Cryo-electron microscopy of extracellular vesicles associated with the marine toxic dinoflagellate Alexandrium minutum.

Extracellular Vesicles (EVs) are likely an important strategy of transport and communication in marine microbial community. Their isolation and characterization from axenic culture of microbial eukaryotes represents a technological challenge not fully solved. Here, for the first time, we isolated EVs from a near-axenic culture of the toxic dinoflagellate Alexandrium minutum. Pictures of the isolated vesicles were done with Cryo TEM (Cryogenic Transmission Electron Microscopy). Based on their morphotype the EVs were clustered in five major groups (rounded, rounded electron-dense, lumen electron-dense, double and irregular) and each EV was measured resulting in an average size of 0.36 µm of diameter. Taking in account that in prokaryotes it has been demonstrated that EVs play an important role in the mechanism of toxicity, this descriptive work aims to be the first step to study the possible role of EVs in the toxicity of dinoflagellates.

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.

Pancreatic cancer, stroma, and exosomes

In the pathogenesis of pancreatic adenocarcinoma, tumor stroma plays a key role in both aggressiveness, immune evasion, resistance to chemotherapy, and the ability to metastasize. Among the elements that characterize the behavior of the stroma, extracellular vesicles and, in particular, exosomes play an important role. These extracellular vesicles carry a wide range of bioactive molecules, from transcription factors to microRNAs, which can substantially alter the phenotype of the cellular components of the stroma. Exosomes are involved in the exchange of signals between tumor cells, tumor-associated macrophages, cancer-associated fibroblasts, and also with the healthy cells surrounding the tumor. They can transfer resistance to chemotherapeutic drugs, promote the epithelial-mesenchymal transition, modify the phenotype of macrophages, or induce the expression of molecules that alter the extracellular matrix to facilitate migration and metastasis. On the other hand, all these characteristics make these vesicles first-rate therapeutic targets, as controlling their functionality could greatly enhance the effectiveness of treatments that, today, are still far from be satisfactory. (AU)

Pancreatic cancer, stroma, and exosomes.

In the pathogenesis of pancreatic adenocarcinoma, tumor stroma plays a key role in both aggressiveness, immune evasion, resistance to chemotherapy, and the ability to metastasize. Among the elements that characterize the behavior of the stroma, extracellular vesicles and, in particular, exosomes play an important role. These extracellular vesicles carry a wide range of bioactive molecules, from transcription factors to microRNAs, which can substantially alter the phenotype of the cellular components of the stroma. Exosomes are involved in the exchange of signals between tumor cells, tumor-associated macrophages, cancer-associated fibroblasts, and also with the healthy cells surrounding the tumor. They can transfer resistance to chemotherapeutic drugs, promote the epithelial-mesenchymal transition, modify the phenotype of macrophages, or induce the expression of molecules that alter the extracellular matrix to facilitate migration and metastasis. On the other hand, all these characteristics make these vesicles first-rate therapeutic targets, as controlling their functionality could greatly enhance the effectiveness of treatments that, today, are still far from be satisfactory.

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.

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.

Targeting REG3ß limits pancreatic ductal adenocarcinoma progression through CTGF downregulation.

The crosstalk between the transformed tumoral cells and their microenvironment is a key aspect for pancreatic ductal adenocarcinoma (PDAC) progression. This molecular dialog is intensively studied because it may result in an efficient therapeutic target. Contrary to this near microenvironment, the stromal portion in direct contact with the transformed cells, a far microenvironment, placed at the periphery of the tumor mass, produces factors signaling tumors. Among these factors, REG3ß, produced by this part of the pancreas, is an important factor in promoting tumor progression. This paper demonstrated that targeting REG3ß protein with specific antibodies limits the PDAC tumor growth in an orthotopic, syngeneic mice model induced by injection of Panc02 cells. Then, we showed that CTGF is over-expressed in response to REG3ß in PDAC-derived cells. Moreover, inactivation of REG3ß by treating tumors with anti-REG3ß antibodies results in a strong decrease of CTGF in PDAC tumors. Lastly, we demonstrated that forced expression of CTGF in xenografted Panc02 cells abolishes the therapeutic effect of the anti-REG3ß antibody treatment. Altogether, these results indicate that the effect of REG3ß in promoting PDAC progression is mediated by CTGF over-activation. Thus, REG3ß is a promising therapeutic target to treat PDAC with an original rationale. In conclusion, we demonstrated that the far microenvironment is essential for PDAC progression by producing active secretory factors, and some of them could be used as therapeutic targets.

Polyethylene Glycol 35 (PEG35) Modulates Exosomal Uptake and Function.

Polyethylene glycols (PEGs) are neutral polymers widely used in biomedical applications due to its hydrophilicity and biocompatibility. Exosomes are small vesicles secreted by nearly all cell types and play an important role in normal and pathological conditions. The purpose of this study was to evaluate the role of a 35-kDa molecular weight PEG (PEG35) on the modulation of exosome-mediated inflammation. Human macrophage-like cells THP-1, epithelial BICR-18, and CAPAN-2 cells were exposed to PEG35 prior to incubation with exosomes of different cellular origins. Exosome internalization was evaluated by confocal microscopy and flow cytometry. In another set of experiments, macrophages were treated with increasing concentrations of PEG35 prior to exposure with the appropriate stimuli: lipopolysaccharide, BICR-18-derived exosomes, or exosomes from acute pancreatitis-induced rats. Nuclear Factor Kappa B (NFκB) and Signal transducer and activator of transcription 3 (STAT3) activation and the expression levels of pro-inflammatory Interleukin 1ß (IL1ß) were determined. PEG35 administration significantly enhanced the internalization of exosomes in both macrophages and epithelial cells. Further, PEG35 ameliorated the inflammatory response induced by acute pancreatitis-derived exosomes by reducing the expression of IL1ß and p65 nuclear translocation. Our results revealed that PEG35 promotes the cellular uptake of exosomes and modulates the pro-inflammatory effect of acute pancreatitis-derived vesicles through inhibition of NFκB, thus emphasizing the potential value of PEG35 as an anti-inflammatory agent for biomedical purposes.

Dietary Fat Patterns and Outcomes in Acute Pancreatitis in Spain.

Background/Objective: Evidence from basic and clinical studies suggests that unsaturated fatty acids (UFAs) might be relevant mediators of the development of complications in acute pancreatitis (AP). Objective: The aim of this study was to analyze outcomes in patients with AP from regions in Spain with different patterns of dietary fat intake. Materials and Methods: A retrospective analysis was performed with data from 1,655 patients with AP from a Spanish prospective cohort study and regional nutritional data from a Spanish cross-sectional study. Nutritional data considered in the study concern the total lipid consumption, detailing total saturated fatty acids, UFAs and monounsaturated fatty acids (MUFAs) consumption derived from regional data and not from the patient prospective cohort. Two multivariable analysis models were used: (1) a model with the Charlson comorbidity index, sex, alcoholic etiology, and recurrent AP; (2) a model that included these variables plus obesity. Results: In multivariable analysis, patients from regions with high UFA intake had a significantly increased frequency of local complications, persistent organ failure (POF), mortality, and moderate-to-severe disease in the model without obesity and a higher frequency of POF in the model with obesity. Patients from regions with high MUFA intake had significantly more local complications and moderate-to-severe disease; this significance remained for moderate-to-severe disease when obesity was added to the model. Conclusions: Differences in dietary fat patterns could be associated with different outcomes in AP, and dietary fat patterns may be a pre-morbid factor that determines the severity of AP. UFAs, and particulary MUFAs, may influence the pathogenesis of the severity of AP.

Involvement of extracellular vesicles in the macrophage-tumor cell communication in head and neck squamous cell carcinoma.

BACKGROUND: Exosomes are cell-derived vesicles that mediate cellular communication in health and multiple diseases, including cancer. However, its role in head and neck cancer has been poorly defined. Here, we investigated the relevance of exosomes in the signaling between larynx cancer cells and macrophages. METHODS: Exosomes from THP1 macrophages and BICR18 cells (a larynx squamous cell carcinoma cell line) were purified and their role in the cancer cell migration, macrophage phenotype and immunosuppressive activity was evaluated. The activation of STAT3 signal transduction in macrophages in response to exosomes obtained from cancer cells was also evaluated. RESULTS: Macrophages foster the cancer cell migration and this effect is mediated by exosome signaling. On the other hand, exosomes also induce the expression of IL-10 in macrophages and PD-L1 in cancer cells, thus resulting in the promotion of an immunosuppressive environment. Moreover, we observed that the effects induced in cancer cells are mediated by the exosome-depending activation of STAT-3 signal transduction pathway. CONCLUSIONS: Our study indicates that exosomes released by both macrophages and cancer cells plays a critical role in tumor progression in larynx cancer and might be a potential target for therapeutic intervention in head and neck cancer.

Fluid resuscitation with lactated Ringer's solution vs normal saline in acute pancreatitis: A triple-blind, randomized, controlled trial.

BACKGROUND: Little is known regarding the optimal type of fluid resuscitation in acute pancreatitis (AP). OBJECTIVE: The objective of this article was to compare the effect of lactated Ringer's solution (LR) vs normal saline (NS) in the inflammatory response in AP. METHODS: We conducted a triple-blind, randomized, controlled trial. Patients ≥ 18 admitted with AP were eligible. Patients were randomized to receive LR or NS. Primary outcome variables were number of systemic inflammatory response syndrome (SIRS) criteria at 24 hours, 48 hours and 72 hours and blood C-reactive protein (CRP) levels at 48 hours and 72 hours. In vitro complementary experiments were performed to further explore the interaction between pH, lactate and inflammation. RESULTS: Nineteen patients receiving LR and 21 receiving NS were analyzed. The median (p25-p75) number of SIRS criteria at 48 hours were 1 (1-2) for NS vs 1 (0-1) for LR, p = 0.060. CRP levels (mg/l) were as follows: at 48 hours NS 166 (78-281) vs LR 28 (3-124), p = 0.037; at 72 hours NS 217 (59-323) vs LR 25 (3-169), p = 0.043. In vitro, LR inhibited the induction of inflammatory phenotype of macrophages and NF-κB activation. This effect was not observed when using Ringer's solution without lactate, suggesting a direct anti-inflammatory effect of lactate. CONCLUSIONS: Lactated Ringer's is associated with an anti-inflammatory effect in patients with acute pancreatitis.

Oleic acid chlorohydrin, a new early biomarker for the prediction of acute pancreatitis severity in humans.

BACKGROUND: The early prediction of the severity of acute pancreatitis still represents a challenge for clinicians. Experimental studies have revealed the generation of specific halogenated lipids, in particular oleic acid chlorohydrin, in the early stages of acute pancreatitis. We hypothesized that the levels of circulating oleic acid chlorohydrin might be a useful early prognostic biomarker in acute pancreatitis in humans. METHODS: In a prospective, multicenter cohort study, plasma samples collected within 24 h after presentation in the emergency room from 59 patients with acute pancreatitis and from 9 healthy subjects were assessed for oleic acid chlorohydrin levels. RESULTS: Pancreatitis was mild in 30 patients, moderately severe in 16 and severe in 13. Oleic acid chlorohydrin levels within 24 h after presentation were significantly higher in patients that later progressed to moderate and severe acute pancreatitis. Using 7.49 nM as the cutoff point, oleic acid chlorohydrin distinguished mild from moderately severe-to-severe pancreatitis with high sensitivity/specificity (96.6/90.0%) and positive/negative predictive values (90.3/96.4%). Using 32.40 nM as the cutoff value sensitivity, specificity, positive and negative predictive values were all 100% for severe acute pancreatitis. It was found to be a better prognostic marker than BISAP score, hematocrit at 48 h, SIRS at admission, persistent SIRS or C-reactive protein at 48 h. CONCLUSIONS: Oleic acid chlorohydrin concentration in plasma is elevated in patients with acute pancreatitis on admission and correlates with a high degree with the final severity of the disease, indicating that it has potential to serve as an early prognostic marker for acute pancreatitis severity.

Factors released by the tumor far microenvironment are decisive for pancreatic adenocarcinoma development and progression.

The REG3ß protein was identified more than 2 decades ago, but its role in PDAC development was only recently reported. In Pancreatic Ductal Adenocarcinoma (PDAC), REG3ß protein is expressed and released by the far microenvironment, which is situated out of the tumor, at the periphery of the tumor mass, and is part of the healthy peri-tumoral region. This compartment is completely unrelated to the classical microenvironment that corresponds to the intra-tumoral stoma. Clinically relevant, the far microenvironment, and the factors released by it, could be novel and original therapeutic targets for treating patients with a PDAC. In this way we recently demonstrated that REG3ß is an essential soluble factor necessary for PDAC development which is able to stimulate several simultaneous pro-tumoral mechanisms. We also find that secreted REG3ß boosts interactions between epithelial cells and immune cells by activating the CXCL12/CXCR4 signaling cascade, which facilitates tumor escape through evasion of immune surveillance, and promotes metastasis. In addition, REG3ß interfere the intercellular communication inside the tumor mediated by extracellular vesicles, resulting in relevant changes in macrophage phenotype or tumor cell migration. Therefore, we are proposing to call as near microenvironment to the classical microenvironment that is constituted by fibroblasts, inflammatory cells and fibers and located into the tumor, and as far microenvironment, which is constituted by the parenchymal non transformed cells located at the periphery of the tumor mass.

REG3ß modifies cell tumor function by impairing extracellular vesicle uptake.

Extracellular vesicles (EVs), including exosomes and microvesicles, are nano-sized membrane vesicles containing proteins and nucleic acids, which act as intercellular messengers. They play an important role in a variety of physiological processes, as well as in pathological situations such as inflammation or cancer. Here, we show that in the case of pancreatic ductal adenocarcinoma (PDAC), the healthy pancreatic tissue surrounding the tumor releases REG3ß, a lectin that binds to the glycoproteins present in the surface of EVs, thus interfering with their uptake and internalization by target cells. In vitro, the disruption of the signaling mediated by EVs due to the presence of REG3ß, prevents the EV-induced phenotypic switch in macrophages, inhibits the increased cell migration of cancer cells and reverses a number of metabolomic changes promoted by EVs. In vivo, the uptake of REG3ß+ EVs by tumor cells is significantly impaired. Furthermore, it results in an increase of circulating REG3ß+ EVs in blood of pancreatic cancer patients. Our findings highlight the effect of a lectin released by the healthy pancreatic tissue surrounding the tumor in modulating the EV-mediated interactions between different cell types in PDAC.

Involvement of exosomes in lung inflammation associated with experimental acute pancreatitis.

A frequent complication of acute pancreatitis is the lung damage associated with the systemic inflammatory response. Although various pro-inflammatory mediators generated at both local and systemic levels have been identified, the pathogenic mechanisms of the disease are still poorly understood. In recent years, exosomes have emerged as a new intercellular communication system able to transfer encapsulated proteins and small RNAs and protect them from degradation. Using an experimental model of taurocholate-induced acute pancreatitis in rats, we aimed to evaluate the role of exosomes in the extent of the systemic inflammatory response. Induction of pancreatitis increased the concentration of circulating exosomes, which showed a different proteomic profile to those obtained from control animals. A series of tracking experiments using PKH26-stained exosomes revealed that circulating exosomes effectively reached the alveolar compartment and were internalized by macrophages. In vitro experiments revealed that exosomes obtained under inflammatory conditions activate and polarize these alveolar macrophages towards a pro-inflammatory phenotype. Interestingly, the proteomic analysis of circulating exosomes during acute pancreatitis suggested a multi-organ origin with a relevant role for the liver as a source of these vesicles. Tracking experiments also revealed that the liver retains the majority of exosomes from the peritoneal cavity. We conclude that exosomes are involved in the lung damage associated with experimental acute pancreatitis and could be relevant mediators in the systemic effects of pancreatitis. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Safety and Tolerability of Alveolar Type II Cell Transplantation in Idiopathic Pulmonary Fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease with limited response to currently available therapies. Alveolar type II (ATII) cells act as progenitor cells in the adult lung, contributing to alveolar repair during pulmonary injury. However, in IPF, ATII cells die and are replaced by fibroblasts and myofibroblasts. In previous preclinical studies, we demonstrated that ATII-cell intratracheal transplantation was able to reduce pulmonary fibrosis. The main objective of this study was to investigate the safety and tolerability of ATII-cell intratracheal transplantation in patients with IPF. METHODS: We enrolled 16 patients with moderate and progressive IPF who underwent ATII-cell intratracheal transplantation through fiberoptic bronchoscopy. We evaluated the safety and tolerability of ATII-cell transplantation by assessing the emergent adverse side effects that appeared within 12 months. Moreover, pulmonary function, respiratory symptoms, and disease extent during 12 months of follow-up were evaluated. RESULTS: No significant adverse events were associated with the ATII-cell intratracheal transplantation. After 12 months of follow-up, there was no deterioration in pulmonary function, respiratory symptoms, or disease extent. CONCLUSIONS: Our results support the hypothesis that ATII-cell intratracheal transplantation is safe and well tolerated in patients with IPF. This study opens the door to designing a clinical trial to elucidate the potential beneficial effects of ATII-cell therapy in IPF.

New Roles for Corticosteroid Binding Globulin and Opposite Expression Profiles in Lung and Liver.

Corticosteroid-binding globulin (CBG) is the specific plasma transport glycoprotein for glucocorticoids. Circulating CBG is mainly synthesized in liver but, its synthesis has been located also in other organs as placenta, kidney and adipose tissue with unknown role. Using an experimental model of acute pancreatitis in cbg-/- mice we investigated whether changes in CBG affect the progression of the disease as well as the metabolism of glucocorticoids in the lung. Lack of CBG does not modify the progression of inflammation associated to pancreatitis but resulted in the loss of gender differences in corticosterone serum levels. In the lung, CBG expression and protein level were detected, and it is noteworthy that these showed a sexual dimorphism opposite to the liver, i.e. with higher levels in males. Reduced expression of 11ß-HSD2, the enzyme involved in the deactivation of corticosterone, was also observed. Our results indicate that, in addition to glucocorticoids transporter, CBG is involved in the gender differences observed in corticosteroids circulating levels and plays a role in the local regulation of corticosteroids availability in organs like lung.

Minocycline inhibits peritoneal macrophages but activates alveolar macrophages in acute pancreatitis

Minocycline is a tetracycline antibiotic that, in addition to its antimicrobial function, has been reported to possess a relevant anti-inflammatory activity. Its effects have been extensively evaluated in inflammatory-related neurological diseases. Here, we evaluate its effect on the systemic inflammatory response in a model of experimental acute pancreatitis. Minocycline treatment significantly reduced the inflammation in pancreas and mesenterium, had no effect on the adipose tissue inflammation, and increased the inflammatory response in the lung. These differences seem to be related with different effects exerted on peritoneal and alveolar macrophages. In vitro, minocycline reduced the expression of IL-1Beta and inhibit the activation of nuclear factor kappa B (NF-kappaB) on peritoneal macrophages, while it had no effect on alveolar macrophages. Our data indicates that although minocycline may be useful as a tool to control some inflammatory processes, differences on its effects depending on the population of macrophages involved in the process can be expected. In the particular case of acute pancreatitis, it could promote or potentiate inflammation in the lung so that its use does not appear to be recommended