Hanjing Zhuo

Mesenchymal stem (stromal) cells for treatment of ARDS: a phase 1 clinical trial

BACKGROUND No effective pharmacotherapy for acute respiratory distress syndrome (ARDS) exists, and mortality remains high. Preclinical studies support the efficacy of mesenchymal stem (stromal) cells (MSCs) in the treatment of lung injury. We aimed to test the safety of a single dose of allogeneic bone marrow-derived MSCs in patients with moderate-to-severe ARDS. METHODS The STem cells for ARDS Treatment (START) trial was a multicentre, open-label, dose-escalation, phase 1 clinical trial. Patients were enrolled in the intensive care units at University of California, San Francisco, CA, USA, Stanford University, Stanford, CA, USA, and Massachusetts General Hospital, Boston, MA, USA, between July 8, 2013, and Jan 13, 2014. Patients were included if they had moderate-to-severe ARDS as defined by the acute onset of the need for positive pressure ventilation by an endotracheal or tracheal tube, a PaO2:FiO2 less than 200 mm Hg with at least 8 cm H2O positive end-expiratory airway pressure (PEEP), and bilateral infiltrates consistent with pulmonary oedema on frontal chest radiograph. The first three patients were treated with low dose MSCs (1 million cells/kg predicted bodyweight [PBW]), the next three patients received intermediate dose MSCs (5 million cells/kg PBW), and the final three patients received high dose MSCs (10 million cells/kg PBW). Primary outcomes included the incidence of prespecified infusion-associated events and serious adverse events. The trial is registered with ClinicalTrials.gov, number NCT01775774. FINDINGS No prespecified infusion-associated events or treatment-related adverse events were reported in any of the nine patients. Serious adverse events were subsequently noted in three patients during the weeks after the infusion: one patient died on study day 9, one patient died on study day 31, and one patient was discovered to have multiple embolic infarcts of the spleen, kidneys, and brain that were age-indeterminate, but thought to have occurred before the MSC infusion based on MRI results. None of these severe adverse events were thought to be MSC-related. INTERPRETATION A single intravenous infusion of allogeneic, bone marrow-derived human MSCs was well tolerated in nine patients with moderate to severe ARDS. Based on this phase 1 experience, we have proceeded to phase 2 testing of MSCs for moderate to severe ARDS with a primary focus on safety and secondary outcomes including respiratory, systemic, and biological endpoints. FUNDING The National Heart, Lung, and Blood Institute.

Human mesenchymal stem cells reduce mortality and bacteremia in gram-negative sepsis in mice in part by enhancing the phagocytic activity of blood monocytes

The potential therapeutic value of cell-based therapy with mesenchymal stem cells (MSC) has been reported in mouse models of polymicrobial peritoneal sepsis. However, the mechanisms responsible for the beneficial effects of MSC have not been well defined. Therefore, we tested the therapeutic effect of intravenous bone marrow-derived human MSC in peritoneal sepsis induced by gram-negative bacteria. At 48 h, survival was significantly increased in mice treated with intravenous MSC compared with control mice treated with intravenous fibroblasts (3T3) or intravenous PBS. There were no significant differences in the levels of TNF-α, macrophage inflammatory protein 2, or IL-10 in the plasma. However, there was a marked reduction in the number of bacterial colony-forming units of Pseudomonas aeruginosa in the blood of MSC-treated mice compared with the 3T3 and PBS control groups. In addition, phagocytic activity was increased in blood monocytes isolated from mice treated with MSC compared with the 3T3 and PBS groups. Furthermore, levels of C5a anaphylotoxin were elevated in the blood of mice treated with MSC, a finding that was associated with upregulation of the phagocytosis receptor CD11b on monocytes. The phagocytic activity of neutrophils was not different among the groups. There was also an increase in alternately activated monocytes/macrophages (CD163- and CD206-positive) in the spleen of the MSC-treated mice compared with the two controls. Thus intravenous MSC increased survival from gram-negative peritoneal sepsis, in part by a monocyte-dependent increase in bacterial phagocytosis.

Randomized Clinical Trial of Activated Protein C for the Treatment of Acute Lung Injury

RATIONALE Microvascular injury, inflammation, and coagulation play critical roles in the pathogenesis of acute lung injury (ALI). Plasma protein C levels are decreased in patients with acute lung injury and are associated with higher mortality and fewer ventilator-free days. OBJECTIVES To test the efficacy of activated protein C (APC) as a therapy for patients with ALI. METHODS Eligible subjects were critically ill patients who met the American/European consensus criteria for ALI. Patients with severe sepsis and an APACHE II score of 25 or more were excluded. Participants were randomized to receive APC (24 microg/kg/h for 96 h) or placebo in a double-blind fashion within 72 hours of the onset of ALI. The primary endpoint was ventilator-free days. MEASUREMENTS AND MAIN RESULTS APC increased plasma protein C levels (P = 0.002) and decreased pulmonary dead space fraction (P = 0.02). However, there was no statistically significant difference between patients receiving placebo (n = 38) or APC (n = 37) in the number of ventilator-free days (median [25-75% interquartile range]: 19 [0-24] vs. 19 [14-22], respectively; P = 0.78) or in 60-day mortality (5/38 vs. 5/37 patients, respectively; P = 1.0). There were no differences in the number of bleeding events between the two groups. CONCLUSIONS APC did not improve outcomes from ALI. The results of this trial do not support a large clinical trial of APC for ALI in the absence of severe sepsis and high disease severity.

Biomarkers of inflammation, coagulation and fibrinolysis predict mortality in acute lung injury

BackgroundAcute lung injury (ALI) is a major cause of acute respiratory failure with high mortality despite lung-protective ventilation. Prior work has shown disordered inflammation and coagulation in ALI, with strong correlations between biomarker abnormalities and worse clinical outcomes. We measured plasma markers of inflammation, coagulation and fibrinolysis simultaneously to assess whether these markers remain predictive in the era of lung-protective ventilation.MethodsPlasma samples and ventilator data were prospectively collected from 50 patients with early ALI. Plasma biomarkers of inflammation (IL-6, IL-8, intercellular adhesion molecule 1), of coagulation (thrombomodulin, protein C) and of fibrinolysis (plasminogen activator inhibitor 1) were measured by ELISA. Biomarker levels were compared between survivors (n = 29) and non-survivors (n = 21) using Mann–Whitney analysis.ResultsThe tidal volume for the study group was 6.6 ± 1.1 ml/kg predicted body weight and the plateau pressure was 25 ± 7 cmH2O (mean ± standard deviation), consistent with lung-protective ventilation. All markers except IL-6 were significantly different between survivors and nonsurvivors. Nonsurvivors had more abnormal values. Three biomarkers – IL-8, intercellular adhesion molecule 1 and protein C – remained significantly different by multivariate analysis that included age, gender, Simplified Acute Physiology Score II and all biomarkers that were significant on bivariate analysis. Higher levels of IL-8 and intercellular adhesion molecule 1 were independently predictive of worse outcomes (odds ratio = 2.0 and 5.8, respectively; P = 0.04 for both). Lower levels of protein C were independently associated with an increased risk of death (odds ratio = 0.5), a result that nearly reached statistical significance (P = 0.06).ConclusionDespite lung-protective ventilation, abnormalities in plasma levels of markers of inflammation, coagulation and fibrinolysis predict mortality in ALI patients, indicating more severe activation of these biologic pathways in nonsurvivors.

Increased mortality of ventilated patients with endotracheal Pseudomonas aeruginosa without clinical signs of infection.

Objective:To investigate the frequency and outcomes of ventilated patients with newly acquired large burdens of Pseudomonas aeruginosa and to test the hypothesis that large quantities of bacteria are associated with adverse patient outcomes. Design:A prospective, single-center, observational, cohort study. Setting:Medical-surgical intensive care units in a tertiary care university hospital. Patients:All adult patients requiring ≥48 hrs of mechanical ventilation and identified as having newly acquired P. aeruginosa in their lower respiratory tracts between October 2002 and April 2006. Interventions:None. Measurements and Main Results:Daily surveillance cultures of endotracheal aspirates were performed on patients intubated ≥48 hrs; 69 patients with newly acquired P. aeruginosa were enrolled. Daily P. aeruginosa quantification of endotracheal aspirates was performed; clinical signs of infection were noted. Of 45 patients with high P. aeruginosa burdens (≥1,000,000 colony-forming units/mL in endotracheal aspirates; ≥10,000 colony-forming units/mL in bronchoalveolar-lavage), 17 (37.8%) patients did not meet clinical criteria for ventilator-associated pneumonia and had a statistically significant higher risk of death (adjusted hazard ratio, 37.53; 95% confidence interval, 3.79–371.96; p = 0.002) when compared with the patients who had P. aeruginosa ventilator-associated pneumonia. When excluding the ten patients who had ventilator-associated pneumonia attributed to bacteria other than P. aeruginosa or attributed to multiple bacteria including P. aeruginosa, the risk of death remained statistically significant (adjusted hazard ratio, 23.98; 95% confidence interval: 2.49–230.53; p = 0.006). Furthermore, more patients with high P. aeruginosa burdens secreted the type III secretion facilitator protein, PcrV (p = 0.01). Conclusions:A group of patients with large burdens of P. aeruginosa who did not meet clinical criteria for ventilator-associated pneumonia had an increased risk of death when compared with patients who had high P. aeruginosa burdens and met ventilator-associated pneumonia criteria. Patients with high P. aeruginosa burden seemed to possess more virulent strains.

Human mesenchymal stem cells reduce the severity of acute lung injury in a sheep model of bacterial pneumonia

Background Human bone marrow-derived mesenchymal stem (stromal) cells (hMSCs) improve survival in mouse models of acute respiratory distress syndrome (ARDS) and reduce pulmonary oedema in a perfused human lung preparation injured with Escherichia coli bacteria. We hypothesised that clinical grade hMSCs would reduce the severity of acute lung injury (ALI) and would be safe in a sheep model of ARDS. Methods Adult sheep (30–40 kg) were surgically prepared. After 5 days of recovery, ALI was induced with cotton smoke insufflation, followed by instillation of live Pseudomonas aeruginosa (2.5×1011 CFU) into both lungs under isoflurane anaesthesia. Following the injury, sheep were ventilated, resuscitated with lactated Ringers solution and studied for 24 h. The sheep were randomly allocated to receive one of the following treatments intravenously over 1 h in one of the following groups: (1) control, PlasmaLyte A, n=8; (2) lower dose hMSCs, 5×106 hMSCs/kg, n=7; and (3) higher-dose hMSCs, 10×106 hMSCs/kg, n=4. Results By 24 h, the PaO2/FiO2 ratio was significantly improved in both hMSC treatment groups compared with the control group (control group: PaO2/FiO2 of 97±15 mm Hg; lower dose: 288±55 mm Hg (p=0.003); higher dose: 327±2 mm Hg (p=0.003)). The median lung water content was lower in the higher-dose hMSC-treated group compared with the control group (higher dose: 5.0 g wet/g dry [IQR 4.9–5.8] vs control: 6.7 g wet/g dry [IQR 6.4–7.5] (p=0.01)). The hMSCs had no adverse effects. Conclusions Human MSCs were well tolerated and improved oxygenation and decreased pulmonary oedema in a sheep model of severe ARDS. Trail registration number: NCT01775774 for Phase 1. NCT02097641 for Phase 2.

Pathogenetic and predictive value of biomarkers in patients with ALI and lower severity of illness: results from two clinical trials

Plasma and bronchoalveolar lavage (BAL) biomarkers related to the pathogenesis of acute lung injury (ALI) have previously been associated with poorer clinical outcomes and increased disease severity among patients with ALI. Whether these biomarkers have predictive value in a less severely ill population that excludes septic patients with high APACHE II scores is currently unknown. We tested the association of plasma and BAL biomarkers with physiological markers of ALI severity or clinically relevant outcomes in a secondary analysis of a clinical trial of activated protein C for the treatment of ALI. Plasma plasminogen activator inhibitor-1 (PAI-1) and mini-BAL protein were both significantly associated with increased oxygenation index (P = 0.02 and 0.01, respectively), whereas there was a trend toward an association between IL-6 and oxygenation index (P = 0.057). High plasma IL-6, thrombomodulin, and mini-BAL protein were all significantly associated with fewer ventilator-free days (VFDs) (P = 0.01, 0.01, and 0.05, respectively); no markers were associated with mortality, but we hypothesized that this was due to the small size of our cohort and the low death rate. To confirm these associations in a larger sample, we identified a restricted cohort of patients from the ARDS Network ALVEOLI study with similar baseline characteristics. We retested the associations of the significant biomarkers with markers of severity and clinical outcomes and studied IL-8 as an additional biomarker given its important predictive value in prior studies. In this restricted cohort, IL-6 was significantly associated with oxygenation index (P = 0.02). Both IL-6 and IL-8 were associated with decreased VFDs and increased 28-day mortality. Future studies should be focused on examining larger numbers of patients with less severe ALI to further test the relative predictive value of plasma and mini-BAL biomarkers for clinically relevant outcomes, including VFDs and mortality, and for their prospective utility in risk stratification for future clinical trials.

The Association Between a Darc Gene Polymorphism and Clinical Outcomes in African American Patients With Acute Lung Injury

BACKGROUND Acute lung injury (ALI) mortality is increased among African Americans compared with Americans of European descent, and genetic factors may be involved. A functional T-46C polymorphism (rs2814778) in the promoter region of Duffy antigen/receptor for chemokines (Darc) gene, present almost exclusively in people of African descent, results in isolated erythrocyte DARC deficiency and has been implicated in ALI pathogenesis in preclinical and murine models, possibly because of an increase in circulating Duffy-binding, proinflammatory chemokines like IL-8. We sought to determine the effect of the functional rs2814778 polymorphism, C/C genotype (Duffy null state), on clinical outcomes in African Americans with acute lung injury. METHODS Clinical data and biologic specimens from African American patients with ALI who enrolled in three randomized controlled trials were analyzed. Multivariate analysis accounted for proportion of African ancestry, sex, cirrhosis, and severity of illness on presentation. RESULTS Among 132 subjects, 88 (67%) were Duffy null (C/C genotype). The Duffy null state was associated with a 17% absolute risk increase (95% CI, 1.4%-33%) in mortality at 60 days, a median of 8 fewer ventilator-free days (95% CI, 1-18.5), and 4.5 fewer organ failure-free days (95% CI, 0-18) compared with individuals with the C/T or T/T genotypes (all P values < .05). Estimates were similar on multivariate analysis. In African Americans without the null variant, clinical outcomes were similar to those in patients of European descent. A subgroup analysis suggested that plasma IL-8 levels are increased in Duffy null individuals. CONCLUSIONS Our results provide evidence that the functional rs2814778 polymorphism in the gene encoding DARC is associated with worse clinical outcomes among African Americans with ALI, possibly via an increase in circulating IL-8.

FGF-23 and PTH levels in patients with acute kidney injury: A cross-sectional case series study

BackgroundFibroblast growth factor-23 (FGF-23), a novel regulator of mineral metabolism, is markedly elevated in chronic kidney disease and has been associated with poor long-term outcomes. However, whether FGF-23 has an analogous role in acute kidney injury is unknown. The goal of this study was to measure FGF-23 levels in critically ill patients with acute kidney injury to determine whether FGF-23 levels were elevated, as in chronic kidney disease.MethodsPlasma FGF-23 and intact parathyroid hormone (PTH) levels were measured in 12 patients with acute kidney injury and 8 control subjects.ResultsFGF-23 levels were significantly higher in acute kidney injury cases than in critically ill subjects without acute kidney injury, with a median FGF-23 level of 1948 RU/mL (interquartile range (IQR), 437-4369) in cases compared with 252 RU/mL (IQR, 65-533) in controls (p = 0.01). No correlations were observed between FGF-23 and severity of acute kidney injury (defined by the Acute Kidney Injury Network criteria); among patients with acute kidney injury, FGF-23 levels were higher in nonsurvivors than survivors (median levels of 4446 RU/mL (IQR, 3455-5443) versus 544 RU/mL (IQR, 390-1948; p = 0.02). Severe hyperparathyroidism (defined as intact PTH >250 mg/dL) was present in 3 of 12 (25%) of the acute kidney injury subjects versus none of the subjects without acute kidney injury, although this result did not meet statistical significance.ConclusionsWe provide novel data that demonstrate that FGF-23 levels are elevated in acute kidney injury, suggesting that FGF-23 dysregulation occurs in acute kidney injury as well as chronic kidney disease. Further studies are needed to define the short- and long-term clinical effects of dysregulated mineral metabolism in acute kidney injury patients.

Increased plasminogen activator inhibitor-1 concentrations in bronchoalveolar lavage fluids are associated with increased mortality in a cohort of patients with Pseudomonas aeruginosa.

Background:Increased plasminogen activator inhibitor-1 (PAI-1) concentrations are found in bronchoalveolar lavage (BAL) fluids from patients with ventilator-associated pneumonia or acute respiratory distress syndrome. The authors hypothesized that PAI-1 concentrations were associated with increased mortality in patients with either Pseudomonas aeruginosa–induced ventilator-associated pneumonia or tracheobronchial colonization. Methods:In a prospective cohort study, daily aspirates from intubated patients were cultured for P. aeruginosa. Positive patients had blind BAL (bBAL) that was processed for biomarker concentrations. Secretion of type III secretion cytotoxins were also analyzed from the P. aeruginosa strains. Results:Thirty-three patients were enrolled. Ten of the 33 patients died. bBAL PAI-1 concentrations were significantly increased in nonsurvivors compared with survivors (31.7 vs. 3.4 ng/ml, P = 0.001 for hospital mortality; 35.9 vs. 4.7 ng/ml, P = 0.02 for 28-day mortality). Even when acute respiratory distress syndrome patients were excluded, there was a significant difference between the survivors and nonsurvivors for bBAL PAI-1 concentrations (P = 0.005). Eighty-three percent of P. aeruginosa strains isolated from patients with high concentrations of bBAL PAI-1 also had strains that secreted cytotoxins. Conclusions:PAI-1 concentrations in bBALs correlated with mortality in ventilated patients with positive cultures for P. aeruginosa. Elevated bBAL PAI-1 concentrations also correlated with the secretion of type III exotoxins by P. aeruginosa.