Peptide inhibition of neutrophil-mediated injury after in vivo challenge with supernatant of Pseudomonas aeruginosa and immune-complexes.

Neutrophils are recognized for their role in host defense against pathogens as well as inflammatory conditions mediated through many mechanisms including neutrophil extracellular trap (NET) formation and generation of reactive oxygen species (ROS). NETs are increasingly appreciated as a major contributor in autoimmune and inflammatory diseases such as cystic fibrosis. Myeloperoxidase (MPO), a key neutrophil granule enzyme mediates generation of hypochlorous acid which, when extracellular, can cause host tissue damage. To better understand the role played by neutrophils in inflammatory diseases, we measured and modulated myeloperoxidase activity and NETs in vivo, utilizing a rat peritonitis model. RLS-0071 is a 15 amino acid peptide that has been shown to inhibit myeloperoxidase activity and NET formation in vitro. The rat model of inflammatory peritonitis was induced with intraperitoneal injection of either P. aeruginosa supernatant or immune-complexes. After euthanasia, a peritoneal wash was performed and measured for myeloperoxidase activity and free DNA as a surrogate for measurement of NETs. P. aeruginosa supernatant caused a 2-fold increase in MPO activity and free DNA when injected IP. Immune-complexes injected IP increased myeloperoxidase activity and free DNA 2- fold. RLS-0071 injection decreased myeloperoxidase activity and NETs in the peritoneal fluid generally to baseline levels in the presence of P. aeruginosa supernatant or immune-complexes. Taken together, RLS-0071 demonstrated the ability to inhibit myeloperoxidase activity and NET formation in vivo when initiated by different inflammatory stimuli including shed or secreted bacterial constituents as well as immune-complexes.

Peptide inhibition of acute lung injury in a novel two-hit rat model.

Acute lung injury (ALI) often causes severe trauma that may progress to significant morbidity and mortality. ALI results from a combination of the underlying clinical condition of the patient (e.g., inflammation) with a secondary insult such as viral pneumonia or a blood transfusion. While the secondary insult may be variable, the rapidly progressive disease process leading to pulmonary failure is typically mediated by an overwhelming innate immunological or inflammatory reaction driven by excessive complement and neutrophil-mediated inflammatory responses. We recently developed a 'two-hit' ALI rat model mediated by lipopolysaccharide followed by transfusion of incompatible human erythrocytes resulting in complement activation, neutrophil-mediated ALI and free DNA in the blood indicative of neutrophil extracellular trap formation. The objective of this study was to evaluate the role of peptide inhibitor of complement C1 (RLS-0071), a classical complement pathway inhibitor and neutrophil modulator in this animal model. Adolescent male Wistar rats were infused with lipopolysaccharide followed by transfusion of incompatible erythrocytes in the presence or absence of RLS-0071. Blood was collected at various time points to assess complement C5a levels, free DNA and cytokines in isolated plasma. Four hours following erythrocyte transfusion, lung tissue was recovered and assayed for ALI by histology. Compared to animals not receiving RLS-0071, lungs of animals treated with a single dose of RLS-0071 showed significant reduction in ALI as well as reduced levels of C5a, free DNA and inflammatory cytokines in the blood. These results demonstrate that RLS-0071 can modulate neutrophil-mediated ALI in this novel rat model.

A complement-mediated rat xenotransfusion model of platelet refractoriness.

BACKGROUND: Platelet refractoriness remains a challenging clinical dilemma although significant advancements have been made in identifying human leukocyte antigen (HLA) matched or HLA compatible units. Antiplatelet antibodies are the major risk factor for immune-mediated platelet refractoriness, yet the role of antibody-initiated complement-mediated platelet destruction remains poorly understood. STUDY DESIGN AND METHODS: Human complement-mediated opsonization and killing of platelets was assayed ex vivo using antibody-sensitized human platelets incubated with complement-sufficient human sera. A new animal model of platelet refractoriness utilizing Wistar rats transfused with human platelets is described. RESULTS: Human platelets sensitized with anti-platelet antibodies were rapidly opsonized with iC3b upon incubation in human sera. This opsonization could be completely blocked with a classical pathway complement inhibitor, PA-dPEG24. Complement activation decreased platelet viability, which was also reversible with complement inhibitor PA-dPEG24. A new rat model of platelet refractoriness was developed that demonstrated some platelet removal from the blood stream was complement mediated. CONCLUSIONS: Complement activation initiated by anti-platelet antibodies leads to complement opsonization and decreased platelet viability. A new rat model of platelet refractoriness was developed that adds a new tool for elucidating the mechanisms of platelet refractoriness.

Incompatible erythrocyte transfusion with lipopolysaccharide induces acute lung injury in a novel rat model.

Acute transfusion reactions can manifest in many forms including acute hemolytic transfusion reaction, allergic reaction and transfusion-related acute lung injury. We previously developed an acute hemolytic transfusion reaction rat model mediated by transfusion of incompatible human erythrocytes against which rats have preexisting antibodies resulting in classical complement pathway mediated intravascular hemolysis. In this study, the acute hemolytic transfusion reaction model was adapted to yield an acute lung injury phenotype. Adolescent male Wistar rats were primed in the presence or absence of lipopolysaccharide followed by transfusion of incompatible erythrocytes. Blood was collected at various time points during the course of the experiment to determine complement C5a levels and free DNA in isolated plasma. At 4 hours, blood and lung tissue were recovered and assayed for complete blood count and histological acute lung injury, respectively. Compared to sham animals or animals receiving increasing amounts of incompatible erythrocytes (equivalent to a 15-45% transfusion) in the absence of lipopolysaccharide, lungs of animals receiving lipopolysaccharide and a 30% erythrocyte transfusion showed dramatic alveolar wall thickening due to neutrophil infiltration. C5a levels were significantly elevated in these animals indicating that complement activation contributes to lung damage. Additionally, these animals demonstrated a significant increase of free DNA in the blood over time suggestive of neutrophil extracellular trap formation previously associated with transfusion-related acute lung injury in humans and mice. This novel 'two-hit' model utilizing incompatible erythrocyte transfusion in the presence of lipopolysaccharide yields a robust acute lung injury phenotype.

Inhibition of complement activation, myeloperoxidase, NET formation and oxidant activity by PIC1 peptide variants.

BACKGROUND: A product of rational molecular design, PA-dPEG24 is the lead derivative of the PIC1 family of peptides with multiple functional abilities including classical complement pathway inhibition, myeloperoxidase inhibition, NET inhibition and antioxidant activity. PA-dPEG24 is composed of a sequence of 15 amino acid, IALILEPICCQERAA, and contains a monodisperse 24-mer PEGylated moiety at its C terminus to increase aqueous solubility. Here we explore a sarcosine substitution scan of the PA peptide to evaluate impacts on solubility in the absence of PEGylation and functional characteristics. METHODS: Sixteen sarcosine substitution variants were synthesized and evaluated for solubility in water. Aqueous soluble variants were then tested in standard complement, myeloperoxidase, NET formation and antioxidant capacity assays. RESULTS: Six sarcosine substitution variants were aqueous soluble without requiring PEGylation. Substitution with sarcosine of the isoleucine at position eight yielded a soluble peptide that surpassed the parent molecule for complement inhibition and myeloperoxidase inhibition. Substitution with sarcosine of the cysteine at position nine improved solubility, but did not otherwise change the functional characteristics compared with the parent compound. However, replacement of both vicinal cysteine residues at positions 9 and 10 with a single sarcosine residue reduced functional activity in most of the assays tested. CONCLUSIONS: Several of the sarcosine PIC1 variant substitutions synthesized yielded improved solubility as well as a number of unanticipated structure-function findings that provide new insights. Several sarcosine substitution variants demonstrate increased potency over the parent peptide suggesting enhanced therapeutic potential for inflammatory disease processes involving complement, myeloperoxidase, NETs or oxidant stress.

Inhibition of Immune Complex Complement Activation and Neutrophil Extracellular Trap Formation by Peptide Inhibitor of Complement C1.

Two major aspects of systemic lupus erythematosus (SLE) pathogenesis that have yet to be targeted therapeutically are immune complex-initiated complement activation and neutrophil extracellular trap (NET) formation by neutrophils. Here, we report in vitro testing of peptide inhibitor of complement C1 (PIC1) in assays of immune complex-mediated complement activation in human sera and assays for NET formation by human neutrophils. The lead PIC1 derivative, PA-dPEG24, was able to dose-dependently inhibit complement activation initiated by multiple types of immune complexes (IC), including C1-anti-C1q IC, limiting the generation of pro-inflammatory complement effectors, including C5a and membrane attack complex (sC5b-9). In several instances, PA-dPEG24 achieved complete inhibition with complement effector levels equivalent to background. PA-dPEG24 was also able to dose-dependently inhibit NET formation by human neutrophils stimulated by PMA, MPO, or immune complex activated human sera. In several instances PA-dPEG24 achieved complete inhibition with NETosis with quantitation equivalent to background levels. These results suggest that PA-dPEG24 inhibition of NETs occurs by blocking the MPO pathway of NET formation. Together these results demonstrate that PA-dPEG24 can inhibit immune complex activation of the complement system and NET formation. This provides proof of concept that peptides can potentially be developed to inhibit these two important contributors to rheumatologic pathology that are currently untargeted by available therapies.

A GAL4-driver line resource for Drosophila neurobiology.

We established a collection of 7,000 transgenic lines of Drosophila melanogaster. Expression of GAL4 in each line is controlled by a different, defined fragment of genomic DNA that serves as a transcriptional enhancer. We used confocal microscopy of dissected nervous systems to determine the expression patterns driven by each fragment in the adult brain and ventral nerve cord. We present image data on 6,650 lines. Using both manual and machine-assisted annotation, we describe the expression patterns in the most useful lines. We illustrate the utility of these data for identifying novel neuronal cell types, revealing brain asymmetry, and describing the nature and extent of neuronal shape stereotypy. The GAL4 lines allow expression of exogenous genes in distinct, small subsets of the adult nervous system. The set of DNA fragments, each driving a documented expression pattern, will facilitate the generation of additional constructs for manipulating neuronal function.