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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., Competing Interests: The authors have read the journal’s policy and the authors of this manuscript have the following competing interests: Alana Sampson, Brittany Lassiter, Pamela Hair, Adrianne Enos, Kenji Cunnion and Neel Krishna are employees of ReAlta Life Sciences. Neel Krishna and Kenji Cunnion have ownership of ReAlta shares and Kenji Cunnion serves as on the Board of Directors of ReAlta Life Sciences. Neel K Krishna and Kenji M Cunnion are listed as inventors on patents that describe PIC1 molecules. The authors would like to declare the following patents/patent applications associated with this research: 8,241,843 methods for regulating complement cascade proteins using astrovirus coat protein and derivatives thereof, 15/738,786 synthetic peptide compounds and methods of use, 16/400,486 synthetic peptide compounds and methods of use, 8,906,845 peptide compounds to regulate the complement system, 9,422,337 peptide compounds to regulate the complement system, 10,005,818 derivative peptide compounds and methods of use, 9,914,753 peptide compounds to regulate the complement system, 10,414,799 peptide compounds to regulate the complement system, 16/534,200 peptide compounds to regulate the complement system, 16/242,550 PIC1 inhibition of myeloperoxidase oxidative activity in an animal model. The commercial affiliation with ReAlta Life Sciences does not alter our adherence to PLOS ONE policies on sharing data and materials.

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., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: In addition to their academic appointments, Neel K Krishna and Kenji M Cunnion are officers of ReAlta Life Sciences, Inc. guiding the development of PIC1 molecules for clinical applications. ReAlta Life Sciences was founded by Eastern Virginia Medical School, Children’s Hospital of the King’s Daughters and Eriko Life Science Ventures. Oversight for potential conflict of interest is provided by Eastern Virginia Medical School Conflict of Interest Committee. Neel K Krishna and Kenji M Cunnion are listed as inventors on patents that describe PIC1 molecules. The authors would like to declare the following patents/patent applications associated with this research: 8,241,843 methods for regulating complement cascade proteins using astrovirus coat protein and derivatives thereof, 15/738,786 synthetic peptide compounds and methods of use,16/400,486 synthetic peptide compounds and methods of use, 8,906,845 peptide compounds to regulate the complement system, 9,422,337 peptide compounds to regulate the complement system, 10,005,818 derivative peptide compounds and methods of use, 9,914,753 peptide compounds to regulate the complement system, 10,414,799 peptide compounds to regulate the complement system, 16/534,200 peptide compounds to regulate the complement system, 16/242,550 pic1 inhibition of myeloperoxidase oxidative activity in an animal model. Additionally, Dr. Cunnion is a Board member for Eriko Life Science Ventures and ReAlta Life Sciences, Inc. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

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., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: Kerry Cunnion also serves as Chief Medical Officer for ReAlta Life Sciences. Neel Krishna also serves as Chief Science Officer for ReAlta Life Sciences. This does not alter our adherence to PLoS ONE policies on sharing data and materials.