [n = 383] at baseline, and 49% [n = 1023] versus 20.6% [n = 321] on day 3). Four hundred and fifty-eight (33%) patients clustered in the first group were clustered in the second group on day 3. In contrast, 638 (27%) patients clustered in the second group were clustered in the first group on day 3. CONCLUSIONS: During the first days, patients can be clustered into two groups and the process of clustering patients may change as they continue to evolve. This means that despite a vast majority of patients remaining in the same cluster, a minority reaching 33% of patients analyzed may be re-categorized into different clusters based on their progress. Such changes can significantly impact their prognosis.

Evaluation of the Kinetics of Pancreatic Stone Protein as a Predictor of Ventilator-Associated Pneumonia.

BACKGROUND: Ventilator-associated pneumonia (VAP) is a severe condition. Early and adequate antibiotic treatment is the most important strategy for improving prognosis. Pancreatic Stone Protein (PSP) has been described as a biomarker that increases values 3-4 days before the clinical diagnosis of nosocomial sepsis in different clinical settings. We hypothesized that serial measures of PSP and its kinetics allow for an early diagnosis of VAP. METHODS: The BioVAP study was a prospective observational study designed to evaluate the role of biomarker dynamics in the diagnosis of VAP. To determine the association between repeatedly measured PSP and the risk of VAP, we used joint models for longitudinal and time-to-event data. RESULTS: Of 209 patients, 43 (20.6%) patients developed VAP, with a median time of 4 days. Multivariate joint models with PSP, CRP, and PCT did not show an association between biomarkers and VAP for the daily absolute value, with a hazard ratio (HR) for PSP of 1.01 (95% credible interval: 0.97 to 1.05), for CRP of 1.00 (0.83 to 1.22), and for PCT of 0.95 (0.82 to 1.08). The daily change of biomarkers provided similar results, with an HR for PSP of 1.15 (0.94 to 1.41), for CRP of 0.76 (0.35 to 1.58), and for PCT of 0.77 (0.40 to 1.45). CONCLUSION: Neither absolute PSP values nor PSP kinetics alone nor in combination with other biomarkers were useful in improving the prediction diagnosis accuracy in patients with VAP. CLINICAL TRIAL REGISTRATION: Registered retrospectively on August 3rd, 2012. NCT02078999.

Bioengineered extracellular vesicles: future of precision medicine for sepsis.

Sepsis is a syndromic response to infection and is frequently a final common pathway to death from many infectious diseases worldwide. The complexity and high heterogeneity of sepsis hinder the possibility to treat all patients with the same protocol, requiring personalized management. The versatility of extracellular vesicles (EVs) and their contribution to sepsis progression bring along promises for one-to-one tailoring sepsis treatment and diagnosis. In this article, we critically review the endogenous role of EVs in sepsis progression and how current advancements have improved EVs-based therapies toward their translational future clinical application, with innovative strategies to enhance EVs effect. More complex approaches, including hybrid and fully synthetic nanocarriers that mimic EVs, are also discussed. Several pre-clinical and clinical studies are examined through the review to offer a general outlook of the current and future perspectives of EV-based sepsis diagnosis and treatment.

Fluorescent PLGA Nanocarriers for Pulmonary Administration: Influence of the Surface Charge.

Nearly four million yearly deaths can be attributed to respiratory diseases, prompting a huge worldwide health emergency. Additionally, the COVID-19 pandemic's death toll has surpassed six million, significantly increasing respiratory disease morbidity and mortality rates. Despite recent advances, it is still challenging for many drugs to be homogeneously distributed throughout the lungs, and specifically to reach the lower respiratory tract with an accurate sustained dose and minimal systemic side effects. Engineered nanocarriers can provide increased therapeutic efficacy while lessening potential biochemical adverse reactions. Poly(lactic-co-glycolic acid) (PLGA), a biodegradable polymer, has attracted significant interest as an inhalable drug delivery system. However, the influence of the nanocarrier surface charge and its intratracheal instillation has not been addressed so far. In this study, we fabricated red fluorescent PLGA nanocapsules (NCs)-Cy5/PLGA-with either positive (Cy5/PLGA+) or negative surface charge (Cy5/PLGA-). We report here on their excellent colloidal stability in culture and biological media, and after cryo-storage. Their lack of cytotoxicity in two relevant lung cell types, even for concentrations as high as 10 mg/mL, is also reported. More importantly, differences in the NCs' cell uptake rates and internalization capacity were identified. The uptake of the anionic system was faster and in much higher amounts-10-fold and 2.5-fold in macrophages and epithelial alveolar cells, respectively. The in vivo study demonstrated that anionic PLGA NCs were retained in all lung lobules after 1 h of being intratracheally instilled, and were found to accumulate in lung macrophages after 24 h, making those nanocarriers especially suitable as a pulmonary immunomodulatory delivery system with a marked translational character.

Anticoagulant Treatment in Severe ARDS COVID-19 Patients.

Patients with COVID-19 may complicate their evolution with thromboembolic events. Incidence of thromboembolic complications are high and also, patients with the critically-ill disease showed evidence of microthrombi and microangiopathy in the lung probably due to endothelial damage by directly and indirectly injured endothelial and epithelial cells. Pulmonary embolism, deep venous thrombosis and arterial embolism were reported in patients with COVID-19, and several analytical abnormal coagulation parameters have been described as well. D-dimer, longer coagulation times and lower platelet counts have been associated with poor outcomes. The use of anticoagulation or high doses of prophylactic heparin is controversial. Despite the use of anticoagulation or high prophylactic dose of heparin have been associated with better outcomes in observational studies, only in patients with non-critically ill disease benefits for anticoagulation was observed. In critically-ill patient, anticoagulation was not associated with better outcomes. Other measures such as antiplatelet therapy, fibrinolytic therapy or nebulized anticoagulants are being studied in ongoing clinical trials.

Inflammatory capacity of exosomes released in the early stages of acute pancreatitis predicts the severity of the disease.

As acute pancreatitis progresses to the severe form, a life-threatening systemic inflammation is triggered. Although the mechanisms involved in this process are not yet well understood, it has been proposed that circulating exosomes may be involved in the progression of inflammation from the pancreas to distant organs. Here, the inflammatory capacity and protein profile of plasma exosomes obtained during the first 24 h of hospitalization of patients diagnosed with acute pancreatitis were characterized and compared with the final severity of the disease. We found that the final severity of the disease strongly correlates with the inflammatory capacity of exosomes in the early stages of acute pancreatitis. Exosomes isolated from patients with mild pancreatitis had no effect on macrophages, while exosomes isolated from patients with severe pancreatitis triggered NFκB activation, TNFα and IL1ß expression, and free radical generation. To delve deeper into the mechanism involved, we performed a proteomic analysis of the different exosomes that allowed us to identify different groups of proteins whose concentration was also correlated with the clinical classification of pancreatitis. In particular, an increase in the amount of S100A8 and S100A9 carried by exosomes of severe pancreatitis suggests that the mechanism of action of exosomes is mediated by the effect of these proteins on NADPH oxidase. This enzyme is activated by S100A8/S100A9, thus generating free radicals and promoting an inflammatory response. Along these lines, we observed that inhibition of this enzyme abolished all the pro-inflammatory effects of exosomes from severe pancreatitis. All this suggests that the systemic effects, and therefore the final severity of acute pancreatitis, are determined by the content of circulating exosomes generated in the early hours of the process. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.