Loss of neutrophil Shp1 produces hemorrhagic and lethal acute lung injury.

The acute respiratory distress syndrome (ARDS) is associated with significant morbidity and mortality and neutrophils are critical to its pathogenesis. Neutrophil activation is closely regulated by inhibitory tyrosine phosphatases including Src homology region 2 domain containing phosphatase-1 (Shp1). Here, we report that loss of neutrophil Shp1 in mice produced hyperinflammation and lethal pulmonary hemorrhage in sterile inflammation and pathogen-induced models of acute lung injury (ALI) through a Syk kinase-dependent mechanism. We observed large intravascular neutrophil clusters, perivascular inflammation, and excessive neutrophil extracellular traps in neutrophil-specific Shp1 knockout mice suggesting an underlying mechanism for the observed pulmonary hemorrhage. Targeted immunomodulation through the administration of a Shp1 activator (SC43) reduced agonist-induced reactive oxygen species in vitro and ameliorated ALI-induced alveolar neutrophilia and NETs in vivo. We propose that the pharmacologic activation of Shp1 has the potential to fine-tune neutrophil hyperinflammation that is central to the pathogenesis of ARDS.

Aspirin reduces lipopolysaccharide-induced pulmonary inflammation in human models of ARDS.

RATIONALE: Platelets play an active role in the pathogenesis of acute respiratory distress syndrome (ARDS). Animal and observational studies have shown aspirin's antiplatelet and immunomodulatory effects may be beneficial in ARDS. OBJECTIVE: To test the hypothesis that aspirin reduces inflammation in clinically relevant human models that recapitulate pathophysiological mechanisms implicated in the development of ARDS. METHODS: Healthy volunteers were randomised to receive placebo or aspirin 75  or 1200 mg (1:1:1) for seven days prior to lipopolysaccharide (LPS) inhalation, in a double-blind, placebo-controlled, allocation-concealed study. Bronchoalveolar lavage (BAL) was performed 6 hours after inhaling 50 µg of LPS. The primary outcome measure was BAL IL-8. Secondary outcome measures included markers of alveolar inflammation (BAL neutrophils, cytokines, neutrophil proteases), alveolar epithelial cell injury, systemic inflammation (neutrophils and plasma C-reactive protein (CRP)) and platelet activation (thromboxane B2, TXB2). Human lungs, perfused and ventilated ex vivo (EVLP) were randomised to placebo or 24 mg aspirin and injured with LPS. BAL was carried out 4 hours later. Inflammation was assessed by BAL differential cell counts and histological changes. RESULTS: In the healthy volunteer (n=33) model, data for the aspirin groups were combined. Aspirin did not reduce BAL IL-8. However, aspirin reduced pulmonary neutrophilia and tissue damaging neutrophil proteases (Matrix Metalloproteinase (MMP)-8/-9), reduced BAL concentrations of tumour necrosis factor α and reduced systemic and pulmonary TXB2. There was no difference between high-dose and low-dose aspirin. In the EVLP model, aspirin reduced BAL neutrophilia and alveolar injury as measured by histological damage. CONCLUSIONS: These are the first prospective human data indicating that aspirin inhibits pulmonary neutrophilic inflammation, at both low and high doses. Further clinical studies are indicated to assess the role of aspirin in the prevention and treatment of ARDS. TRIAL REGISTRATION NUMBER: NCT01659307 Results.

Incidence and clinical characteristics of transfusion-associated circulatory overload using an active surveillance algorithm.

BACKGROUND: The concordance of haemovigilance criteria developed for surveillance of transfusion-associated circulatory overload (TACO) with its clinical diagnosis has not been assessed. In a pilot study to evaluate an electronic screening algorithm, we sought to examine TACO incidence and application of haemovigilance criteria in patients with post-transfusion pulmonary oedema. STUDY DESIGN AND METHODS: From June to September 2014, all transfused adult inpatients at four academic hospitals were screened with an algorithm identifying chest radiographs ordered within 12 h of blood component release. Patients with post-transfusion pulmonary oedema underwent case adjudication by an expert panel. TACO incidence was calculated, and clinical characteristics were compared with other causes of post-transfusion pulmonary oedema. RESULTS: Among 4932 transfused patients, there were 3412 algorithm alerts, 50 cases of TACO and 47 other causes of pulmonary oedema. TACO incidence was 1 case per 100 patients transfused. TACO classification based on two sets of haemovigilance criteria (National Healthcare Safety Network and proposed revised International Society for Blood Transfusion) was concordant with expert panel diagnosis in 57% and 54% of reviewed cases, respectively. Although the majority of clinical parameters did not differentiate expert panel adjudicated TACO from other cases, improved oxygenation within 24 h of transfusion did (P = 0·01). CONCLUSIONS: The incidence of TACO was similar to that observed in prior studies utilizing active surveillance. Case classification by haemovigilance criteria was frequently discordant with clinical diagnoses of TACO in patients with post-transfusion pulmonary oedema. Improvements in oxygenation within 24 h of transfusion merit further evaluation in the diagnosis of TACO.

Inhibiting Integrin αvß5 Reduces Ischemia-Reperfusion Injury in an Orthotopic Lung Transplant Model in Mice.

Primary graft dysfunction after lung transplantation is the leading cause of morbidity and mortality in the immediate posttransplant period and is characterized by endothelial and epithelial barrier disruption and the leakage of protein-rich edema fluid. Integrins are cell surface receptors that have an important role in maintenance of the cell barrier, and inhibition of integrins, such as αvß5, can diminish alveolar flooding in lung injury models. We hypothesized that inhibition of αvß5 during donor lung cold ischemia would reduce endothelial permeability during reperfusion. Using an orthotopic single lung transplantation model with and without cold ischemia, donor lungs were perfused with αvß5-blocking antibody (ALULA) or control antibody at the time of collection, followed by transplantation, 8 h of reperfusion, and the measurement of lung injury parameters. Prolonged cold ischemia (18 h) produced increases in extravascular lung water, protein permeability, and neutrophilic alveolitis and decreased oxygenation compared with lungs without cold ischemia. Perfusion of lungs with αvß5 antibody versus control antibody protected donor lungs from injury and significantly improved oxygenation. In summary, αvß5 integrin blockade protects from the development of ischemia-reperfusion lung injury and is a promising approach to preventing primary graft dysfunction in human lung transplant procedures.

Role of coagulation pathways and treatment with activated protein C in hyperoxic lung injury.

BACKGROUND: Activated protein C (APC) significantly decreases mortality in severe sepsis, but its role in acute lung injury from non-infectious aetiologies is unclear. The role of APC in hyperoxic acute lung injury was tested by studying the physiology of lung injury development, measurement of key coagulation proteins and treatment with murine APC (mAPC). METHODS: Mice were continuously exposed to >95% oxygen and lung injury was assessed by extravascular lung water, lung vascular protein permeability and alveolar fluid clearance. Coagulation proteins were measured in bronchoalveolar lavage (BAL) fluid and plasma. Recombinant mAPC was administered in preventive and treatment strategies. RESULTS: Hyperoxia produced dramatic increases in lung vascular permeability and extravascular lung water between 72 and 96 h. Lung fluid balance was also adversely affected by progressive decreases in basal and cAMP-stimulated alveolar fluid clearance. Plasma levels of APC decreased at 72 h and were 90% depleted at 96 h. There were significant increases in BAL fluid levels of thrombomodulin, thrombin-antithrombin complexes and plasminogen activator inhibitor-1 at later time points of hyperoxia. Lung thrombomodulin expression was severely decreased during late hyperoxia and plasma levels of APC were not restored by excess thrombin administration. Administration of recombinant mAPC failed to improve indices of lung injury. CONCLUSIONS: Hyperoxic acute lung injury produces procoagulant changes in the lung with a decrease in plasma levels of APC due to significant endothelial dysfunction. Replacement of mAPC failed to improve lung injury.

Transfusion-related acute lung injury in the paediatric patient: Two case reports and a review of the literature.

Transfusion-related acute lung injury (TRALI) is increasingly recognized as a major complication of transfusion therapy; it was the leading cause of transfusion-related fatalities in the United States in 2003. Most cases of TRALI that have been reported are in adult patients. We present two cases of TRALI that occurred in children and review the existing literature of paediatric TRALI. The paediatric TRALI case reports highlight two laboratory findings that can help in the diagnosis of TRALI: transient leucopenia and an elevated pulmonary oedema fluid/plasma protein ratio. These two simple diagnostic tests can help rule out other diagnoses and add confidence to the clinical diagnosis of TRALI. Finally, our first case also highlights the potential danger of directed maternal blood donations, which may increase the risk of paediatric TRALI.

Synaptophysin immunoreactivity in temporal lobe epilepsy-associated hippocampal sclerosis.

We have previously devised a semiquantitative grading system for hippocampal sclerosis (HS) in specimens resected for intractable temporal lobe epilepsy. The grades range from zero to four based on the amount and distribution of neuronal loss and gliosis. In the present study hippocampal sections from 25 patients who had temporal lobe epilepsy and had previously been assigned a grade were examined with synaptophysin immunohistochemistry, and the synaptic content in specific hippocampal fields was correlated with the results of the HS grading system. There was evidence of both significant synaptic loss and increased synaptic density in different fields of the hippocampus with increasing HS. A marked decrement of synaptic inmmunostaining was present in fields CA1 and CA4 that were highly correlated with HS grade. Sector CA4 seemed to respond in a more graded or continuous way to the pathological insults occurring in temporal lobe epilepsy than did CA1, which appeared to exhibit an all or nothing response. Also, while the width of the outer part of the molecular layer of the dentate (mid) gyrus decreased with increasing HS grade, the inner part of the mid became wider and showed an increased synaptic density so that the overall width of the mid was increased in the high-grade group. We conclude that quantitative measurement of synaptic loss in CAI and CA4 using synaptophysin immunohistochemistry is a sensitive method for detecting HS and correlates well with the empirically derived HS grading scale, with CA4 exhibiting a more graded response than CA1. In addition, a plasticity response in the inner part of the mld in patients with high-grade HS has been confirmed and quantitated.

Synaptophysin immunohistochemistry densitometry measurement in resected human hippocampus: implication for the etiology of hippocampal sclerosis.

Synaptophysin (SY) is a protein expressed at presynaptic vesicles. SY immunohistochemistry (IHC) was undertaken in surgically resected hippocampal specimens from 25 patients with intractable epilepsy. All were investigated with chronic ictal EEG videotelemetry, which showed a temporal onset in each case, and all had normal magnetic resonance imaging (MRI). The density of reaction product of SY IHC was used to generate optical density (OD) measurements as an estimate of synaptic density in CA1 and CA4 fields (ODCA1 and ODCA4), and inner third and outer two-thirds of the molecular layer of the dentate gyrus (ODIML and ODOML). There was significant correlation between ODCA1 (r=0.619, P=0.001) and ODCA4 (r=0.639, P=0.001) and onset age of epilepsy. There was no correlation between ODCA1 and duration of epilepsy. There was correlation between ODCA4 and duration (r=-0.412, P=0.041), but partial correlations showed no significant correlation between ODCA4 and duration when controlling for onset, although correlation between ODCA4 and onset when controlling for duration remained significant (r=0.536, P < 0.01). Univariate ANOVA revealed onset age to be the only predictor of ODCA1 and ODCA4. Chronological age and duration were not predictors. There was no correlation between ODIML or ODOML and either onset age or duration. ODIML and ODOML were not predicted by onset age, duration or chronological age. These findings support the hypothesis that the major cause of hippocampal sclerosis is an age specific insult rather than the result of repeated seizures.