Antoine Monsel

Human Mesenchymal Stem Cell Microvesicles for Treatment of Escherichia coli Endotoxin‐Induced Acute Lung Injury in Mice

We previously found that human mesenchymal stem cells (MSC) or its conditioned medium restored lung protein permeability and reduced alveolar inflammation following Escherichia coli endotoxin‐induced acute lung injury (ALI) in an ex vivo perfused human lung in part through the secretion of soluble factors such as keratinocyte growth factor (KGF). Recently, MSC were found to release microvesicles (MVs) that were biologically active because of the presence of mRNA or miRNA with reparative properties. MVs are circular fragments of membrane released from the endosomal compartment as exosomes or shed from the surface membranes. These studies were designed to determine if MVs released by human bone marrow derived MSCs would be effective in restoring lung protein permeability and reducing inflammation in E. coli endotoxin‐induced ALI in C57BL/6 mice. The intratracheal instillation of MVs improved several indices of ALI at 48 hours. Compared to endotoxin‐injured mice, MVs reduced extravascular lung water by 43% and reduced total protein levels in the bronchoalveolar lavage (BAL) fluid by 35%, demonstrating a reduction in pulmonary edema and lung protein permeability. MVs also reduced the influx of neutrophils and macrophage inflammatory protein‐2 levels in the BAL fluid by 73% and 49%, respectively, demonstrating a reduction in inflammation. KGF siRNA‐pretreatment of MSC partially eliminated the therapeutic effects of MVs released by MSCs, suggesting that KGF protein expression was important for the underlying mechanism. In summary, human MSC‐derived MVs were therapeutically effective following E. coli endotoxin‐induced ALI in mice in part through the expression of KGF mRNA in the injured alveolus. Stem Cells 2014;32:116–125

Intermittent Subglottic Secretion Drainage and Ventilator-associated Pneumonia

RATIONALE Ventilator-associated pneumonia (VAP) causes substantial morbidity and mortality. The influence of subglottic secretion drainage (SSD) in preventing VAP remains controversial. OBJECTIVES To determine whether SSD reduces the overall incidence of microbiologically confirmed VAP. METHODS Randomized controlled clinical trial conducted at four French centers. A total of 333 adult patients intubated with a tracheal tube allowing drainage of subglottic secretions and expected to require mechanical ventilation for ≥48 hours was included. Patients were randomly assigned to undergo intermittent SSD (n = 169) or not (n = 164). MEASUREMENTS AND MAIN RESULTS Primary outcome was the overall incidence of VAP based on quantitative culture of distal pulmonary samplings performed after each clinical suspicion. Other outcomes included incidence of early- and late-onset VAP, duration of mechanical ventilation, and hospital mortality. Microbiologically confirmed VAP occurred in 67 patients, 25 of 169 (14.8%) in the SSD group and 42 of 164 (25.6%) in the control group (P = 0.02), yielding a relative risk reduction of 42.2% (95% confidential interval, 10.4-63.1%). Using the Day 5 threshold, the beneficial effect of SSD in reducing VAP was observed in both early-onset VAP (2 of 169 [1.2%] patients undergoing SSD vs. 10 of 164 [6.1%] control patients; P = 0.02) and late-onset VAP (23 of 126 [18.6%] patients undergoing SSD vs. 32 of 97 [33.0%] control patients; P = 0.01). VAP was clinically suspected at least once in 51 of 169 (30.2%) patients undergoing SSD and 60 of 164 (36.6%) control patients (P = 0.25). No significant between-group differences were observed in duration of mechanical ventilation and hospital mortality. CONCLUSIONS Subglottic secretion drainage during mechanical ventilation results in a significant reduction in VAP, including late-onset VAP. Clinical trial registered with www.clinicaltrials.gov (NCT00219661).

Therapeutic Effects of Human Mesenchymal Stem Cell–derived Microvesicles in Severe Pneumonia in Mice

RATIONALE Microvesicles (MVs) are anuclear fragments of cells released from the endosomal compartment or shed from surface membranes. We and other investigators demonstrated that MVs released by mesenchymal stem cells (MSCs) were as effective as the cells themselves in inflammatory injuries, such as after endotoxin-induced acute lung injury. However, the therapeutic effects of MVs in an infectious model of acute lung injury remain unknown. OBJECTIVES We investigated the effects of human MSC MVs on lung inflammation, protein permeability, bacterial clearance, and survival after severe bacterial pneumonia. METHODS We tested the effects of MVs derived from human MSCs on Escherichia coli pneumonia in mice. We also studied the interactions between MVs and human monocytes and human alveolar epithelial type 2 cells. MEASUREMENTS AND MAIN RESULTS Administration of MVs derived from human MSCs improved survival in part through keratinocyte growth factor secretion and decreased the influx of inflammatory cells, cytokines, protein, and bacteria in mice injured with bacterial pneumonia. In primary cultures of human monocytes or alveolar type 2 cells, the uptake of MVs was mediated by CD44 receptors, which were essential for the therapeutic effects. MVs enhanced monocyte phagocytosis of bacteria while decreasing inflammatory cytokine secretion and increased intracellular ATP levels in injured alveolar epithelial type 2 cells. Prestimulation of MSCs with a toll-like receptor 3 agonist further enhanced the therapeutic effects of the released MVs. CONCLUSIONS MVs derived from human MSCs were as effective as the parent stem cells in severe bacterial pneumonia.

Cell-based therapy for acute organ injury: preclinical evidence and ongoing clinical trials using mesenchymal stem cells.

Critically ill patients often suffer from multiple organ failures involving lung, kidney, liver, or brain. Genomic, proteomic, and metabolomic approaches highlight common injury mechanisms leading to acute organ failure. This underlines the need to focus on therapeutic strategies affecting multiple injury pathways. The use of adult stem cells such as mesenchymal stem or stromal cells (MSC) may represent a promising new therapeutic approach as increasing evidence shows that MSC can exert protective effects following injury through the release of promitotic, antiapoptotic, antiinflammatory, and immunomodulatory soluble factors. Furthermore, they can mitigate metabolomic and oxidative stress imbalance. In this work, the authors review the biological capabilities of MSC and the results of clinical trials using MSC as therapy in acute organ injuries. Although preliminary results are encouraging, more studies concerning safety and efficacy of MSC therapy are needed to determine their optimal clinical use. (ANESTHESIOLOGY 2014; 121:1099-121)

Aerosolized antibiotics for ventilator-associated pneumonia: lessons from experimental studies.

The aim of this review is to perform a critical analysis of experimental studies on aerosolized antibiotics and draw lessons for clinical use in patients with ventilator-associated pneumonia. Ultrasonic or vibrating plate nebulizers should be preferred to jet nebulizers. During the nebulization period, specific ventilator settings aimed at decreasing flow turbulence should be used, and discoordination with the ventilator should be avoided. The appropriate dose of aerosolized antibiotic can be determined as the intravenous dose plus extrapulmonary deposition. If these conditions are strictly respected, then high lung tissue deposition associated with rapid and efficient bacterial killing can be expected. For aerosolized aminoglycosides and cephalosporins, a decrease in systemic exposure leading to reduced toxicity is not proven by experimental studies. Aerosolized colistin, however, does not easily cross the alveolar–capillary membrane even in the presence of severe lung infection, and high doses can be delivered by nebulization without significant systemic exposure.

Differential down-regulation of HLA-DR on monocyte subpopulations during systemic inflammation

IntroductionDecreased expression of human leukocyte antigen class II (HLA-DR) on monocytes is a hallmark of altered immune status in patients with a systemic inflammatory response syndrome (SIRS). So far, the analyses were mainly performed without taking into account monocytes subpopulations.MethodsWe studied this modification on CD14HIGH and CD14LOW monocytes of 20 SIRS patients undergoing abdominal aortic surgery (AAS), 20 patients undergoing carotid artery surgery (CAS), and 9 healthy controls, and we investigated mediators and intracellular molecules that may be involved in this process.ResultsHLA-DR on CD14HIGH monocytes started to decrease during surgery, after blood reperfusion, and was further reduced post-surgery. In contrast, HLA-DR expression on CD14LOW cells only decreased after surgery, and to a lesser extent than on CD14HIGH monocytes. Negative correlations were found between the reduction of HLA-DR expression and the change in cortisol levels for both subpopulations, whereas a negative correlation between interleukin-10 (IL-10) levels and HLA-DR modulation was only observed for CD14HIGH cells. In accordance with these ex vivo results, HLA-DR on CD14HIGH and CD14LOW monocytes of healthy donors was reduced following incubation with hydrocortisone, whereas IL-10 only acted on CD14HIGH subpopulation. Furthermore, flow cytometry revealed that the expression of IL-10 receptor was higher on CD14HIGH versus CD14LOW monocytes. In addition, hydrocortisone, and to a lesser extent IL-10, reversed the up-regulation of HLA-DR induced by bacterial products. Finally, membrane-associated RING-CH-1 protein (MARCH1) mRNA, a negative regulator of MHC class II, was up-regulated in monocytes of AAS patients on Day 1 post-surgery, and in those of healthy subjects exposed to hydrocortisone.ConclusionsThis study reveals that HLA-DR expression is modulated differently on CD14HIGH (classical) versus CD14LOW (inflammatory) monocytes after systemic inflammation.

Microvesicles Derived From Human Mesenchymal Stem Cells Restore Alveolar Fluid Clearance in Human Lungs Rejected for Transplantation

The need to increase the donor pool for lung transplantation is a major public health issue. We previously found that administration of mesenchymal stem cells “rehabilitated” marginal donor lungs rejected for transplantation using ex vivo lung perfusion. However, the use of stem cells has some inherent limitation such as the potential for tumor formation. In the current study, we hypothesized that microvesicles, small anuclear membrane fragments constitutively released from mesenchymal stem cells, may be a good alternative to using stem cells. Using our well established ex vivo lung perfusion model, microvesicles derived from human mesenchymal stem cells increased alveolar fluid clearance (i.e. ability to absorb pulmonary edema fluid) in a dose‐dependent manner, decreased lung weight gain following perfusion and ventilation, and improved airway and hemodynamic parameters compared to perfusion alone. Microvesicles derived from normal human lung fibroblasts as a control had no effect. Co‐administration of microvesicles with anti‐CD44 antibody attenuated these effects, suggesting a key role of the CD44 receptor in the internalization of the microvesicles into the injured host cell and its effect. In summary, microvesicles derived from human mesenchymal stem cells were as effective as the parent mesenchymal stem cells in rehabilitating marginal donor human lungs.

Cell-Based therapy for traumatic brain injury

Traumatic brain injury is a major economic burden to hospitals in terms of emergency department visits, hospitalizations, and utilization of intensive care units. Current guidelines for the management of severe traumatic brain injuries are primarily supportive, with an emphasis on surveillance (i.e. intracranial pressure) and preventive measures to reduce morbidity and mortality. There are no direct effective therapies available. Over the last fifteen years, pre-clinical studies in regenerative medicine utilizing cell-based therapy have generated enthusiasm as a possible treatment option for traumatic brain injury. In these studies, stem cells and progenitor cells were shown to migrate into the injured brain and proliferate, exerting protective effects through possible cell replacement, gene and protein transfer, and release of anti-inflammatory and growth factors. In this work, we reviewed the pathophysiological mechanisms of traumatic brain injury, the biological rationale for using stem cells and progenitor cells, and the results of clinical trials using cell-based therapy for traumatic brain injury. Although the benefits of cell-based therapy have been clearly demonstrated in pre-clinical studies, some questions remain regarding the biological mechanisms of repair and safety, dose, route and timing of cell delivery, which ultimately will determine its optimal clinical use.

Study of Bone Marrow and Embryonic Stem Cell-Derived Human Mesenchymal Stem Cells for Treatment of Escherichia coli Endotoxin-Induced Acute Lung Injury in Mice

Mesenchymal stem cells (MSCs) can be derived from multiple tissue sources. However, the optimal source of MSCs for cell‐based therapy for acute lung injury (ALI) is unclear. In the present experiments, we studied bone marrow (BM)‐derived and embryonic stem cell‐derived human MSC (ES‐MSCs) as a therapeutic agent in Escherichia coli endotoxin‐induced ALI in mice. We hypothesized that ES‐MSCs would be more potent than BM‐MSCs owing to its more primitive source of origin. ALI was induced by the intratracheal instillation of endotoxin at 4 mg/kg into 10–12‐week‐old C57BL/6 mice with or without BM‐MSCs, ES‐MSCs, or normal human lung fibroblasts as a cellular control. Compared with the endotoxin‐injured mice at 48 hours, the administration of ES‐MSCs provided results similar to those of BM‐MSCs, significantly reducing the influx of white blood cells and neutrophils and decreasing the secretion of the inflammatory cytokines, macrophage inflammatory protein‐2 and tumor necrosis factor‐α, in the injured alveolus. BM‐MSCs also reduced extravascular lung water, a measure of pulmonary edema, by 60% and the total protein levels, a measure of lung permeability, by 66%. However, surprisingly, ES‐MSCs did not have these protective effects, which was partially explained by the increased secretion of matrix metallopeptidase 9 by ES‐MSCs, an enzyme known to increase lung protein permeability. In conclusion, both BM‐MSCs and ES‐MSCs markedly decreased endotoxin‐induced inflammation. However, ES‐MSCs did not show any beneficial effect on reducing pulmonary edema and lung protein permeability compared with BM‐MSCs, suggesting that not all MSCs behave in a similar fashion. Our results highlight the need perhaps for a disease‐specific potency assay for MSCs.

Adult stem cells for acute lung injury: Remaining questions and concerns

Acute lung injury (ALI) or acute respiratory distress syndrome remains a major cause of morbidity and mortality in hospitalized patients. The pathophysiology of ALI involves complex interactions between the inciting event, such as pneumonia, sepsis or aspiration, and the host immune response resulting in lung protein permeability, impaired resolution of pulmonary oedema, an intense inflammatory response in the injured alveolus and hypoxemia. In multiple preclinical studies, adult stem cells have been shown to be therapeutic due to both the ability to mitigate injury and inflammation through paracrine mechanisms and perhaps to regenerate tissue by virtue of their multi‐potency. These characteristics have stimulated intensive research efforts to explore the possibility of using stem or progenitor cells for the treatment of lung injury. A variety of stem or progenitor cells have been isolated, characterized and tested experimentally in preclinical animal models of ALI. However, questions remain concerning the optimal dose, route and the adult stem or progenitor cell to use. Here, the current mechanisms underlying the therapeutic effect of stem cells in ALI as well as the questions that will arise as clinical trials for ALI are planned are reviewed.