De novo design of highly selective miniprotein inhibitors of integrins αvß6 and αvß8.

The RGD (Arg-Gly-Asp)-binding integrins αvß6 and αvß8 are clinically validated cancer and fibrosis targets of considerable therapeutic importance. Compounds that can discriminate between homologous αvß6 and αvß8 and other RGD integrins, stabilize specific conformational states, and have high thermal stability could have considerable therapeutic utility. Existing small molecule and antibody inhibitors do not have all these properties, and hence new approaches are needed. Here we describe a generalized method for computationally designing RGD-containing miniproteins selective for a single RGD integrin heterodimer and conformational state. We design hyperstable, selective αvß6 and αvß8 inhibitors that bind with picomolar affinity. CryoEM structures of the designed inhibitor-integrin complexes are very close to the computational design models, and show that the inhibitors stabilize specific conformational states of the αvß6 and the αvß8 integrins. In a lung fibrosis mouse model, the αvß6 inhibitor potently reduced fibrotic burden and improved overall lung mechanics, demonstrating the therapeutic potential of de novo designed integrin binding proteins with high selectivity.

De novo design of highly selective miniprotein inhibitors of integrins αvß6 and αvß8.

The RGD (Arg-Gly-Asp)-binding integrins αvß6 and αvß8 are clinically validated cancer and fibrosis targets of considerable therapeutic importance. Compounds that can discriminate between the two closely related integrin proteins and other RGD integrins, stabilize specific conformational states, and have sufficient stability enabling tissue restricted administration could have considerable therapeutic utility. Existing small molecules and antibody inhibitors do not have all of these properties, and hence there is a need for new approaches. Here we describe a method for computationally designing hyperstable RGD-containing miniproteins that are highly selective for a single RGD integrin heterodimer and conformational state, and use this strategy to design inhibitors of αvß6 and αvß8 with high selectivity. The αvß6 and αvß8 inhibitors have picomolar affinities for their targets, and >1000-fold selectivity over other RGD integrins. CryoEM structures are within 0.6-0.7Å root-mean-square deviation (RMSD) to the computational design models; the designed αvß6 inhibitor and native ligand stabilize the open conformation in contrast to the therapeutic anti-αvß6 antibody BG00011 that stabilizes the bent-closed conformation and caused on-target toxicity in patients with lung fibrosis, and the αvß8 inhibitor maintains the constitutively fixed extended-closed αvß8 conformation. In a mouse model of bleomycin-induced lung fibrosis, the αvß6 inhibitor potently reduced fibrotic burden and improved overall lung mechanics when delivered via oropharyngeal administration mimicking inhalation, demonstrating the therapeutic potential of de novo designed integrin binding proteins with high selectivity.

The bioactivity of soluble Fas ligand is modulated by key amino acids of its stalk region.

We have previously reported that the 26-amino acid N-terminus stalk region of soluble Fas ligand (sFasL), which is separate from its binding site, is required for its biological function. Here we investigate the mechanisms that link the structure of the sFasL stalk region with its function. Using site-directed mutagenesis we cloned a mutant form of sFasL in which all the charged amino acids of the stalk region were changed to neutral alanines (mut-sFasL). We used the Fas-sensitive Jurkat T-cell line and mouse and human alveolar epithelial cells to test the bioactivity of sFasL complexes, using caspase-3 activity and Annexin-V externalization as readouts. Finally, we tested the effects of mut-sFasL on lipopolysaccharide-induced lung injury in mice. We found that mutation of all the 8 charged amino acids of the stalk region into the non-charged amino acid alanine (mut-sFasL) resulted in reduced apoptotic activity compared to wild type sFasL (WT-sFasL). The mut-sFasL attenuated WT-sFasL function on the Fas-sensitive human T-cell line Jurkat and on primary human small airway epithelial cells. The inhibitory mechanism was associated with the formation of complexes of mut-sFasL with the WT protein. Intratracheal administration of the mut-sFasL to mice 24 hours after intratracheal Escherichia coli lipopolysaccharide resulted in attenuation of the inflammatory response 24 hours later. Therefore, the stalk region of sFasL has a critical role on bioactivity, and changes in the structure of the stalk region can result in mutant variants that interfere with the wild type protein function in vitro and in vivo.

Comparison of direct and indirect models of early induced acute lung injury.

BACKGROUND: The animal experimental counterpart of human acute respiratory distress syndrome (ARDS) is acute lung injury (ALI). Most models of ALI involve reproducing the clinical risk factors associated with human ARDS, such as sepsis or acid aspiration; however, none of these models fully replicates human ARDS. AIM: To compare different experimental animal models of ALI, based on direct or indirect mechanisms of lung injury, to characterize a model which more closely could reproduce the acute phase of human ARDS. MATERIALS AND METHODS: Adult male Sprague-Dawley rats were subjected to intratracheal instillations of (1) HCl to mimic aspiration of gastric contents; (2) lipopolysaccharide (LPS) to mimic bacterial infection; (3) HCl followed by LPS to mimic aspiration of gastric contents with bacterial superinfection; or (4) cecal ligation and puncture (CLP) to induce peritonitis and mimic sepsis. Rats were sacrificed 24 h after instillations or 24 h after CLP. RESULTS: At 24 h, rats instilled with LPS or HCl-LPS had increased lung permeability, alveolar neutrophilic recruitment and inflammatory markers (GRO/KC, TNF-α, MCP-1, IL-1ß, IL-6). Rats receiving only HCl or subjected to CLP had no evidence of lung injury. CONCLUSIONS: Rat models of ALI induced directly by LPS or HCl-LPS more closely reproduced the acute phase of human ARDS than the CLP model of indirectly induced ALI.

Alveolar CCN1 is associated with mechanical stretch and acute respiratory distress syndrome severity.

The cellular communication network factor 1 (CCN1) is a matricellular protein that can modulate multiple tissue responses, including inflammation and repair. We have previously shown that adenoviral overexpression of Ccn1 is sufficient to cause acute lung injury in mice. We hypothesized that CCN1 is present in the airspaces of lungs during the acute phase of lung injury, and higher concentrations are associated with acute respiratory distress syndrome (ARDS) severity. We tested this hypothesis by measuring 1) CCN1 in bronchoalveolar lavage fluid (BALF) and lung homogenates from mice subjected to ventilation-induced lung injury (VILI), 2) Ccn1 gene expression and protein levels in MLE-12 cells (alveolar epithelial cell line) subjected to mechanical stretch, and 3) CCN1 in BALF from mechanically ventilated humans with and without ARDS. BALF CCN1 concentrations and whole lung CCN1 protein levels were significantly increased in mice with VILI (n = 6) versus noninjured controls (n = 6). Ccn1 gene expression and CCN1 protein levels were increased in MLE-12 cells cultured under stretch conditions. Subjects with ARDS (n = 77) had higher BALF CCN1 levels compared with mechanically ventilated subjects without ARDS (n = 45) (P < 0.05). In subjects with ARDS, BALF CCN1 concentrations were associated with higher total protein, sRAGE, and worse [Formula: see text]/[Formula: see text] ratios (all P < 0.05). CCN1 is present in the lungs of mice and humans during the acute inflammatory phase of lung injury, and concentrations are higher in patients with increased markers of severity. Alveolar epithelial cells may be an important source of CCN1 under mechanical stretch conditions.

A 68-Year-Old Man With Skin Rash and a Pleural Effusion.

CASE PRESENTATION: A 68-year-old man developed an erythematous, papular, pruritic rash on his right thigh 1 month prior to presentation. It subsequently spread to his other extremities and trunk. He also endorsed fevers of > 38.3°C, night sweats, fatigue, shortness of breath, and a dry cough. He was prescribed triamcinolone 0.1% cream for his rash and azithromycin for presumed community-acquired pneumonia, with no improvement in symptoms. He had a history of relapsing polychondritis for which he was prescribed infliximab and low-dose prednisone. He had never smoked tobacco, did not use alcohol or illicit substances, and had no significant travel history.

Fluid restriction reduces pulmonary edema in a model of acute lung injury in mechanically ventilated rats.

Experimental acute lung injury models are often used to increase our knowledge on the acute respiratory distress syndrome (ARDS), however, existing animal models often do not take into account the impact of specific fluid strategies on the development of lung injury. In contrast, the current literature strongly suggests that fluid management strategies have a significant impact on clinical outcome of patients with ARDS. Thus, it is important to characterize the role of fluid management strategies in experimental models of lung injury. In this study we investigated the effect of two different fluid strategies on commonly used outcome variables in a short-term model of acute lung injury, in relation to age. Infant (2-3 weeks) and adult (3-4 months) Wistar rats received intratracheal instillations of lipopolysaccharide and 24 hours later were mechanically ventilated for 6 hours. During mechanical ventilation, rats from both age groups were randomized to either a standard or conservative intravenous fluid strategy. We found that the hemodynamic response in infant and adult rats was similar in both fluid strategies. Lung wet-to-dry ratios were lower in adult, but not in infant rats receiving the conservative fluid strategy as compared to the standard fluid strategy. There were age-related differences in markers of alveolar capillary barrier disruption and alveolar fluid clearance, yet these were unaffected by fluid strategy. Finally, we found significantly higher IL-1ß and TNF-α concentrations in the adult rats treated with the conservative as compared to the standard fluid regimen. In conclusion, the choice of fluid strategy in mechanically ventilated rats with experimental LPS-induced acute lung injury has a significant effect on pulmonary extravascular water, an important and well-recognized lung injury marker, and on the local pro-inflammatory cytokine profiles. We advocate the use of a more uniform, conservative, fluid strategy regimen in experimental models of acute lung injury.

Fas activation alters tight junction proteins in acute lung injury.

Background:The acute respiratory distress syndrome (ARDS) is characterized by protein-rich oedema in the alveolar spaces, a feature in which Fas-mediated apoptosis of the alveolar epithelium has been involved. Objective:To determine whether Fas activation increases protein permeability by mechanisms involving disruption of the paracellular tight junction (TJ) proteins in the pulmonary alveoli. Methods: Protein permeability and the expression of TJ proteins were assessed in vivo in wild-type and Fas-deficient lpr mice 16 hours after the intratracheal instillation of recombinant human soluble Fas ligand (rh-sFasL), and at different time points in vitro in human pulmonary alveolar epithelial cells (HPAEpiC) exposed to rh-sFasL Results:Activation of the Fas pathway increased protein permeability in mouse lungs and altered the expression of the TJ proteins occludin and zonula occludens-1 in the alveolar-capillary membrane in vivo and in human alveolar epithelial cell monolayers in vitro. Blockade of caspase-3, but not inhibition of tyrosine kinase dependent pathways, prevented the alterations in TJ protein expression and permeability induced by the Fas/FasL system in human alveolar cell monolayers in vitro. We also observed that both the Fas-induced increase of protein permeability and disruption of TJ proteins occurred before cell death could be detected in the cell monolayers in vitro. Conclusion:Targeting caspase pathways could prevent the disruption of TJs and reduce the formation of lung oedema in the early stages of ARDS.

Acute Respiratory Distress Syndrome and Diffuse Alveolar Damage. New Insights on a Complex Relationship.

Acute respiratory distress syndrome (ARDS) is a major clinical problem with high morbidity and mortality. Diffuse alveolar damage (DAD) is considered the histological hallmark for the acute phase of ARDS. DAD is characterized by an acute phase with edema, hyaline membranes, and inflammation, followed by an organizing phase with alveolar septal fibrosis and type II pneumocyte hyperplasia. Given the difficulties in obtaining a biopsy in patients with ARDS, the presence of DAD is not required to make the diagnosis. However, biopsy and autopsy studies suggest that only one-half of patients who meet the clinical definition of ARDS also have DAD. The other half are found to have a group of heterogeneous disorders, including pneumonia. Importantly, the subgroup of patients with ARDS who also have DAD appears to have increased mortality. It is possible that the response of these patients to specific therapies targeting the molecular mechanisms of ARDS may differ from patients without DAD. Therefore, it may be important to develop noninvasive methods to identify DAD. A predictive model for DAD based on noninvasive measurements has been developed in an autopsy cohort but must be validated. It would be ideal to identify biomarkers or imaging techniques that help determine which patients with ARDS have DAD. We conclude that additional studies are needed to determine the effect of DAD on outcomes in ARDS, and whether noninvasive techniques to identify DAD should be developed with the goal of determining whether this population responds differently to specific therapies targeting the molecular mechanisms of ARDS.

Symptom profiles and inflammatory markers in moderate to severe COPD.

BACKGROUND: Physical and psychological symptoms are the hallmark of patients' subjective perception of their illness. The purpose of this analysis was to determine if patients with COPD have distinctive symptom profiles and to examine the association of symptom profiles with systemic biomarkers of inflammation. METHODS: We conducted latent class analyses of three physical (dyspnea, fatigue, and pain) and two psychological symptoms (depression and anxiety) in 302 patients with moderate to severe COPD using baseline data from a longitudinal observational study of depression in COPD. Systemic inflammatory markers included IL1, IL8, IL10, IL12, IL13, INF, GM-CSF, TNF-α (levels >75thcentile was considered high); and CRP (levels >3 mg/L was considered high). Multinominal logistic regression models were used to examine the association between symptom classes and inflammation while adjusting for key socio-demographic and disease characteristics. RESULTS: We found that a 4-class model best fit the data: 1) low physical and psychological symptoms (26%, Low-Phys/Low-Psych), 2) low physical but moderate psychological symptoms (18%, Low-Phys/Mod Psych), 3) high physical but moderate psychological symptoms (25%, High-Phys/Mod Psych), and 4) high physical and psychological symptoms (30%, High-Phys/High Psych). Unadjusted analyses showed associations between symptom class with high levels of IL7, IL-8 (p ≤ .10) and CRP (p < .01). In the adjusted model, those with a high CRP level were less likely to be in the High-Phys/Mod-Psych class compared to the Low-Phys/Low-Psych (OR: 0.41, 95%CI 0.19, 0.90) and Low-Phys/Mod-Psych classes (OR: 0.35, 95%CI 0.16, 0.78); elevated CRP was associated with in increased odds of being in the High-Phys/High-Psych compared to the High-Phys/Mod-Psych class (OR: 2.22, 95%CI 1.08, 4.58). Younger age, having at least a college education, oxygen use and depression history were more prominent predictors of membership in the higher symptom classes. CONCLUSIONS: Patients with COPD can be classified into four distinct symptom classes based on five commonly co-occurring physical and psychological symptoms. Systemic biomarkers of inflammation were not associated with symptom class. Additional work to test the reliability of these symptom classes, their biological drivers and their validity for prognostication and tailoring therapy in larger and more diverse samples is needed. TRIAL REGISTRATION: Clinicaltrials.gov, NCT01074515 .

IVIG-mediated protection against necrotizing pneumonia caused by MRSA.

New therapeutic approaches are urgently needed to improve survival outcomes for patients with necrotizing pneumonia caused by Staphylococcus aureus One such approach is adjunctive treatment with intravenous immunoglobulin (IVIG), but clinical practice guidelines offer conflicting recommendations. In a preclinical rabbit model, prophylaxis with IVIG conferred protection against necrotizing pneumonia caused by five different epidemic strains of community-associated methicillin-resistant S. aureus (MRSA) as well as a widespread strain of hospital-associated MRSA. Treatment with IVIG, either alone or in combination with vancomycin or linezolid, improved survival outcomes in this rabbit model. Two specific IVIG antibodies that neutralized the toxic effects of α-hemolysin (Hla) and Panton-Valentine leukocidin (PVL) conferred protection against necrotizing pneumonia in the rabbit model. This mechanism of action of IVIG was uncovered by analyzing loss-of-function mutant bacterial strains containing deletions in 17 genes encoding staphylococcal exotoxins, which revealed only Hla and PVL as having an impact on necrotizing pneumonia. These results demonstrate the potential clinical utility of IVIG in the treatment of severe pneumonia induced by S. aureus.

Endogenous secreted phospholipase A2 group X regulates cysteinyl leukotrienes synthesis by human eosinophils.

BACKGROUND: Phospholipase A2s mediate the rate-limiting step in the formation of eicosanoids such as cysteinyl leukotrienes (CysLTs). Group IVA cytosolic PLA2α (cPLA2α) is thought to be the dominant PLA2 in eosinophils; however, eosinophils also have secreted PLA2 (sPLA2) activity that has not been fully defined. OBJECTIVES: To examine the expression of sPLA2 group X (sPLA2-X) in eosinophils, the participation of sPLA2-X in the formation of CysLTs, and the mechanism by which sPLA2-X initiates the synthesis of CysLTs in eosinophils. METHODS: Peripheral blood eosinophils were obtained from volunteers with asthma and/or allergy. A rabbit polyclonal anti-sPLA2-X antibody identified sPLA2-X by Western blot. We used confocal microscopy to colocalize the sPLA2-X to intracellular structures. An inhibitor of sPLA2-X (ROC-0929) that does not inhibit other mammalian sPLA2s, as well as inhibitors of the mitogen-activated kinase cascade (MAPK) and cPLA2α, was used to examine the mechanism of N-formyl-methionyl-leucyl-phenylalanine (fMLP)-mediated formation of CysLT. RESULTS: Eosinophils express the mammalian sPLA2-X gene (PLA2G10). The sPLA2-X protein is located in the endoplasmic reticulum, golgi, and granules of eosinophils and moves to the granules and lipid bodies during fMLP-mediated activation. Selective sPLA2-X inhibition attenuated the fMLP-mediated release of arachidonic acid and CysLT formation by eosinophils. Inhibitors of p38, extracellular-signal-regulated kinases 1/2 (p44/42 MAPK), c-Jun N-terminal kinase, and cPLA2α also attenuated the fMLP-mediated formation of CysLT. The sPLA2-X inhibitor reduced the phosphorylation of p38 and extracellular-signal-regulated kinases 1/2 (p44/42 MAPK) as well as cPLA2α during cellular activation, indicating that sPLA2-X is involved in activating the MAPK cascade leading to the formation of CysLT via cPLA2α. We further demonstrate that sPLA2-X is activated before secretion from the cell during activation. Short-term priming with IL-13 and TNF/IL-1ß increased the expression of PLA2G10 by eosinophils. CONCLUSIONS: These results demonstrate that sPLA2-X plays a significant role in the formation of CysLTs by human eosinophils. The predominant role of the enzyme is the regulation of MAPK activation that leads to the phosphorylation of cPLA2α. The sPLA2-X protein is regulated by proteolytic cleavage, suggesting that an inflammatory environment may promote the formation of CysLTs through this mechanism. These results have important implications for the treatment of eosinophilic disorders such as asthma.

The caspase inhibitor zVAD increases lung inflammation in pneumovirus infection in mice.

Severe respiratory syncytial virus (RSV) disease is a frequent cause of acute respiratory distress syndrome (ARDS) in young children, and is associated with marked lung epithelial injury and neutrophilic inflammation. Experimental studies on ARDS have shown that inhibition of apoptosis in the lungs reduces lung epithelial injury. However, the blockade of apoptosis in the lungs may also have deleterious effects by hampering viral clearance, and importantly, by enhancing or prolonging local proinflammatory responses. The aim of this study was to determine the effect of the broad caspase inhibitor Z-VAD(OMe)-FMK (zVAD) on inflammation and lung injury in a mouse pneumovirus model for severe RSV disease. Eight- to 11-week-old female C57BL/6OlaHsd mice were inoculated with the rodent-specific pneumovirus pneumonia virus of mice (PVM) strain J3666 and received multiple injections of zVAD or vehicle (control) during the course of disease, after which they were studied for markers of apoptosis, inflammation, and lung injury on day 7 after infection. PVM-infected mice that received zVAD had a strong increase in neutrophil numbers in the lungs, which was associated with decreased neutrophil apoptosis. Furthermore, zVAD treatment led to higher concentrations of several proinflammatory cytokines in the lungs and more weight loss in PVM-infected mice. In contrast, zVAD did not reduce apoptosis of lung epithelial cells and did not affect the degree of lung injury, permeability, and viral titers in PVM disease. We conclude that zVAD has an adverse effect in severe pneumovirus disease in mice by enhancing the lung proinflammatory response.